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TITLE: Effects of avoiding neuromuscular blocking agents during maintenance of anaesthesia on recovery characteristics in patients undergoing craniotomy for supratentorial lesions: A randomised controlled study ABSTRACT.BACKGROUND AND AIMS:: Neuromuscular blocking agents have been one of the cornerstones of anaesthesia. With the advent of newer surgical, anaesthetic and neurological monitoring techniques, their utility in neuroanaesthesia practice seems dispensable. The aim of this prospective, comparative, randomised study was to determine whether neuromuscular blocking agents are required in patients undergoing supratentorial surgery when balanced anaesthesia with desflurane, dexmedetomidine and scalp block is used. ABSTRACT.METHODS:: Sixty patients with the American Society of Anesthesiologists physical status I or II, aged between 18 and 60 years were included in the study. All patients received anaesthesia including desflurane, dexmedetomidine and scalp block. The patients were randomly allocated to receive no neuromuscular blocking agent (Group A) or atracurium infusion to keep train-of-four count 2 (Group B). The two groups were compared with respect to haemodynamic stability, brain relaxation scores and recovery characteristics. Haemodynamic parameters and time taken to achieve Aldrete score >9 and other secondary outcomes were analysed using Student's t-test. Non-parametric data were analysed using the Mann–Whitney test. ABSTRACT.RESULTS:: The mean arterial pressure was comparable between the groups. The intraoperative heart rate was comparable; however, in the post-operative period, it remained higher in Group B for 30 min after extubation (P = 0.02). The brain relaxation scores were comparable among the two groups (P = 0.27). Tracheal extubation time, time taken for orientation and time required to reach Aldrete score ≥9 were comparable among the two groups. ABSTRACT.CONCLUSION:: The present study suggests that balanced anaesthesia using desflurane, dexmedetomidine and scalp block can preclude the use of neuromuscular blocking agents in patients undergoing supratentorial surgery under intense haemodynamic monitoring. BODY.INTRODUCTION: The goals during anaesthesia for neurosurgery are the maintenance of haemodynamic stability, optimal operative conditions, ensuring early emergence for neurological assessment and allowing electrophysiological monitoring. Keeping these goals in mind, the necessity of neuromuscular blocking agents may be debatable. Residual effects of neuromuscular blocking agents can produce post-operative respiratory complications with increased chances of hypoxia and hypercarbia, thereby increasing intracranial pressure (ICP). The analysis of electrophysiological monitoring can be confounding when neuromuscular blocking drugs are used. Neuromuscular blocking agents are required during maintenance of anaesthesia to keep the patient immobile, facilitate mechanical ventilation and ensure optimal operating conditions by paralysis of muscles. All these goals can be achieved using balanced anaesthesia with inhalational agents or target controlled infusion of propofol, alpha 2 agonists such as dexmedetomidine and regional blocks such as scalp block. Furthermore, there are no muscles in the scalp which require to be paralysed to improve surgical conditions. Desflurane, a volatile anaesthetic with low blood solubility, allows for early recovery after anaesthesia. In neurosurgery, it may facilitate early post-operative neurological assessment.[12] Scalp block can blunt the haemodynamic response to noxious stimuli, decrease the amount of opioids and reduce post-operative pain[34] facilitating smooth and early emergence. Dexmedetomidine provides good perioperative haemodynamic stability, decreases the requirement of intraoperative opioid and has minimal effect on ICP. This makes it a suitable adjuvant to neurosurgical anaesthesia.[56] Dexmedetomidine has been shown to reduce the minimum alveolar concentration (MAC) of inhalation anaesthetics and the need of perioperative fentanyl.[789] The aim of our study was to assess the time taken for discharge from the Post-Anaesthesia Care Unit (PACU) by measuring the time taken to achieve an Aldrete score of 9 as the primary end-point and testing the hypothesis that omitting neuromuscular blocking agents can result in faster discharge from PACU. BODY.METHODS: After the Institutional Ethics committee approval and written informed consent, sixty patients (30 in each group) of the American Society of Anesthesiologists (ASA) physical status I and II, age ranging from 18 to 60 years of either sex with a Glasgow Coma Score of 15, undergoing elective craniotomy for supratentorial lesions, were enrolled in this prospective randomised study in a tertiary care hospital. The patients were randomly allocated to either Group A (without use of neuromuscular blocking agent) or to Group B (with atracurium infusion to keep train-of-four count 2). Randomisation was done by picking chits allocating the patient to either study group by a person not involved in the study. The patients, neurosurgeons assessing brain relaxation, nurses in the PACU and outcome assessors were blinded to the group allocation. The use of neuromuscular monitoring prevented intraoperative blinding of the anaesthesiologist managing the case. The criteria for exclusion were baseline heart rate (HR) <50 beats/min, pregnant and lactating patients, patient on β blockers, sick sinus syndrome and known hypersensitivity to drugs being used and/or refusal to consent. The criteria for withdrawal were defined as severe intraoperative bleeding and elective ventilation due to the surgical cause. Pre-induction monitoring included electrocardiogram, oxygen saturation and non-invasive blood pressure. Infusion of dexmedetomidine (loading dose of 0.5 μg/kg over 10 min followed by maintenance dose 0.2–0.7 μg/kg/min) was started as soon as intravenous (IV) access was established. IV preservative-free 2% lignocaine 1.5 mg/kg, midazolam 0.02 mg/kg and fentanyl 2 μg/kg were administered before induction of anaesthesia with propofol 2 mg/kg. Tracheal intubation was assisted with atracurium 0.75 mg/kg. End-tidal CO2, respiratory gas monitoring with MAC monitoring and neuromuscular monitoring were initiated after induction. Anaesthesia was maintained on O2, N2O, desflurane (MAC 0.8–1.0) and dexmedetomidine in Group A and O2, N2O, desflurane (MAC 0.8–1.0) dexmedetomidine and atracurium infusion (4–6 μg/kg/min) to keep train-of-four count (TOF) of 2 in Group B. Scalp block with 0.25% bupivacaine was given to patients in both groups. In case of patient movement, propofol was started at 25 μg/kg/min and titrated as per effect. Vital parameters were monitored every 10 min. After opening the dura, surgeon satisfaction with brain relaxation was gauged by Brain Relaxation Scoring Scale on a scale from 1 to 4 [Table 1]. Table 1 Brain relaxation scoring scale Propofol and atracurium infusions were stopped at the end of dura closure. Dexmedetomidine and desflurane were discontinued at the end of skin closure. Paracetamol 15 mg/kg was given IV at the time of dura closure. Only patients in Group B were reversed with glycopyrrolate 0.008 mg/kg and neostigmine 0.06 mg/kg at TOF count ≥3. Patients were extubated when no fade was detected on double burst stimulus. The time taken for eye opening and extubation was noted. The total amount of dexmedetomidine and propofol used was calculated. The time taken for orientation and ability to call out name and city of residence were noted. Vital parameters were monitored every 15 min for 1st h and then at 60 min, 90 and 120 min after extubation. The time taken to reach modified Aldrete score of 9 was recorded. Bradycardia (HR <50/min) was treated with atropine 0.6 mg IV. Hypertension (>20% increase from baseline value) and tachycardia (HR >100/min) were first treated with increasing dose of dexmedetomidine and one dose of fentanyl 1 μg/kg, if required, after 5 min. For persistent hypertension, 10 mg aliquots of esmolol were given, or nitroglycerine infusion was started depending on HR. Hypotension (defined as a decrease in mean arterial pressure [MAP] <60 mm Hg for >1 min) was managed with increased rate of infusion of crystalloids. If this was not effective, 6 mg aliquots of ephedrine were used. Recovery from anaesthesia was the primary outcome and was assessed by measuring the time taken for reaching a modified Aldrete score of 9 after stopping desflurane and dexmedetomidine. The Aldrete score was assessed every 15 min after extubation in the PACU. The secondary end-points included intraoperative and post-operative HR and MAP, tracheal extubation time, time taken for eye opening and orientation, surgeon's satisfaction with the intraoperative degree of brain swelling and requirement of dexmedetomidine, fentanyl and other drugs to maintain haemodynamic stability. Brain relaxation scoring scale was used for assessing the surgeon's satisfaction with the operative field. Sample size calculation, to achieve 80% power of the study, was on the basis of the previous study investigating the recovery charecteristics of desflurane, which demonstrated the mean time to achieve Aldrete score ≥8 as 9.58 ± 6.13 min.[1] We determined that 27 patients would be required for each group to detect 80% difference (8 min) in the time taken to achieve an Aldrete score of 9, using α and β values of 0.05 and 0.2. We chose to detect 80% difference as a smaller change would be clinically insignificant. P < 0.05 was considered statistically significant. Gender, ASA grade and brain relaxation score were analysed using Chi-square test. Haemodynamic parameters and time taken to achieve Aldrete score >9 and other secondary outcomes were analysed using student t-test using Microsoft Excel 2010. Non-parametric data were analysed using the Mann–Whitney test. BODY.RESULTS: Sixty patients were identified and randomised for the study. No patient had to be excluded from the study on the basis of withdrawal criteria. The age (Group A - 41.17 ± 11.83 years, Group B - 35.87 ± 11.58 years), sex (13 males and 17 females in both groups), weight (Group A - 54.47 ± 10.94 kg, Group B - 55.57 ± 10.62 kg), ASA status, type of lesion as well as duration of anaesthesia (Group A - 225.17 ± 47.86 min, Group B 231.67 ± 41.03 min) were comparable among the two groups. There was no significant difference in the HR for most part of the intraoperative and post-operative period. The HR was significantly higher in Group B intraoperatively between 210 min and 260 min. The HR was significantly higher in Group B in the immediate post-operative period for first 30 min (P = 0.024) [Figure 1]. There was no significant difference between the MAP in the intraoperative as well as post-operative period [Figure 2]. No significant difference was found between the requirement of propofol, dexmedetomidine, fentanyl, atropine and ephedrine [Table 2]. The time taken for opening eyes, for extubation, for orientation and for achieving Aldrete score >9 was comparable among the two groups [Table 3]. Brain relaxation scores were also comparable (P = 0.271) [Figure 3]. One patient in each group was administered nitroglycerine for control of blood pressure. Two patients in Group A and four patients in Group B had transient fall in MAP up to 44 mm of Hg, which responded to fluid challenge and aliquots of ephedrine. Dexmedetomidine was stopped in four patients in Group A and three patients in Group B due to hypotension. However, hypotension was corrected promptly after stopping dexmedetomidine. No patient required inotropic support. All patients were extubated at the end of surgery without any neurological deficit attributable to hypotension. The incidence of minimal patient movement was same in both groups (three patients in each group). None of the patients desaturated or had convulsions in the post-operative period. One patient in Group B has post-operative nausea vomiting. Figure 1Comparison of mean heart rate Figure 2Comparison of mean arterial blood pressure Table 2 Comparison of dose requirement Table 3 Comparison of recovery time Figure 3Comparison of brain relaxation score BODY.DISCUSSION: We observed from our study that there was no difference between the two groups in the recovery profiles, haemodynamic parameters or surgical conditions. The Aldrete score of 9 was achieved at 15 min (time of first assessment) in both groups and is similar to the time (9.58 min) obtained previously.[1] The time taken for eye opening and orientation in both groups was not significantly different and it matched the time recorded (8.1 ± 7.0 min and 12.0 ± 8.2 min, respectively) in the previous study where a much higher dose of dexmedetomidine (0.9 μg/kg/h) was used.[5] Desflurane-dexmedetomidine anaesthesia offers faster eye opening, response to verbal commands and post-operative neurologic assessment than sevoflurane-dexmedetomidine or isoflurane-dexmedetomidine anaesthesia.[5] Dexmedetomidine infusion provides faster recovery after anaesthesia without inducing respiratory depression after extubation as compared to fentanyl.[10] Dexmedetomidine, when administered as a single dose IV before the induction of anaesthesia, shortens the period for recovery from the anaesthesia.[11] Balanced anaesthesia without neuromuscular blocking agents appears to achieve the goals of neuroanaesthesia as seen in our study. The average age-adjusted MAC value to maintain BIS between 45 and 50 for desflurane has been found to be 0.65 ± 0.04.[12] Hence, desflurane with nitrous oxide with a MAC between 0.8 and 1.0 was chosen for our study. Maintenance of perioperative haemodynamic stability is an important goal of neuroanaesthesia practice. Hypertension can result in intracranial haemorrhage and cerebral oedema and hypotension may exacerbate neuronal injury in hypoperfused areas of the brain. In our study, the haemodynamic profile was similar in both groups at most points of observation during the study period. The HR was significantly higher in Group B intraoperatively between 210 min and 260 min. However, only 11 patients in Group A and 20 patients in Group B had surgeries lasting beyond 210 min. The sample size of the study was not calculated to detect changes in HR and MAP as the primary end-point. Hence, there is a possibility of error in evaluation of the significance of haemodynamic results. The HR in the immediate post-operative period is higher in Group B. This can be attributed to the effect on glycopyrrolate administered at the time of reversal only in Group B. The absence of significant difference in the MAP among the two groups indicates the efficacy of this combination in maintaining target haemodynamic parameters. Scalp block with 0.25% bupivacaine is an effective adjuvant for maintaining stable haemodynamics for patients undergoing craniotomy during general anaesthesia.[13] Dexmedetomidine administered during neuroanaesthesia reduces the requirement of opioids, antihypertensive treatments and offers better haemodynamic stability.[6] Similarly, low concentration of desflurane combined with remifentanil or fentanyl provides good control of blood pressure in patients who underwent intracranial surgery for vascular or space-occupying lesions.[14] A slack brain provides a good surgical field and reduces the need for brain retraction. We used the brain relaxation scoring scale as a surrogate for ICP monitoring as described earlier.[1] We did not find any significant difference between the scores of the two groups. Dexmedetomidine has been shown to provide good operating conditions.[15] Propofol, sevoflurane and desflurane have the same and acceptable effects on the brain relaxation scores at various stages of surgery.[16] Dexmedetomidine is not sufficient to suppress haemodynamic responses but decreases the requirement of inhalation agents and provides brain relaxation and good surgical field exposure conditions without any side effect in patients undergoing supratentorial craniotomy.[5] The necessity of neuromuscular blockade in supratentorial surgeries has not yet been studied extensively. To the best of our knowledge, there has been no study which has analysed the use of desflurane-dexmedetomidine without using neuromuscular blocking agents in neurosurgical cases. The incidence of patient movement was same between both groups and could have been possibly due to inadequate depth of anaesthesia. The movement did not occur during the main surgical procedure with the microscope. Maintaining adequate depth of anaesthesia eliminated the problem in both groups. There were few limitations of the study; intraoperative blinding could not be established due to neuromuscular blockade monitoring. The major concern with omitting neuromuscular blocking agents is that the higher concentration of anaesthetic agents required to maintain anaesthetic depth may lead to haemodynamic instability/depression requiring inotropic support.[17] However, we did not encounter any major haemodynamic issue. We did not have depth of anaesthesia monitoring. Use of Bispectral Index® or Entropy® could have guided the titration of anaesthetic agents better. The study was not designed to study the effect of desflurane–dexmedetomidine combination on intraoperative haemodynamic parameters. Hence, there can be an error in evaluating the incidence of hypotension that has been encountered. Furthermore, we did not have the facilities for evoked potential monitoring. The exact utility of omitting neuromuscular blocking agents in the presence of such monitoring could not be assessed. Further research utilising the protocol with evoked potential monitoring will render more information as it has been previously reported that with evoked potential monitoring, no neuromuscular blockade is preferred over the partial neuromuscular blockade.[18] BODY.CONCLUSION: Goals of anaesthesia for supratentorial surgeries can be achieved with balanced anaesthesia with desflurane-dexmedetomidine and scalp block. We would like to highlight that supratentorial craniotomies can be performed by avoiding relaxants. This may help anaesthesiologists in the decision regarding the use of neuromuscular blocking agents during neurosurgery. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Comparative Evaluation of Crystalloid Resuscitation Rate in a Human Model of Compensated Haemorrhagic Shock ABSTRACT.ABSTRACT.INTRODUCTION:: The most effective rate of fluid resuscitation in haemorrhagic shock is unknown. ABSTRACT.ABSTRACT.METHODS:: We performed a randomized crossover pilot study in a healthy volunteer model of compensated haemorrhagic shock. Following venesection of 15 mL/kg of blood, participants were randomized to 20 mL/kg of crystalloid over 10 min (FAST treatment) or 30 min (SLOW treatment). The primary end point was oxygen delivery (DO2). Secondary end points included pressure and flow-based haemodynamic variables, blood volume expansion, and clinical biochemistry. ABSTRACT.ABSTRACT.RESULTS:: Nine normotensive healthy adult volunteers participated. No significant differences were observed in DO2 and biochemical variables between the SLOW and FAST groups. Blood volume was reduced by 16% following venesection, with a corresponding 5% reduction in cardiac index (CI) (P < 0.001). Immediately following resuscitation the increase in blood volume corresponded to 54% of the infused volume under FAST treatment and 69% of the infused volume under SLOW treatment (P = 0.03). This blood volume expansion attenuated with time to 24% and 25% of the infused volume 30 min postinfusion. During fluid resuscitation, blood pressure was higher under FAST treatment. However, CI paradoxically decreased in most participants during the resuscitation phase; a finding not observed under SLOW treatment. ABSTRACT.ABSTRACT.CONCLUSION:: FAST or SLOW fluid resuscitation had no significant impact on DO2 between treatment groups. In both groups, changes in CI and blood pressure did not reflect the magnitude of intravascular blood volume deficit. Crystalloid resuscitation expanded intravascular blood volume by approximately 25%. BODY.INTRODUCTION: Haemorrhagic shock is a clinical syndrome resulting from decreased perfusion of vital organs secondary to a loss of blood volume. The World Health Organization estimated that approximately 5 million people die annually from injury, with haemorrhage contributing to more than one-third of the deaths (1). The current Advanced Trauma Life Support (ATLS) guidelines for the management of haemorrhagic shock advocate rapid infusion of 1–2 L of crystalloid solution in the absence of the matched blood products (2). This traditional teaching of resuscitation appears to be based on expert opinion with minimal supporting evidence from human clinical trials (3). Furthermore, there is no current consensus regarding exactly what defines a "fluid bolus" and the most effective rate of fluid resuscitation in haemorrhagic shock is unknown. Although animal studies have shown that resuscitation with a fast fluid infusion had worse outcome compared with a slow fluid infusion in various models of haemorrhagic shock (4–10), prospective research in humans on the same comparison is lacking. While fast crystalloid resuscitation has been associated with increased mortality and complications in patients with severe sepsis (11), there are no human studies to date comparing the rate of crystalloid resuscitation for patients in haemorrhagic shock. Therefore, we evaluated, in a model of compensated haemorrhagic shock, whether a slow (SLOW treatment) or fast (FAST treatment) rate of resuscitation with crystalloid infusion would differ in terms of oxygen delivery (DO2), pressure- and flow-based haemodynamic variables, and clinical biochemistry including N-terminal pro B-type natriuretic peptide (NT-BNP). BODY.PATIENTS AND METHODS: The Austin Health Research and Ethics Committee approved this study (HREC no: HREC/14/Austin/458) and written informed consent was obtained from all participants. The study was prospectively registered with the Australian New Zealand Clinical Trials Registry (ACTRN: 12615000100594). Inclusion criteria included normotensive healthy adults, 18 to 50 years of age, BMI <35 kg/m2, normal haemoglobin levels for sex, and no regular medication for comorbid disease. BODY.PATIENTS AND METHODS.STANDARDIZATION OF STUDY PROTOCOL: Nine participants were randomized into either a FAST or SLOW fluid resuscitation protocol based on a random allocation sequence generated by a computer-based randomization programme. All participants were fasted for 6 h for solids and encouraged to maintain adequate hydration with clear fluids for 2 h prior to each experiment. The study was conducted in a dedicated Anaesthesia Research Laboratory with standardization of illumination intensity and noise, and with ambient air temperature set to 21°C to avoid distractions that could alter haemodynamics during the continuous measurements. Participants were placed in a supine position on a standard hospital bed with their heads raised at 45 degrees and resting on a pillow for comfort. The Clearsite advanced haemodynamic monitor (Edwards Lifesciences, Irvine, CA) was used for continuous beat-by-beat haemodynamic monitoring. Baseline haemodynamic measurements were obtained after a 10-min resting period. A 16-gauge and 20-gauge cannula were then established aseptically using local anaesthetic solution (Topical Emla Gel and subcutaneous 2% lignocaine and adrenaline) for fluid resuscitation, blood sampling, and venesection. After the insertion of the cannula, blood pressure was allowed to stabilize to baseline values over a further 10-min period. Once haemodynamic variables had returned to precannulation baseline values, 15 mL/kg of blood ideal body weight (12) was venesected over 25 min into purpose venesection bags (Fenwal, PL146 Citrate Phosphate Dextrose 63 mL, Lake Zurich, IL), in accordance with the Australian Red Cross and hospital blood bank venesection protocols. Blood was crosschecked by two study investigators, and immediately placed in a dedicated blood fridge set at 4oC until re-infusion. A total of 450 mL (± 10%) of blood was placed in each bag, in accordance with the manufactures instructions. For incomplete bags, the 63 mL ratio of Citrate Phosphate Dextrose to venesected blood was adjusted accordingly to keep the anticoagulant ratio of blood returned consistent. Calcium chloride was not infused with the return of the venesected blood. After 30 min of continuous haemodynamic monitoring, Plasmalyte (1.3 × total venesected blood volume in milliliter) was infused over 10 min (FAST) or 30 min (SLOW) using dedicated volumetric pumps to control the infusion rate and volume infused. After 2 h of monitoring, and completion of the study protocol, the venesected blood was re-infused over 30 min. Participants were observed for a further 60 min to ensure no adverse effects of the reinfusion blood were reported. After a 2-week period participants returned for the alternate arm of the crossover study. BODY.PATIENTS AND METHODS.OUTCOMES AND DATA COLLECTED.PRIMARY OUTCOME: OXYGEN DELIVERY: The primary outcome variable was change in oxygen delivery (ΔDO2), comparing DO2 at 120-min post-resuscitation (T6) with the baseline DO2 value. The difference in ΔDO2 under the FAST and SLOW treatments, ΔDO2Fast-Slow, allowed assessment of resuscitation strategy effectiveness. Additionally, we examined the overall difference between treatments in DO2 over the 120-min monitoring period that followed fluid resuscitation. We assumed that the contribution of free oxygen to the total oxygen delivery was negligible and DO2 was calculated using the following formula (12): DO2 = CI × 1.34 × [Hb] × SaO2 whereDO2 is arterial oxygen delivery (mLO2/min/m2)Hb is concentration of haemoglobin (g/dL)SaO2 is haemoglobin oxygen saturationCI is cardiac index (L/min) BODY.PATIENTS AND METHODS.OUTCOMES AND DATA COLLECTED.SECONDARY OUTCOMES: Secondary outcomes included pressure-based haemodynamic variables: mean arterial pressure (MAP), systolic blood pressure (sBP), diastolic blood pressure (dBP); flow-based haemodymanic variables: cardiac index (CI), stroke volume index (SVI), systemic vascular resistance index (SVRI), and pulse rate; blood volume (BV); biochemical variables including, haemoglobin, venous blood gas variables (pH, pCO2, bicarbonate, standard base excess, glucose, and lactate), electrolytes (sodium, potassium, calcium, magnesium, phosphate, and albumin), and N-terminal pro B-type natriuretic peptide (NT-BNP). BODY.PATIENTS AND METHODS.OUTCOMES AND DATA COLLECTED.HAEMODYNAMIC VARIABLES: Haemodynamic variables were measured continuously beat-to-beat with the noninvasive Edwards Life Science ClearSight monitor that is based on the Nexfin system technology. The Nexfin system gained approval from the Association for the Advancement of Medical Instrument and has been validated against other intermittent noninvasive (13) and continuous invasive haemodynamic monitoring methods (14). This system uses the volume clamp method (15) and the physiocal method (16) to formulate an accurate real-time construction of the pressure based haemodynamic waveform. Using the blood pressure measurements, the Nexfin system derives CI, SVI, and SVRI through validated algorithms. Similar to the measurements of DO2, haemodynamic variables at 120-min post-resuscitation (T6) were compared to baseline values for both the FAST and SLOW groups. This comparison between the groups is denoted as ΔHaemodynamic variablefast-slow. We also examined the overall difference between the two groups in blood pressure and flow-based haemodynamic variables for the 120-min post-resuscitation monitoring period. BODY.PATIENTS AND METHODS.OUTCOMES AND DATA COLLECTED.BLOOD VOLUME: The blood volume at baseline (BVo) was estimated by assuming that the blood volume amounts to 7% of the absolute body weight. The blood volume change (ΔBV) at time t when no bleeding occurred was calculated as follows: ΔBVT = BVo ([Hbo]/ [HbT]) − BVo where the subscript "o" denotes baseline values, and subscript "T" denotes values at a later time. The amount of fluid retained in the blood (efficacy of the fluid) is given by: fluid retained (%) = 100 × ΔBVT/infused volume To account for withdrawn blood, the total Hb content (total Hb) of BVo was first calculated, and then all losses of Hb were subtracted from this product. BVT was then obtained dividing the remaining Hb mass by HbT: Total Hbo = BVo × [Hbo] BVT = (Total Hbo – Hbloss)/[Hbt] ΔBVT = BVT – BVo BODY.PATIENTS AND METHODS.OUTCOMES AND DATA COLLECTED.BIOCHEMISTRY AND BLOOD GASES: A total of 20 mL of blood was collected for the measurements of serum biochemistry and venous blood gases at seven time points: baseline, post-venesection, preresuscitation, 0-min post-resuscitation, 60-min post-resuscitation, 120-min post-resuscitation, and post-blood re-infusion. Serum biochemistry was measured at each time point at 37°C. Calcium, magnesium, phosphate, and albumin were measured with a Cobas analyzer (Roche Diagnostics, Rotkreuz, Switzerland), using a standardized photometric module, linear and non-linear multipoints, and a two-point calibration. Measurements of venous pH, CO2, bicarbonate, glucose, lactate, and routine electrolytes in venous blood were completed on an ABL 800 Blood Gas Analyzer (Radiometer, Copenhagen, Denmark) with a fully automated micromode eliminating risk of user-induced bias or loss of accuracy with very small samples, and an interference-protected lactate analysis. The machine calculates the bicarbonate concentration using the Henderson–Hasselbalch equation and the standard base excess using the Van Slyke Eq. (17). BODY.PATIENTS AND METHODS.OUTCOMES AND DATA COLLECTED.N-TERMINAL PRO B-TYPE NATRIURETIC PEPTIDE: The venous blood samplings from each experiment were also used for the measurements of NT-pro B-type natriuretic peptide at four time points: baseline, 0-min post-resuscitation, 60-min post-resuscitation, 120-min post-resuscitation with Cobas e602 immunoassay analyzer (Roche Diagnostics). BODY.PATIENTS AND METHODS.OUTCOMES AND DATA COLLECTED.STATISTICS: Sample calculations were based on comparing two dependent sample populations where a sample size of nine participants provided the desired power of 0.8 and allowed a detection of 75 mL/min/m2 difference in DO2 between the two groups. In the primary outcome analysis to compare ΔDO2 (the difference between DO2 at 120-min post-resuscitation (T6) and the baseline DO2 value) and all the haemodynamic variables under FAST and SLOW treatments, a Wilcoxon signed-rank test was used to test the hypothesis that there was no difference between the two treatments, i.e., that ΔDO2Fast-Slow was 0. For DO2 and the haemodynamic variables, we also used a longitudinal analysis. Due to the cross-over nature of the study, where all the patients have undergone both FAST and SLOW treatments and multiple repeated measures over time were taken under both treatment regimes, there were two levels of "nestedness" in the data: the treatment data was "nested" in the individual time points and the time data was "nested" within individual participants. To appropriately account for this multilevel data structure, we utilized three-level random effect generalized linear regression models with DO2 or the haemodynamic variables as the dependent variable, FAST/SLOW treatment as independent variable, order of treatment and time as adjustment covariates, and time point and participant being random effects. Given the exploratory nature of this study, no formal adjustment for multiplicity of testing was undertaken, and P = 0.05 was regarded as significant for every outcome. This may yield a potential increase in Type 1 error rate, which is acceptable given the context of the study. BODY.RESULTS: Nine participants consented and received crystalloid at the designated rate according to the study protocol. There were no breaches or violations in the study. One participant had a decrease in ionized calcium from their baseline value (1.1–0.86 mmol/L) immediately after crystalloid resuscitation (attributed to haemodilution). The participant complained of mild tingling of the lips that resolved over 5 min. There were no other adverse events during the experiment or during the 60 min observation period post reinfusion of the blood. One participant noted mild bruising over the cannula sight the following day, which resolved within 72 h. Seven of the participants were male. The median (IQR) age was 27 years (24–39 years). The mean (SD) height and the IBW were 177.6 cm (1.5 cm) and 74.7 kg (14.5 kg), respectively. The mean (SD) venesection volume was 1069 mL (326 mL). The mean crystalloid infusion volume was 1390 mL (424 mL). The mean (SD) infusion rate for the FAST and the SLOW group was 139 mL/min (42.4 mL/min) and 46.3 mL/min (14.1 mL/min), respectively. BODY.RESULTS.PRIMARY END POINT: OXYGEN DELIVERY (DO: Changes in oxygen delivery (DO2) at baseline, post-venesection, preresuscitation, 0-min, 60-min and 120-min post-resuscitation are summarized in Table 1. The median (IQR) of ΔDO2Fast-Slow from baseline values at 120-min was 55 mLO2/min/m2 (−192 to 13 mLO2/min/m2; P = 0.250) (Table 2). No statistically significant changes in DO2 between the treatments over the duration of the study were identified. Even after adjusting for gender, BMI, treatment order and time points, no statistically significant differences were observed. BODY.RESULTS.SECONDARY END POINTS.PRESSURE-BASED HAEMODYNAMIC VARIABLES (MAP, SBP, AND DBP): Changes in haemodynamic variables at baseline, post-venesection, preresuscitation, 0, 60, and 120-min post-resuscitation are summarized in Table 1. Comparing the 120-min post-resuscitation haemodynamic values to the baseline values, the median (IQR) of ΔMAPFast-Slow, ΔsBPFast-Slow, and ΔdBPFast-Slow was 3 mm Hg (−5 to 8 mm Hg; P = 0.516); 3 mm Hg (−7 to 12 mm Hg; P = 0.289); and 3 mm Hg (−4 to 6 mm Hg; P = 0.621), respectively (Table 2). Longitudinal analysis showed that MAP, sBP, and dBP were all significantly higher under the FAST treatment compared with the SLOW treatment (P = 0.035; P = 0.046; P = 0.035) (Table 2). BODY.RESULTS.SECONDARY END POINTS.FLOW-BASED HAEMODYNAMIC VARIABLES (CI, SVRI, SVI): Changes in these haemodynamic variables at baseline, post-venesection, preresuscitation, 0, 60, and 120-min post-resuscitation are summarized in Table 1. At 120-min post-resuscitation, compared with baseline values, the median (IQR) of ΔCIFast-Slow and ΔSVRIFast-Slow was 0 L/min/m2 (−0.4 to 0.3 L/min/m2; P = 0.883) and 150 dyne s m2/cm5 (−172 to 296 dyne s m2/cm5; P = 0.426), respectively (Table 2). There were no differences in CI or SVRI between the two groups based on our longitudinal analysis (P = 0.432; 0.964). Changes in cardiac index from baseline to 120-min post-resuscitation are presented graphically in Figure 1. During the resuscitation phase (time period between Shock and Resus Complete in Fig. 1), CI paradoxically declined in eight participants (89%) under the FAST treatment in the last quarter of this phase. These findings were coupled with reporting of abdominal discomfort, chest, and facial fullness during resuscitation in six participants (67%) under the FAST treatment, compared with none under the SLOW treatment. Similar to the pattern change found in CI during the resuscitation phase, four participants (44%) under the FAST treatment also had a decrease in SVI during during the final stages of resuscitation. This is in contrast to the SLOW treatment group where all participants had a consistent increase in the SVI throughout the resuscitation phase, which peaked within 30 min post-resuscitation. Fig. 1Box-and-Whisker plot showing median changes in cardiac index observed during FAST (20 mL/kg crystalloid over 10 min) and SLOW (20 mL/kg crystalloid over 30 min) resuscitation in a human model of compensated haemorrhagic shock. BODY.RESULTS.SECONDARY END POINTS.PULSE RATE: Changes in pulse rate at baseline, post-venesection, preresuscitation, 0, 60, and 120-min post-resuscitation are summarized in Table 1. At 120-min post-resuscitation, compared with baseline values, the median (IQR) ΔHRFast-Slow and ΔSVIFast-Slow was −4 bpm (−11 to 3 bpm) and −1 mL/b/m2 (−6 to 10 mL/b/m2), respectively, (P = 0.191; P = 0.562) (Table 2). Longitudinal analysis indicated that the pulse rate under the FAST treatment was significantly higher than under the SLOW treatment (P = 0.022). The pulse rate was uniformly increased under the FAST treatment during the resuscitation period, compared with an increase in <50% of the participants under the SLOW treatment. BODY.RESULTS.SECONDARY END POINTS.INTRAVASCULAR BLOOD VOLUME (BV): The estimated mean (SD) BVo was 5.1 (1.0) L before venesection commenced. Figure 2 shows the change in BV with the change in CI over the period of the study. In both groups, venesection reduced the BV by 16%, while CI decreased by only 5% (P < 0.0011). During the resuscitation phase, the SLOW and FAST infusions increased BV by 720 (210) and (920) (223) mL, respectively (P < 0.03). This increase in BV corresponds to 54 (7%) and 69 (16%) of the infused volume of crystalloid fluid. The immediate BV expansion was attenuated with time, and represented 24% and 25% of the infused volume 30 min post-resuscitation. The retransfusion of the venesected blood increased CI by a similar magnitude as the infused volume (1.05 L). Fig. 2Changes in percentage in cardiac index in relation to blood volume in a human model of compensated haemorrhagic shock.A, fast infusion. B, slow infusion. T1 indicates baseline; T2, post-venesection; T3, preresuscitation; T4, 0-min post-resuscitation; T5, 60-min post-resuscitation; T6, 120-min post-resuscitation; T7, post-blood transfusion. BODY.RESULTS.SECONDARY END POINTS.BIOCHEMISTRY, OXYGEN SATURATIONS, VENOUS BLOOD GAS, AND N-TERMINAL PRO B-TYPE NATRIURETIC PEPTIDE (NT-BNP): Changes in the above variables are summarized in Tables 3 and 4. No statistically significant differences were found in venous acid base variables, electrolytes, oxygen saturations, haemoglobin, glucose, lactate, or BNP-NT values between the FAST and SLOW treatment regimes at any time point BODY.DISCUSSION.KEY STUDY FINDINGS: To date, there are no human studies that compare rates of crystalloid resuscitation in a haemorrhagic shock model. In this randomized crossover study to compare the effects of FAST and SLOW crystalloid resuscitation in a model of compensated haemorrhagic shock, we found that despite higher pulse rates and blood pressure values in the FAST group, there were no differences in DO2, flow-based haemodynamic parameters, BV, and clinical biochemistry. However, during the resuscitation phase, CI and SVI decreased under the FAST treatment before resuscitation was complete, suggesting a degree of impaired myocardial performance. Throughout the experiment we observed important physiological findings. During venesection under both treatments, the amount of withdrawn blood was not fully accounted for by the observed decrease in blood volume. This is likely attributed to physiological compensation by capillary refill observed in hypovolaemia induced by exsanguination (18). This result is consistent with findings in conscious sheep, where one-third of the lost blood volume is compensated by capillary refill within the first 30 min after haemorrhage, after which compensation diminishes (19). This spontaneous compensation observed in animals is consistent with our study, where approximately 300 mL of the venesected blood was promptly replaced by capillary refill by the end of the venesection, after which restoration slowed until the resuscitation commenced. While colloid infusion is thought to be more effective in intravascular expansion compared with crystalloid infusion (20), we observed that the immediate plasma volume expanding effect of crystalloid fluid is close to what is expected from a colloid fluid (21). By 30 min after the crystalloid infusion, fluid kinetics between the intravascular and the interstitial space reached equilibrium, where the final plasma volume expansion was only 25% of the transfused volume, a finding consistent with most medical textbooks. Our results also show that the crystalloid fluid, after complete distribution, expands the blood volume by 25% even when volunteers are hypovolaemic. In contrast, hydroxyethyl starch (22) and autologous plasma (23) given to replace blood that has just been withdrawn from volunteers seem to remain there. We did not find any significant correlation between changes in CI and blood volume. Importantly, the CI did not decrease to the same proportion as the blood volume in response to venesection. In addition, CI increased to 20% above baseline in response to the crystalloid resuscitation despite the failure to restore hypovolaemia to baseline. The discrepancy between the change in CI and blood volume is likely related to the stress response induced by a single hypovolaemic episode, evident by the mild tachycardia, hyperglycaemia, and improvement in cardiac contractility (20% increase in CI) for several hours. Unlike animal studies that have shown that the MAP is higher and more effectively maintained in a SLOW fluid resuscitation (6, 9), we found that the pressure-based haemodynamic variables were better preserved in the FAST group in our study. In accordance with the increasing evidence suggesting that blood pressures does not accurately reflect oxygen delivery and tissue perfusion (24), we found that the higher blood pressures resulted from the FAST resuscitation did not yield superiority in DO2. The discrepancies between the findings in animal studies and our findings are not surprising. In contrast to our controlled compensated haemorrhagic shock model, most of the animal studies adopted haemorrhagic shock model that involved uncontrolled bleeding and more significant blood loss secondary to large vessels and solid organ injuries (4–10). Moreover, a variety of resuscitation fluids were infused at arbitrarily predetermined rates in these animal studies. Unfortunately, the combination of the differences in physiology and in the experimental protocols precludes direct comparison. The higher blood pressures observed under the FAST treatment can be attributed to the rapid increase in ventricular preload, increased SV, and higher pulse rate compared with the SLOW treatment. This increase in pulse rate may be a result of an increase in body temperature due to the warm crystalloid being rapidly infused during the resuscitation period in addition to the participants' anxiety associated with receiving the FAST resuscitation. Despite the pressure-based haemodynamic variables being higher under the FAST treatment, a more critical analysis of haemodynamic variables during the actual resuscitation period showed that CI peaked late during resuscitation and then decreased before the resuscitation was complete (Fig. 1). This finding was not observed under the SLOW treatment, where CI and SVI continued to increase throughout the resuscitation period. The decline in CI might be simply reflective of a baroreceptor response to a rise in blood pressure, which would fit with our observations that there were no elevations in NT-BNP. The stretching of the myocardial wall from the increased venous return is directly responsible for the release of NT-BNP. Studies have reported that the level of NT-BNP increases as the resuscitation volume increases, suggesting that NT-BNP may be a useful marker of fluid overload from resuscitation (25, 26). Following fluid resuscitation NT-BNP levels may increase at least 12 h after the fluid intervention; therefore, our study might not have allowed sufficient time (2 h) to capture the accurate level of NT-BNP. The initial choice between BNP and NT-BNP to detect the stretching of myocardial wall induced by fluid overload from resuscitation was based on assay availability at our institution and the sensitivity of these two hormones. BNP has a shorter half-life (22 min) than that of NT-BNP (120 min) (27) and might have been sufficient to detect any measurable responses in our study; however, we preferred NT-BNP for its higher sensitivity and stability (28). Nevertheless, not extending the collection of NT-BNP for at least 12 h is a limitation of the present study. The decrease in SVI and CI during the end of the resuscitation phase under the FAST treatment could also be due to over resuscitation from aggressive fluid intervention. Stretching of the myocardial walls by the FAST resuscitation might have exceeded the optimal contractility point on the Frank–Starling curve. Following the Frank–Starling theory, a rapid increase in venous return theoretically should have increased the LVEDV, which should in turn have increased the SV. In our study, during the resuscitation period this was not observed. Instead, there was a decline in both SVI and CI (despite a significant increase in HR) in the FAST group most noticeable before the end of the resuscitation. These clinical observations in hemodynamics changes support the postulation that the myocardial wall of the ventricle was possibly overfilled and stretched beyond the optimal contractile response, consequently leading to under-performance by the myocardium. Finally, a rapid dilution of serum calcium in the FAST group could also have affected the changes observed in CI at the end of the resuscitation. We consider this to be less likely, as the magnitudes of the decreases in serum calcium from baseline were similar in both groups. BODY.DISCUSSION.STUDY IMPLICATIONS: Our findings contradict current resuscitation practices, which are based on presumption that FAST crystalloid resuscitation is superior to SLOW crystalloid resuscitation (2, 3). There were no significance differences in DO2 between the FAST and SLOW treatments groups, and changes in CI and blood pressure did not reflect the magnitude of intravascular blood volume deficit. Crystalloid resuscitation expanded intravascular blood volume by approximately 25%. This was associated with a reduction in CI and SVI during the resuscitation phase, again supporting the theory of over resuscitation and suboptimal myocardial performance. In the view of the increasing prevalence of cardiovascular disease with advancing age, the impact of FAST resuscitation on the CI observed in this study, which is of most relevance to combat casualties, might be more profound in the current aging population or patients with pre-existing cardiac dysfunction. Older subjects have a more poorly functioning adrenergic system, although stress hormones are generally higher than in younger subjects, which makes them more sensitive to changes in preloading and more prone to hypotension in hypovolaemia. Finally, the discrepancy between changes in blood volume and CI could mislead the clinician who tries to maintain the blood volume by monitoring CI alone. Neither did the reduction in CI proportionally reflect the degree of blood loss from the venesection, nor did the increase in CI accurately reflect the persisting hypovolemic state. There are several limitations to our study. We did not use invasive techniques to measure arterial blood pressure, CVP, and cardiac output. However, the accuracy of the Nexfin cardiac output technology has been validated against pulmonary thermodilution, transpulmonary thermodilution (29), transoesophageal and transthoracic echocardiography (30, 31), and inert gas rebreathing (32). Percentage errors range from 23% to 39%, comparable to more invasive methods (33, 34). Measurement of central venous pressure via a central venous catheter would have been useful in identifying the effects of resuscitation on preload and in more accurately calculating SVRI, but was considered too invasive in a healthy volunteer population. Use of transthoracic echocardiography would have allowed valuable noninvasive and focused estimations of cardiac function, preload, and fluid responsiveness. However, its continuous use over the entire duration of each experiment (>3 h) was not pragmatic. Finally, Plasma-Lyte was the choice of crystalloid solution used in this study for its physiological-like properties and its accessibility at our institution. The effects of FAST and SLOW infusion on DO2, pressure- and flow-based haemodynamic variables, biochemistry, and NT-BNP are likely to be similar from those yielded from any crystalloid solutions of different electrolyte compositions, e.g., Hartmann's solution or saline 0.9%. BODY.CONCLUSION: In a healthy volunteer model of compensated haemorrhagic shock, FAST or SLOW fluid resuscitation had no significant impact on DO2 between treatments groups. In both groups, changes in CI and blood pressure did not reflect the magnitude of intravascular blood volume deficit. Crystalloid resuscitation expanded intravascular blood volume by approximately 25%.

TITLE: The Effect of Adjuvant Zinc Therapy on Recovery from Pneumonia in Hospitalized Children: A Double-Blind Randomized Controlled Trial ABSTRACT: Objectives. Pneumonia is one of the common mortality causes in young children. Some studies have shown beneficial effect of zinc supplements on treatment of pneumonia. The present study aimed to investigate the effects of short courses of zinc administration on recovery from this disease in hospitalized children. Methods. In a parallel Double-Blind Randomized Controlled Trial at Ayatollah Golpaygani Hospital in Qom, 120 children aged 3–60 months with pneumonia were randomly assigned 1 : 1 to receive zinc or placebo (5 mL every 12 hours) along with the common antibiotic treatments until discharge. Primary outcome was recovery from pneumonia which included the incidence and resolving clinical symptoms and duration of hospitalization. Results. The difference between two groups in all clinical symptoms at admittance and the variables affecting the disease such as age and sex were not statistically significant (P < 0.05) at baseline. Compared to the placebo group, the treatment group showed a statistically significant decrease in duration of clinical symptoms (P = 0.044) and hospitalization (P = 0.004). Conclusions. Supplemental administration of zinc can expedite the healing process and results in faster resolution of clinical symptoms in children with pneumonia. In general, zinc administration, along with common antibiotic treatments, is recommended in this group of children. It can also reduce the drug resistance caused by multiple antibiotic therapies. This trial is approved by Medical Ethic Committee of Islamic Azad University in Iran (ID Number: 8579622-Q). This study is also registered in AEARCTR (The American Economic Association's Registry for Randomized Controlled Trials). This trial is registered with RCT ID: AEARCTR-0000187. BODY.1. INTRODUCTION : Acute lower respiratory tract infection is one of the most important and common diseases among children, which is accompanied by high mortality rate, especially in young children. This infection is the most important cause of mortality among children under 5 in developing countries, accounting for nearly one-third of the cases [1–4]. Pneumonia is one of the most common implications of lower respiratory tract involvement. The World Health Organization estimates that of approximately 4 million annual deaths due to pneumonia, half of the cases occur in children less than 1 year of age [2, 3, 5]. On the other hand, malnutrition plays a significant role in the increased prevalence, severity, and prognosis of pneumonia, especially among children [3]. Zinc and iron deficiency is one of the most common nutritional problems in Iran and many developed countries. According to statistics, about 50% of the common nutritional problems are due to a combined deficiency of the two elements, though the beneficial role of zinc compared to iron has been forgotten in Iran [6]. Zinc is an essential nutritional element, with a broad spectrum of biological activities in humans. This element plays an important and vital role in the physical development of digestive and immune systems. Zinc deficiency in children can cause stunted growth and increased incidence of infections (pneumonia, gastroenteritis) through weakening the immune system and changing neural and behavioral actions [1, 7]. Numerous studies on therapeutic and prophylactic effects of zinc administration in infectious diseases indicate that administration of zinc compounds significantly reduces the incidence of gastroenteritis and pneumonia, and its deficiency could cause immune system deficiencies and increase the risk of serious infectious diseases such as diarrhea and malaria [1, 8–10]. Another study showed that serum zinc level in children with pneumonia and gastroenteritis was lower than in those of the same age [11]. It should be noted that inadequate intakes of zinc in the diet were the main cause of zinc deficiency. The annual report of the World Health Organization in 2003 has emphasized on the importance of adding zinc as a food supplement to the diet. The clinical symptoms of zinc deficiency during early childhood include acute or chronic diarrhea accompanied by malnutrition, psychiatric disorders, and behavioral problems. A chronic zinc deficiency could cause alopecia, stunted growth, skin lesions, and common childhood infections such as pneumonia [12]. Zinc supplements can prevent and decrease the incidence of pneumonia. It can also shorten diarrhea episodes and resolve them [10, 13, 14]. The World Health Organization (WHO) and the United Nations Children's Fund (UNICEF) recommend that the children living in developing countries should take zinc supplement for 10 to 12 days as follows: 10 mg daily for infants younger than 6 months and 20 mg daily for infants older than 6 months. The purpose of this treatment is to reduce the severity of acute diarrhea episodes and hasten recovery from severe pneumonia in developing countries [7]. Given what was previously mentioned about the beneficial effects of administrating zinc compounds for prevention or treatment of pneumonia among children, and knowing that only a few studies have been conducted in this field, especially in Iran [15], the present study aimed to determine the effect of zinc on clinical course of 3 to 60-month children hospitalized due to pneumonia. It was assumed that this element (zinc) was effective in resolving clinical symptoms and duration of hospitalization. BODY.2. MATERIALS AND METHODS: This study was a parallel, double-blind, randomized controlled clinical trial conducted on 120 children aged 3–60 months suffering from pneumonia, after obtaining permission from the University Research Council and Ethics Committee. The participants were all patients hospitalized at Ayatollah Golpaygani Hospital in Qom, Iran, from June 2012 to June 2013. Moreover, the children's parents completed and signed the informed written consent forms for conducting the study and the relevant experiments before entering the study. The participants were randomly assigned, following simple randomization procedures (1 : 1), to receive zinc or placebo (5 mL every 12 hours) along with the common antibiotic treatments until discharge. Participants and their parents and those assessing the outcomes were blinded to group assignment. Patient assigned to placebo received the same solution as zinc supplement syrup but without zinc. Primary outcome was recovery from pneumonia which included the incidence and remission of clinical symptoms and duration of hospitalization. Diagnostic criteria of pneumonia and signs and symptoms were recorded according to Nelson Textbook of Pediatrics [7]. Family history of respiratory diseases, infections leading to hospitalization, duration of symptoms and taking medication at home, and influenza vaccination in the participants' families were recorded. Moreover, symptoms such as cough, fever, tachypnea, tachycardia, respiratory distress at admittance, and pleural effusion and bronchopneumonia on chest X-ray were evaluated. Then disease symptoms, duration of hospitalization, and duration of the resolution of clinical symptoms were investigated. The children taking zinc compounds, those suffering from severe malnutrition, and those with the symptoms of gastroenteritis, and other diseases, were excluded from the study. The data were analyzed by running the Chi-square and Fisher's tests using SPSS 16. The level of significance was considered 0.05. BODY.3. RESULTS: A total of 120 pneumonia patients were hospitalized from June 2012 to June 2013; 60 were randomized to treatment and 60 to placebo. None of them loss and exclude after randomization. The baseline characteristics were well balanced in the 2 study groups. As shown in Table 1, there was no statistically significant difference between the two groups in terms of the age and sex variables. The family history of respiratory diseases in the treatment and control groups was 18.3 and 13.3, respectively. About the administration of influenza vaccine in family, 3.3% of the subjects in the treatment group had a history of injection in the family, while the control group had no history of vaccination. In the treatment group, the rate of infection leading to hospitalization as well as pneumonia (for both variables) was estimated as 30%. In the control group, the rate of corresponding infections was 43.3% and 40%, respectively. There was no statistically significant difference between all these proportions. In the treatment group, the incidence of symptoms such as cough, fever, tachypnea, tachycardia, and respiratory distress at admittance was reported as 96.7, 36.7, 10, 6.7, and 15%, respectively. In the control group, this incidence was reported as 100, 25, 8.3, 3.3, and 16.7%, respectively. There was no statistically significant difference between these proportions in both groups. Moreover, there was no significant difference between the evaluation of chest X-ray in the two groups in terms of pleural effusion and bronchopneumonia. The prevalence of radiological findings in the treatment group was 50% and 26.7%, respectively, and the prevalence of corresponding values in the control group was 48.3% and 25%, respectively. Table 2 shows the duration of symptoms and taking medication at home as well as the duration of hospitalization and remission of clinical symptoms. As can be seen, there was a significant difference between the two groups in terms of the duration of symptoms and hospitalization. BODY.4. DISCUSSION: The present study aimed to evaluate the effect of zinc on the clinical course of pneumonia in 3 to 60-month-old children hospitalized in pediatric wards. Implementation of a random allocation might be a possible reason for the fact that in this trial, there was no statistically significant difference between the two groups in terms of some factors affecting the disease such as age, sex, family history of respiratory infections, and infections leading to hospitalization. Similar symptoms such as cough, fever, tachypnea, tachycardia, and respiratory distress in the above-mentioned groups at admittance and before the intervention indicate that the severity of the disease has been almost the same. Therefore, the major difference between the children in the treatment group can be found in the element of zinc, which is administered along with the standard antimicrobial therapy for pneumonia. In this study and compared to the comparison group, a significant decrease was found in the duration of hospitalization and recovery from pneumonia symptoms in zinc-receiving children. This indicates the effect of zinc therapy and a change in the clinical course of pneumonia among the children under investigation. This finding is consistent with the results of most studies in this field [5, 16–18], some of which are mentioned below. Brooks et al. reported that prescription of zinc in 2 to 23-month children suffering from severe pneumonia leads to significant reduction in the severity of tachypnea, anorexia, restlessness, and duration of hospitalization [5]. A similar study in India (2007) on 299 children aged 2–23 months and hospitalized due to severe pneumonia showed that, compared to the comparison group, disease symptoms were improved faster and the duration of hospitalization decreased significantly in the zinc-receiving patients [17]. In another similar study in India that was conducted on 153 children aged 2–24 months, who were hospitalized due to acute lower respiratory infection and divided into two groups (one taking 10 mg of zinc plus vitamin A daily, and the other taking placebo plus vitamin A), it was shown that the recovery time was significantly faster in the treatment group than in the control group. Overall, zinc therapy can reduce the duration of symptoms and acute clinical condition [18]. This finding is consistent with the findings of our study. Moreover, another field trial with similar implementation in India (2003), however, on 2482 healthy children aged 6–30 months, showed that the prevalence of pneumonia was lower in the treatment group than in the control group. This indicates that in addition to reducing the duration of symptoms and expediting the healing process in patients suffering from pneumonia, zinc can also prevent this disease [16]. In a study at the University of Mashhad on 200 early school age children (2009), it was found that, compared to the control group, the number of cold attacks reduced in the zinc-treated group. This shows that zinc can be useful in the prevention of respiratory diseases among children through improving their nutritional status [14]. BODY.5. CONCLUSION: According to the results of the present study and comparing them with other similar studies in this field, it can be inferred that zinc can hasten the recovery from pneumonia and quickly resolve its symptoms in children suffering from this disease. Overall, using zinc along with antibiotic therapies is recommended in this group of children. Zinc therapy can also reduce the drug resistance caused by multiple antibiotic therapies. Hence, in order to improve the clinical course and duration of symptoms, it is recommended to administer zinc supplementation to the children with suspected respiratory symptoms on their arrival at the hospital. We recommended that further studies with larger sample sizes would be useful in confirming the results of this study and reaching a conclusive opinion in this field.

TITLE: Comparative Evaluation of Continuous Lumbar Paravertebral Versus Continuous Epidural Block for Post-Operative Pain Relief in Hip Surgeries ABSTRACT.BACKGROUND:: Effective control of postoperative pain remains one of the most important and pressing issues in the field of surgery and has a significant impact on our health care system. In too many patients, pain is treated inadequately, causing them needless suffering and they can develop complications as an indirect consequence of pain. Analgesic modalities, if properly applied, can prevent or at least minimize this needless suffering and these complications. ABSTRACT.OBJECTIVES:: The aim of this study was to compare the efficacy of continuous infusions of local anesthetic drugs by paravertebral and epidural routes in controlling postoperative pain in patients undergoing hip surgeries. ABSTRACT.PATIENTS AND METHODS:: The study involved 60 patients who were undergoing hip surgery under the subarachnoid block. They were randomly divided into 2 groups of 30 patients. Group I (paravertebral group) received a single dose of spinal anesthesia with 2.5 mL 0.5% bupivacaine (heavy) + a continuous infusion of 0.125% bupivacaine at 5 mL/h in the paravertebral space. Group II (epidural group) received a single dose of spinal anesthesia with 0.5% bupivacaine (heavy) + a continuous infusion of 0.125% bupivacaine at a rate of 5 mL/hr in the epidural space for 48 hours in the postoperative period. Visual analogue scale (VAS) score, vital statistics, rescue analgesia, and procedure time were compared with the corresponding times between the 2 groups by student's t-test and repeated measures ANOVA with post hoc Bonferroni. P < 0.05 was considered significant. There were no statistically significant differences between the 2 groups regarding mean pain score in the first 48 hours. ABSTRACT.RESULTS:: Mean arterial pressure was significantly lower in the epidural group compared with the paravertebral group from 2 hours after start of the infusion until 48 hrs. Regional anesthesia procedure time was significantly longer in the epidural group (P < 0.001). There was no significant difference between the 2 groups regarding frequency of postoperative complications and catheter-related problems. ABSTRACT.CONCLUSIONS:: The results of our study indicate that for patients who are scheduled for hip surgery, both continuous paravertebral and continuous epidural analgesia are effective in controlling postoperative pain but that the former has several crucial advantages. BODY.1. BACKGROUND: The conquest of pain that is inherent to surgery is the most important event in the history of mankind. Pain following surgery is a universal phenomenon; yet, it is often underestimated and undertreated. This is likely due to difficulties in balancing an effective postoperative pain treatment regimen with acceptable side effects. Effective control of postoperative pain remains one of the most important and pressing issues in the field of surgery and has a significant impact on our health care system. Most of the hundreds of millions of people worldwide who undergo operations each year experience pain of varying intensity. In too many patients, pain is treated inadequately, causing them needless suffering, and they can develop complications as an indirect consequence of pain. Analgesic modalities, if properly applied, can prevent or at least minimize this needless suffering and these complications. Pain following hip surgery, which constitutes the majority of operations (1), especially in elderly with other comorbid conditions, is particularly severe, as this major joint operation entails massive nociceptive inputs from richly innervated joint tissue that produces continuous deep somatic pain and bouts of severe reflex spasm of the muscle that is supplied by the same and adjacent spinal cord segment and are superimposed onto the incision pain. The incidence, intensity (severity), and duration following hip surgery hare estimated to be moderate in 30% to 40% and severe in 40% to 50%, and the duration ranges from 2-4 days (2). Adequate postoperative pain relief improves the surgical outcome in terms of reduced morbidity, reduced hospital stay in the postoperative period (3), and reduced postoperative organ dysfunction (4) due to lower surgical stress. Various pharmacological and nonpharmacological methods have been used to provide postoperative pain relief. Such agents include oral analgesics, intramuscular (IM) or intravenous (IV) narcotics, nonsteroidal anti-inflammatory drugs, subanesthetic doses of ketamine, sublingual buprenorphine hydrochloride, intrathecal and epidural drugs, non-narcotics like clonidine, midazolam, ketamine, intra pleural local anesthetics or opiods, local anesthetic infiltration at the line of incision, inhalation agents (such as nitrous oxide), cryoanalgesia, transcutaneous nerve stimulation, and acupuncture. However, they effect varying results. Some of these techniques are also associated with unpredictable responses and complications. When compared with other techniques, many regional analgesic techniques, such as epidural and paravertebral blocks, provide superior pain relief, may favorably influence outcomes with regard to blood loss and thromboembolic events (5), and can lead to substantial reductions in surgical stress responses (6). BODY.2. OBJECTIVES: The purpose of this study was to compare the efficacy of postoperative pain relief after hip surgery using lumbar epidural and lumbar paravertebral anesthetic (Psoas compartment block) with a continuous infusion of 0.125% bupivacaine. BODY.3. PATIENTS AND METHODS: A randomized, controlled comparative study of 60 patients who were scheduled for hip surgery was performed. After the ethical committee approved the study design, we enrolled American society of anesthesiologist (ASA) class I, II, and III patients who were scheduled for unilateral hip surgery. Written informed consent was obtained. Patients with the following criteria were excluded from the study: Lack of patient consentSepsis over lumbar vertebraPatient on chronic analgesic/anticoagulant therapyPatient with a neurological disorderKnown allergy to any local anestheticDementia that prevented proper comprehension Sixty such patients formed the total sample size and were randomly allocated into groups of 30 subjects. Group I: 5 mL/h, 0.125% bupivacaine, continuous paravertebral groupGroup II: 5 mL /h, 0.125% bupivacaine, continuous epidural group Informed written consent was obtained from each patient after the study protocol and use of the visual analog scale were explained and after we gained their confidence during the preanesthetic assessment visit on the night before surgery. BODY.3. PATIENTS AND METHODS.3.1. PREANESTHETIC ASSESSMENT: During the preanesthetic assessment, a detailed history of each patient was taken and recorded in Performa. A thorough clinical examination was performed. Detected to be having any of the conditions mentioned in exclusion criteria were excluded from the study. Hematological measures, such as hemoglobin, blood count, bleeding time, and clotting time, were recorded, and routine and microscopic urine analysis was performed. Chest X-ray and electrocardiogram (ECG) were taken whenever necessary. All patients were visited the night before the surgery and had the procedure explained to them; after being advised and reassured, their confidence was gained. All patients were advised to fast after 2200 hours the night before the surgery. BODY.3. PATIENTS AND METHODS.3.2. PREOPERATIVE MEDICATION: All patients were given tablet alprazolam 0.25 mg orally at 10 pm in the night before surgery. BODY.3. PATIENTS AND METHODS.3.3. TECHNIQUE: Patients pulse rate, ECG, and noninvasive blood pressure were recorded, and a wide-bore intravenous line was established. Patients were preloaded with Ringer's solution at 15 mL/kg body weight 30-60 minutes before intrathecal drug administration. Patients were positioned in the sitting position, supported, and chin-flexed on the chest; those who were unable to sit were positioned in the lateral decubitus position. The back was prepared using Ioprep and wiped with methylated spirit, and the area was draped with a sterile towel. BODY.3. PATIENTS AND METHODS.3.3. TECHNIQUE.3.3.1. GROUP I: To locate the puncture site, a point was made on the upper border of the spinous process of the L2 vertebra, and we identified the punctured site 3 cm lateral to the first point on the target side. Local anesthetic was infiltrated at the puncture site, and a 16-G Tuohy needle was advanced perpendicular to all planes until it made contact with the transverse process of the L2 vertebra. It was then redirected slightly caudal to the transverse process and advanced 1-2 cm to reach the Psoas compartment. The stylet was removed, and 10 mL of saline was injected to expand the compartment. Next, an 18-G catheter was inserted through the Tuohy needle and advanced approximately 4 cm caudally within the compartment. Initially, a 3-mL test dose solution containing 2% lidocaine and 1:200,00 epinephrine was injected via the paravertebral catheter. Then, a lumbar puncture was performed through the L3-L4 interspace, and 2.5 to 3.0 mL of 0.5% bupivacaine was injected. BODY.3. PATIENTS AND METHODS.3.3. TECHNIQUE.3.3.2. GROUP II: The L3-L4 interspace was identified and infiltrated with local anesthetic. A 16-G Tuohy needle was inserted through the the L3-L4 interspace, and the epidural space was located by loss of resistance technique. The stylet was removed, and a 3-mL test dose solution containing 2% lidocaine and 1:200,00 epinephrine was injected. Then, a lumbar puncture was performed with a 25-G spinal needle, which was passed through the Tuohy needle, and 2.5-3.0 mL of 0.5% bupivacaine was injected. The catheter was then advanced approximately 2-3 cm cephalad and secured. BODY.3. PATIENTS AND METHODS.3.4. MONITORING: The cephalad spread of analgesia and the degree of motor blockade of the lower limb were recorded. The level of sensory blockade was assessed using a 25-gauze short-bevel needle and recorded as analgesia to loss of sensation to a pin prick (4). The following parameters were monitored continuously in all patients and recorded. Heart rate and ECGNoninvasive blood pressureSPO2 (pulse oximetry)Blood lossUrine outputIV fluid input Patients were observed for any discomfort, nausea, vomiting, shivering, pain, bradycardia, and any other side effects, and the need for any additional medications was recorded. IV fluids were administered in the form of Ringer's lactate, dextrose normal saline, and colloids in calculated doses, depending on the weight of the patient, and further adjusted as per blood loss during surgery. Hypotension of more than 30% of the preanesthetic value was treated with rapid infusion of fluids and an injection of mephenterine intravenously. Bradycardia (heart rate less than 60/mt) was treated with intravenous atropine sulphate. BODY.3. PATIENTS AND METHODS.3.5. POSTOPERATIVE OBSERVATION: At the end of the operation, each patient was connected to an infuser set to deliver an infusion of 0.125% bupivacaine at a rate of 5 mL per hour for 48 hours. All patients were observed in the postanesthesia care unit and then in the ward. Immediately after surgery, each patient started identical physical therapy regimens. From postoperative Day 1, patients performed active and assisted hip flexion and extension exercises against gravity twice daily. Getting up from bed was encouraged as soon as possible, followed by ambulation with a walker. Several parameters were assessed postoperatively. Regional anesthesia procedure time; i.e., the period from positioning the patient for technique to catheter fixation.Operation timeSeverity of pain using the 10-cm visual analog scale and vital parameters, such as pulse, mean arterial pressure (MAP), and respiratory rate (RR) at 2, 4, 8, 12, 18, 24, 32, 40, and 48 hoursSupplemental analgesic requirementCatheter-related problems and any complications in first 48 postoperative hoursRescue analgesia was provided by Inj. diclofenac 1 mg/kg IM BODY.4. RESULTS: A total of 60 patients were studied. They were randomly divided into 2 groups of 30 patients each. Six patients were excluded from the study. The data that we present are from the remaining 54 patients (Table 1). Table 1. Patients’ Charecterestics Group I, (n = 28) Group II, (n = 26) Age a , y, Mean ± SD 70.26 ± 10.25 65.8 ± 12.65 Sex, No. b Male 16 16 Female 12 10 Weight c , kg, Mean ± SD 63.96 ± 9.04 63.40 ± 7.64 Height d , e , cm, Mean ± SD 155.47 ± 2.54 156.27 ± 3.07 ASA Status f ASA-I 9 9 ASA-II 14 13 ASA-III 5 4 Type of Surgery, No. Hemiarthroplasty g 14 12 ORIF k with DHS g , k 11 10 THR g , k 2 3 External fixator g 1 1 Duration of surgery g , Mean ± SD 92.83 ± 15.1 94.5 ± 16.93 Regional anesthesia procedure Time, min h , Mean ± SD 10.633 ± 1.29 15.66 ± 1.52 Attempts, Mean ± SD 1.3 ± 0.5 1.6 ± 0.9 Injection of diclofenac sodium i , No. 2.88 2.66 Side effects j , d , No. Nausea and vomiting 2 3 Hypotension 0 2 Dislocated catheter 2 1 a Analysis of the data showed no significant statistical difference between them. b Analysis of the data showed no significant statistical difference between them. c Comparison of mean body weight of the two groups showed no Significant. d P value is not significant (P > 0.05). e The mean body height between two groups showed no significant statistical difference. f Patients of ASA-I, ASA -II and ASA -III are included in the study and their distribution is given. g P value is not significant (P > 0.05). h Procedure time was significantly longer in epidural group (P < 0.001). i Rescue analgesia in 48 hours. j There was no significant difference between the two groups regarding frequency of postoperative complication and catheter related problems. k Abbreviations: DHS, dynamic hip screw fixation; ORIF, open reduction and internal fixation; THR, total hip replacement Group I (paravertebral group)- 28 patientsGroup II (epidural group)- 26 patients 5 BODY.4. RESULTS.4.1. HEMODYNAMIC CHANGES: The hemodynamic parameters that we measured were heart rate and mean arterial pressure. The preoperative heart rate (HR) and MAP of all patients were recorded before the procedure was performed. Subsequent readings were taken every 5 minutes. Intraoperatively after spinal anesthesia and thereafter recorded at 2, 4, 8, 12, 18, 24, 32, 40 and 48 hours. BODY.4. RESULTS.4.1. HEMODYNAMIC CHANGES.4.1.1. MEAN ARTERIAL PRESSURE: Figure 1 shows the mean MAP in both groups. There mean arterial pressure was significantly lower in he epidural group compared with the paravertebral group from 2 hours after the infusion was begun to 48 hours. Figure 1.Comparison of Mean Arterial Pressure Between Group I and Group II BODY.4. RESULTS.4.1. HEMODYNAMIC CHANGES.4.1.2. HEART RATE: Figure 2 shows the mean HR in each group. The mean heart rate was slightly higher in the epidural group throughout the study period, albeit insignificantly. Figure 2.Comparison of Mean Heart Rate Between Group I and Group II BODY.4. RESULTS.4.1. HEMODYNAMIC CHANGES.4.1.3. VISUAL ANALOGUE SCALE: Figure 3 shows mean visual analogue scale (VAS) in each group. There were no statistically significant differences between the groups with regard to mean pain scores in the first 48 hours. Figure 3.Comparison of Visual Analogue Scale Between Group I and Group II BODY.5. DISCUSSION: The results of our study indicate that for patients who are scheduled for hip surgery, both continuous paravertebral and continuous epidural analgesia are effective in controlling postoperative pain but that the former has several crucial advantages. We modified the posterior approach technique of Deckrey et al., Chayen et al. (7), and Winnie et al. (8) slightly, as follows. To locate the puncture site, a point was made on the upper border of the spinous process of the L2 vertebra and identified the punctured site 3 cm lateral to the first point on the target side. Local anesthetic was infiltrated at the puncture site, and a 16-G Tuohy needle was advanced perpendicular to all planes until it made contact with the transverse process of the L2 vertebra. It was then redirected slightly caudal to the transverse process and advanced 1-2 cm to reach the Psoas compartment. The stylet was removed, and 10 mL saline was injected to expand the compartment. Next, the 18-G catheter was inserted through the Tuohy needle and advanced approximately 4 cm caudally within the compartment to reach the vicinity of the L4 vertebra, based on the finding that most branches of the lumbar plexus are in close proximity in the region of the 4th lumbar vertebra in the Psoas compartment. The efficacy of this postoperative pain management was assessed using the VAS. Six patients were excluded from the study. In 2 patients, the epidural catheter could not be sited; both patients were aged above 70 years and had mild scoliosis. Three patients-2 in the paravertebral group and 1 in the epidural group—were excluded due to accidental removal of the catheter while transferring them to the ward. One patient from the epidural group withdrew due to persistent hypotension. Vascular puncture was encountered during the procedure in 2 patients; the needle was redirected cephalad to the transverse process, and the block was administered. The data are from the remaining 54 patients. There was no significant difference in mean age, sex, height, weight, or ASA class distribution between patients in this study. The various surgeries that were performed and the duration of surgery were comparable between them. We measured the VAS at 2, 4, 8, 12, 18, 24, 32, 40, and 48 postoperatively. We found that both routes were effective in controlling postoperative pain and did not differ significantly, reflecting good postoperative pain control in both groups. The results of our study are consistent with those of of P.J. Mathews (9), who did not observe any significant difference in analgesia by VAS score over 24 hours. Perttunen et al. (10) demonstrated good postoperative pain relief at rest in paravertebral and extradural groups 1 hour after surgery and found comparable segmental analgesia in both groups up to 20 hours. The rescue analgesia was provided with Inj. voveran 1 mg/kg IM, and there was no significant difference in the number of doses that was required in both groups. However, the first analgesic requirement was earlier in the paravertebral group between 8-10 hours versus 8-12 hours in the epidural group. Intraoperative heart rate, mean arterial pressure, and SPO2 were monitored and were similar in both groups (data not reported). The mean arterial pressure at 2, 4, 8, 12, 18, 24, 32, 40, and 48 hours after starting the continuous infusion with 0.125% bupivacaine at 5 mL/h fell significantly between two groups. The paravertebral block effects a predominantly unilateral sympathetic blockade, whereas an epidural block is usually bilateral; the extent of the spread of the drugs is also greater. These differences might explain the disparities in the incidence of hypotension between the 2 groups. This conquer with the study G Turker et al. (11), White and Chappell (12), Richardson et al. (13). The mean heart rate, although not statistically significant, was slightly higher in the epidural group throughout the period. This finding may be explained by the hypotension in the epidural group. The regional anesthesia procedure time (i.e., time from positioning the patient to fixation of the catheter) was significantly longer in the epidural group. Our data also showed that the epidural block required more attempts than the paravertebral block. These results might be attributed to positioning difficulties, as the epidural block requires a strict midline position, and to spinal calcification and degeneration, which are frequently encountered in elderly patients. In our study, both continuous paravertebral and epidural analgesia provides effective postoperative pain relief after hip surgery, but the paravertebral block is technically simple and easy to learn with few contraindications, provides hemodynamic stability, and has a low complication rate and is therefore a safe and effective technique in controlling postoperative pain after unilateral hip surgery.

TITLE: The Higher Response of Vascular Endothelial Growth Factor and Angiotensin-II to Human Chorionic Gonadotropin in Women with Polycystic Ovary Syndrome ABSTRACT.BACKGROUND: This research investigated the response of vascular active factors, vascular endothelial growth factor (VEGF) and angiotensin-II (AT-II) to ovarian stimulation during 24 hours in patients with polycystic ovary syndrome (PCOS). ABSTRACT.MATERIALS AND METHODS: In this clinical trial study, 52 patients with PCOS and 8 control cases were stimulated with human chorionic gonadotropin (HCG) on the 4th to 7th day of the patients' natural or induced menstrual cycles. We measured VEGF and AT-II by radioimmunoassay before the injection (0 hour) and 3, 8, 12, 18 and 24 hours after the stimulation. ABSTRACT.RESULTS: After ovarian stimulation, there was substantially higher level of VEGF in typical PCOS patients than the other three groups at the 3 hour time point (p<0.05), while there were no significant differences in VEGF at all the other time points among the four groups. As for AT-II, before and at all time points after the ovarian stimulation, it seemed that the AT-II levels in patients' sera with different phenotypes of PCOS by the Rotterdam criteria were all higher than in the control group although the differences were not statistically significant. The level of AT-II in typical PCOS patients was also significantly higher than the other three groups at the 3 hour time point (p<0.05), while no significant differences at all the other time points among the four groups were observed. ABSTRACT.CONCLUSION: The response to the stimulation varied among patients with different phenotypes of PCOS according to the Rotterdam criteria. Serum VEGF and AT-II were possible contributors to an increased risk of developing ovarian hyperstimulation syndrome (OHSS) in patients with typical PCOS during the early follicular phase (3 hours) after ovarian stimulation (Registration Number: NCT02265861). BODY.INTRODUCTION: Polycystic ovary syndrome (PCOS) is a complex and common gynecological endocrine disorder that occurs in 5-10% of reproductive women (1). In China, this disorder is found in 50-60% of outpatients in gynecologic endocrinopathy clinics (2). It is characterized by a polycystic ovary, ovarian hyperandrogenism (HA), and anovulation although there are several different phenotypes of PCOS according to the Rotterdam criteria (3). A key pathophysiological feature of PCOS is an increase in ovarian mass caused by new blood vessel proliferation in the stroma and theca interna (4). Ultrasonographic assessment of the stromal area and blood flow is currently used as a diagnostic test (5). In ovarian hyperstimulation syndrome (OHSS), massive ovarian enlargement has been reported with PCOS as one of its risk factors (6). Vascular active factors, such as vascular endothelial growth factor (VEGF), angiotensin-II (AT-II), insulin-like growth factor-1 (IGF-1), and some cytokines may be involved which have certain relevance for PCOS pathophysiology (7). Ovarian stimulation may change the production of vascular active factors and different phenotypes of PCOS may have different responses to the stimulation. VEGF is a 46 kd dimeric protein. Its expression is increased where vascular proliferation is active. The most important sources of VEGF in the female reproductive system are local macrophages and granulosa cells, and the production of VEGF can be increased by human chorionic gonadotropin (HCG) (8). VEGF is one of the most likely candidates for promoting angiogenesis in PCOS and OHSS. The serum VEGF level is increased both in the patients with PCOS and OHSS (9). The increased number of actively secreting granulosa lutein cells and the increased secretory capacity of each granulosa cell both contribute to excessive VEGF production (10). In PCOS, the Renin-Angiotensin System (RAS) is accentuated. The roles of AT-II have been proposed in growth and atresia of follicles, oocyte maturation, ovulation, corpus luteum formation, steroidogenesis, and corpus luteum regression (11). The level of AT-II in peripheral blood is higher in PCOS patients than in non-PCOS women. AT-II concentration is positively correlated with the level of testosterone (T) in PCOS patients (12). Therefore, components of the RAS, such as AT-II, may be involved in PCOS pathophysiology. VEGF and AT-II belong to different molecular systems, however both can act as vascular active factors. The aim of this study is to investigate whether ovarian stimulation increases serum vascular active factors such as VEGF and AT-II within 24 hours after stimulation in patients with PCOS during the early follicular phase. BODY.MATERIALS AND METHODS: This clinical trial study enrolled women with or without PCOS who entered an in vitro fertilization (IVF) program at the hospital affiliated to the Medical School of Nanjing University. This research was approved by the Medical Ethics Committee of the school and informed consent was obtained from the participating subjects. A total of 60 women were recruited and divided into four groups by the Rotterdam criteria according to three typical characteristics: 1. biochemical characteristics of HA, 2. chronic anovulation, and 3. polycystic ovarian morphology (PCO). Group 1 (typical PCOS) was composed of 21 women who had all three of the above features, group 2 (PCOS without PCO) included 14 women with both biochemical characteristics of HA and chronic anovulation, group 3 (PCOS without HA) included 17 women with both chronic anovulation and PCO, and group 4 consisted of 8 volunteers without any biochemical characteristics of HA, chronic anovulation or PCO. This group served as the controls for the three PCOS groups. No patients were prescribed any hormonal prescriptions during the three months preceding the study. The clinical manifestations and the basic data are shown in table 1. An ovarian stimulation test, which mimicked the common ovulation protocol (10) was performed by the administration of a single intramuscular (i.m.) injection of HCG (5000 IU, The First Biochemical Pharmapeutic, Shanghai, China) during the early follicular phase of the same menstrual cycle (4th to 7th day of the cycle). Blood samples from all subjects were collected immediately prior to the injection (H0), and at 3 (H1), 8 (H2), 12 (H3), 18 (H4), and 24 (H5) hours after the injection. The serum was stored at -80 ̊C until further analyses of VEGF and AT-II levels by immunoradioassay. Concentrations of androstenedione (A), T and estradiol (E2) at H0 were also measured. Table 1 Basic clinical and hormonal profiles in the four groups Group 1 Group 2 Group 3 Group 4 N 21 14 17 8 Age (Y) 30.1± 4.03 30.5± 3.69 31.0 ± 4.12 29.5 ± 5.12 BMI (kg/m 2 ) 24.5± 1.29 23.5± 0.8 22.3 ± 0.99 22.4 ± 0.76 WHR 0.82± 0.02 0.80± 0.02 0.80 ± 0.02 0.78 ± 0.02 T (nmol/L) 2.09± 0.14 a, b 2.09± 0.12 a , b 1.22 ± 0.13 0.73 ± 0.14 FTI 10.76 ± 1.57 a, b 6.44± 0.7 3.52 ± 0.62 1.58 ± 0.48 A (nmol/L) 9.56± 0.63 a, b 8.07± 0.8 a 6.97 ± 0.62 4.47 ± 0.36 E 2 (pg/ml) 34.9± 5.09 37.5± 5.48 29.2 ± 2.57 30.8 ± 3.26 E 2 /T 17.8± 2.73 a 18.8± 5.76 a 33.9 ± 8.15 78.0 ± 37.8 Note: A total of 60 women were recruited and divided into four groups by the Rotterdam criteria. a; P<0.05 vs. group 4, b; P<0.05 vs. group 3, BMI; Body mass index, WHR; Waist to hip ratio, T; Testosterone, FTI; Free testosterone index, A; Androstenedione and E 2 : Estradiol. Among the groups there were no differences in age, BMI and WHR. The T, A, and E 2 levels of the polycystic ovary syndrome (PCOS) groups were significantly higher than the control group. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Tests were two-sided and conducted at α <0.05. In general, the methods for the multiple comparisons were based on analysis of variance (ANOVA) and analysis of covariance (ANCOVA) to identify group differences with the Bonferroni correction when appropriate. Logarithmic transformation of data was performed for data that were not normally distributed. For all analyses, a two-tailed p<0.05 was considered statistically significant. Unless otherwise noted, all results were described as mean ± SD. Correlations were examined by linear regression analysis. Statistical analysis was performed using the SPSS 11.0 software package for Windows. BODY.RESULTS: The subject characteristics of the four groups are summarized in table 1. Among the groups there were no differences in age, body mass index (BMI) and waist to hip ratio (WHR). There were significantly higher T, A, and E2 levels of the PCOS groups compared to the control group (Table 1). Before ovarian stimulation, there were no significant differences in VEGF between the four groups (Fig 1A). After ovarian stimulation, the level of VEGF in the typical PCOS patients (Group 1) was substantially higher than the other three groups at the 3 hour time point (p<0.05), while there were no significant differences in VEGF at the other time points among the four groups (Fig 1A). As for AT-II, before and at all time points after ovarian stimulation, it appeared that AT-II levels in the patients' sera with different phenotypes of PCOS according to the Rotterdam criteria were all higher than in the control group (Fig 1B), however these differences were not statistically significant. The level of AT-II in typical PCOS patients (Group 1) was also significantly higher than the other three groups at the 3 hour time point (p<0.05), while no significant differences at all the other time points among the four groups (Fig 1B) were observed. Fig 1Vascular endothelial growth factor (VEGF) (A) and angiotensin II (AT- II) (B) response to stimuli during the ovarian stimulation test.The level of VEGF and AT- II in typical polycystic ovary syndrome (PCOS) patients (group 1) was substantially higher than the other three groups at the 3 hour time point.Note: Logarithmic transformation of data was performed for measures that were not normally distributed. All results are expressed as Log10 (x ± s). *; P<0.05, PCO; Polycystic ovarian morphology and HA; Hyperandrogenism. BODY.DISCUSSION: Abnormal expression of VEGF, accompanied by abnormal angiogenesis and vascular permeability has been suggested as a cause of several diseases including PCOS and OHSS (13-16). Increased secretion of VEGF in the serum of patients with PCOS may be induced as a result of an increased number of actively secreting granulosa lutein cells that have both increased secretory capacity and upregulated gene expression level (17). The expression level of VEGF is increased in the hyper-echogenic stroma of PCOS and it induces subsequent stroma growth by promoting microvascular permeability (18, 19). Androgen is secreted in the theca interna and the latter will grow abnormally where vascular proliferation is active. VEGF can affect vascular endothelial cell proliferation and vascular permeability (20). PCOS is one of the risk factors of OHSS (21). The significance of VEGF is its contribution to the induction and progression of OHSS during ovarian induction. VEGF has also been suggested to be responsible for OHSS which is an iatrogenic and potentially life-threatening complication of ovulation induction for the treatment of infertility. Our findings have demonstrated that ovarian stimulation increased VEGF expression during the early follicular phase of typical PCOS patients, which indicated that VEGF might be directly involved in OHSS pathogenesis (22). Despite the fact that OHSS occurs in the luteal phase after ovarian induction, we have proposed that VEGF should be monitored during the early follicular phase. However, in this study, the level of VEGF in the PCOS without PCO or HA did not significantly increase. This phenomenon has demonstrated that the responsiveness of patients with typical PCOS to ovarian stimulation was greater than that of the PCOS without PCO or HA. As we know, women of both typical PCOS and PCOS without HA are at higher risks for OHSS (23). However according to our observation, the VEGF response patterns in these two groups differed; group 1 was more sensitive than group 3. This phenomenon may suggest that the typical PCOS is more severe than the phenotypic PCOS. Further research is needed to clarify this phenomenon. AT-II plays an important role in RAS and it affects the reproduction cycle at different stages (24). Disturbances in ovarian RAS can be the cause or the result of such reproductive disorders as PCOS and OHSS. Our data have demonstrated that ovarian stimulation led to AT-II expression in patients with typical PCOS. Consistent with a previous report, AT-II might play an important role in PCOS pathophysiology (25). AT-II has been shown to modulate the local functions of ovaries, such as ovarian steroidogenesis and formation of the corpus luteum, in addition to stimulation of oocyte maturation and ovulation via AT-II receptors on granulosa cells (26). In addition to the circulating RAS, the ovary has been recently demonstrated to exhibit its own RAS products and activities. Such an intrinsic RAS can modulate the local functions of ovaries such as follicular development, ovulation, and formation of the corpus luteum (27). As AT-II is mainly produced by follicular theca cells and granulosa lutein cells (28), patients with PCOS have a higher risk of developing OHSS because of exogenous gonadotropin for ovulation induction. Results of these studies indicate that RAS is more active in the ovaries of PCOS patients. Although OHSS occurs during the luteal phases, the current study was performed during the follicular phase. Ovarian stimulation increased the AT-II levels during the follicular phase in PCOS patients. These results, taken together with other parallel studies (29), led us to consider serum AT-II as a possible contributor to a greater risk of OHSS in patients with PCOS during ovulation induction. Inhibition of RAS, commensurate with the changes of serum AT-II concentrations, might be used as a therapeutic approach. BODY.CONCLUSION: In conclusion, the response of the ovaries to HCG stimulation differs in patients with different phenotypes of PCOS according to the Rotterdam criteria. The typical PCOS is more severe than the phenotypic PCOS. Further research is needed to clarify this phenomenon. Serum VEGF and AT-II levels may be considered as biomarkers to predict risks of developing OHSS in patients with typical PCOS during the early follicular phase, at 3 hours after ovarian stimulation.

TITLE: The effects of sulodexide on both clinical and molecular parameters in patients with mixed arterial and venous ulcers of lower limbs ABSTRACT.BACKGROUND: Mixed venous and arterial ulcers account for approximately 15%–30% of all venous leg ulcerations. Several studies have shown that matrix metalloproteinases (MMPs) and neutrophil gelatinase-associated lipocalin (NGAL) play a central role in the pathophysiology of venous and arterial diseases. Some studies have shown the efficacy of glycosaminoglycans, such as sulodexide (SDX), in treating patients with leg ulcers. The aim of this study was to evaluate clinical effects of SDX and its correlation with MMPs and NGAL expression in patients with mixed arterial and venous leg ulcers. ABSTRACT.METHODS: Patients eligible for this study were of both sexes, older than 20 years, and with a clinical and instrumental diagnosis of mixed ulcer. ABSTRACT.RESULTS: Fifty-three patients of both sexes were enrolled and divided into two groups by means of randomization tables. Group A (treated group) comprised 18 females and ten males (median age: 68.7 years) treated with standard treatment (compression therapy and surgery) + SDX (600 lipoprotein lipase-releasing units/day intramuscularly) for 15 days followed by SDX 250 lipase-releasing units every 12 hours day orally for 6 months as adjunctive treatment. Group B (control group) comprised 17 females and eight males (median age: 64.2 years) treated with standard treatment only (compression therapy and surgery). The type of surgery was chosen according to anatomical level of vein incompetence: superficial venous open surgery and/or subfascial endoscopic perforating surgery. In all enrolled patients, blood samples were collected in order to evaluate the plasma levels of MMPs and NGAL through enzyme-linked immunosorbent assay. These results were compared to another control group (Group C) of healthy individuals. Moreover, biopsies of ulcers were taken to evaluate the tissue expression of MMPs and NGAL through Western blot analysis. Our results revealed that SDX treatment is able to reduce both plasma levels and tissue expression of MMPs improving the clinical conditions in patients with mixed ulcers. ABSTRACT.CONCLUSION: Inhibition of MMPs could represent a possible therapeutic intervention to limit the progression of leg ulceration. In particular, our findings demonstrate the efficacy of SDX in patients with mixed arterial and venous chronic ulcers of the lower limbs. BODY.INTRODUCTION: Chronic ulceration of the lower limbs is a serious clinical condition that induces pain and loss of limb function along with an impairment of quality of life and an increase in health care costs.1,2 In Western countries, the incidence of ulceration is rising in the population due to an increase in both life expectancy and risk factors for atherosclerotic stenosis, ie, smoking, obesity, and diabetes.3–5 Chronic venous ulceration (CVU), the pathophysiologic evolution of chronic venous disease, affects 1% of the adult population and is associated with a marked decrease in the quality of life and an increase in economic burden.6–8 Around 15%–30% of patients with CVU have signs of arterial impairment presenting with a reduced ankle-brachial pressure index ([ABPI] lower than 0.8).9–12 Mixed ulcers are characterized by edema, eczema, hyperkeratotic skin, maceration, inadequate presence of granulation tissue, rolled wound edges, and delayed healing.12,13 The biomolecular substrate of these manifestations is the change in both structure and function of the extracellular matrix (ECM). ECM is a network of interlacing macromolecules that forms a supporting structure for vascular wall and skin integrity and is maintained by the action of matrix metalloproteinases (MMPs) (which degrade ECM proteins) and their inhibitors (tissue inhibitors of MMPs).14 Several studies have shown that MMPs play a central role in the pathophysiology of venous and arterial diseases15–26 and in related diseases.27 Neutrophil gelatinase-associated lipocalin (NGAL) is a protein belonging to the lipocalin family and is expressed by activated neutrophils. NGAL has the ability to positively modulate the activity of MMP-9 in particular, by forming the NGAL/MMP-9 complex, protecting MMP-9 from proteolytic degradation. Inhibition of MMPs could represent a possible therapeutic intervention to limit the progression of leg ulceration. In particular, some authors have documented the efficacy of glycosaminoglycans in patients with chronic ulcers of the lower limbs.28,29 The term "glycosaminoglycan" refers to a category of related molecules that share common biologic properties, including heparin, low-molecular-weight heparin, heparan sulfate, and mixed glycosaminoglycan formulations, such as sulodexide (SDX). In particular, the role of SDX in vascular disease and its inhibitory effect on the proteolytic activity has been reported.30–34 The aim of this study was to evaluate clinical effects of SDX and its correlation with MMPs and NGAL expression in patients with mixed arterial and venous leg ulcers. BODY.MATERIALS AND METHODS.STUDY DESIGN: We performed an open-label, parallel-groups study, which was conducted between January 2010 and December 2012 in four clinical departments (Catanzaro 1, Catanzaro 2, Messina, and Naples) and with prior approval from the investigational review board of CIFL at University Magna Graecia of Catanzaro, in accordance with the Declaration of Helsinki. Before the beginning of the study, all participants provided written informed consent. In all patients, at the time of admission, the medical history was recorded and clinical examination, laboratory findings, and duplex ultrasonography were performed. During debridement, biopsies of the ulcers were taken and frozen (−80°C) for Western blot evaluation of MMPs and NGAL expression. Venous diseases were classified according to Clinical, Etiology, Anatomy, Pathophysiology (CEAP) classification.35 Superficial and deep vein systems and severity of venous reflux by duplex ultrasound and computed hemodynamic mapping were evaluated, as previously described.36,37 Arterial diseases were classified according to the ABPI:38 normal arteries (ABPI >0.80); moderate arterial disease (0.5< ABPI <0.85); and severe arterial disease (ABPI <0.5). BODY.MATERIALS AND METHODS.PATIENTS: Patients eligible for this study were of both sexes, older than 20 years, with a clinical and instrumental diagnosis of mixed ulcer, presence of venous reflux flow, ABPI >0.5 and <0.8, ulcer duration >6 weeks, ulcer size 2.5–10 cm2, and >50% granulation tissue on the wound bed. Patients were excluded for the presence of diabetes mellitus; rheumatoid arthritis; malignancy; blood disorders; systemic disease; no current episode of ulceration; wound infection; ABPI <0.5 (patients with severe arterial disease at presentation were considered for arterial imaging with a view to revascularization) or >0.8; systolic ankle pressure <60 mmHg; presence of necrotic tissue on the wound bed; use of medications that may impair wound healing; pain at rest; sensory loss (neuropathy); cardiac insufficiency; and medial calcinosis. BODY.MATERIALS AND METHODS.HEALING EVALUATION: The healing was assessed in agreement with previous studies.16,20 Briefly, healing was calculated by means of computed planimetry at T1 and T2 of the study compared to initial measurement at T0. The result was divided by the number of weeks that the patient has been observed to obtain the total area healed per week. For wound healing evaluation, we considered as rapid-healing ulcers those with a healing speed rate ≥1 cm2/week and slow-healing ulcers those with a healing speed rate <1 cm2/week. BODY.MATERIALS AND METHODS.EXPERIMENTAL PROTOCOL: Blood samples were collected at the time of admission (T0) and 1 month (T1), 3 months (T2), and 6 months later (T3) in all enrolled patients, in order to evaluate plasma levels of MMPs and NGAL through enzyme-linked immunosorbent assay (ELISA). Moreover, at the time of surgery, biopsies of ulcers were taken to evaluate the expression of MMPs and NGAL through Western blot analysis. BODY.MATERIALS AND METHODS.ELISA TEST: In order to evaluate plasma MMPs and NGAL levels, blood samples were collected at the time of the admission in accordance with our previous studies.20–26 ELISA testing was performed with a commercially available generic ELISA kit (EMD Millipore, Billerica, MA, USA), using anti-MMP-2, MMP-8, MMP-9, and anti-NGAL monoclonal antibodies (monoclonal antibody kit against activated form of MMPs; EMD Millipore) that recognized only activated MMPs. For both MMPs and NGAL, the results were evaluated with respect to a control group without ulcers. BODY.MATERIALS AND METHODS.WESTERN BLOT EVALUATION: Wounds were biopsied at the time of the surgery (T1) under a 1% lidocaine local anesthesia and with full sterile precautions. The biopsy was made at a point equidistant from the center and edge of the ulcer. Our experience with biopsies in these patients indicates that the biopsy is well tolerated by the subject and does not influence healing outcomes in venous ulcers. Biopsy was immediately placed into a sterile collection container and sent for quantitative (microbiology) culture. The biopsies obtained at the time of wound bed preparation (T1 and T2) were lysed for Western blot analysis in 2 mL of tissue protein extraction reagent (25 mM Bicine, 150 mM sodium chloride, pH 7.6; Thermo Fisher Scientific, Waltham, MA, USA). The extracts were stored at −80°C. Immunoblotting was performed using anti-MMP-2, MMP-8, MMP-9, and anti-NGAL monoclonal antibodies (monoclonal antibody kit against activated form of MMPs; Millipore Corporation) that recognized only activated MMPs and NGAL, and results have been expressed as arbitrary units, as recently described.20–26 All experiments were performed in triplicate. BODY.MATERIALS AND METHODS.QUALITY OF LIFE MEASUREMENT: The EQ-5DTM questionnaire39,40 was used in order to measure health outcomes and quality of life of study patients. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: All data are expressed as mean ± standard error of the mean. Student's t-test was performed in order to analyze the difference between each group and the control. Analysis of variance (ANOVA) was used to evaluate the differences among the groups. Differences identified by ANOVA were pinpointed by unpaired Student's t-test. The threshold of statistical significance was set at P<0.05. SPSS software (version 21.0; IBM Corporation, Armonk, NY, USA) was used for the statistical analyses. We defined this study as exploratory, therefore we did not determine a power calculation. In this light, the results can only be labeled as exploratory. BODY.RESULTS.PATIENTS: During the study period, 53 patients of both sexes were enrolled and divided into two groups by means of randomization tables. Group A (treated group) comprised 18 females and ten males (median age: 68.7 years) with mixed ulcers and evidence of venous reflux at duplex scanning, treated with standard treatment + SDX (600 lipoprotein lipase-releasing units [LRUs]/day intramuscularly) for 15 days followed by SDX 250 LRU every 12 hours orally for 6 months as adjunctive treatment.Group B (control group) comprised 17 females and eight males (median age: 64.2 years) with mixed ulcers treated with standard treatment only. All patients were subjected to the most appropriate surgical treatment (defined as standard treatment), considering also the patient's wishes. The type of surgery, when it was accepted, was chosen according to anatomical level of vein incompetence: superficial venous surgery (Cure Conservatrice et Haemodinamique de l'Insuffisance Veineuse en Ambulatorie [CHIVA] procedure was used for the correction of superficial venous reflux) and/or subfascial endoscopic perforating surgery after computed hemodynamic mapping, as previously described.15–17,20–22,36,37 All patients received the application of a multicomponent, multilayer, compression bandage with pressure of 20–30 mmHg. Patient characteristics are reported in Table 1. BODY.RESULTS.WOUND HEALING: Our results revealed a nonsignificant difference between groups A and B in both median ulcer area and mean area heal/week at admission (T1). In contrast, at the end of the treatment (T2), we documented a significant improvement in wound healing in Group A with respect to Group B (P<0.01) (Table 2). BODY.RESULTS.MMPS AND NGAL PLASMA EVALUATION: Using ELISA testing, we documented significantly higher levels (P<0.01) of plasma MMP-2, MMP-8, MMP-9, and NGAL in wound patients (Group A and B) with respect to control patients (Group C) (data not shown). Group C consisted of 14 healthy volunteer patients (seven male and seven female, age range 50–80 years, median age: 66 years). In these patients, blood samples were taken in order to evaluate through the ELISA test both MMPs and NGAL values (Table 3). We detected significantly lower levels of MMP-2, MMP-9, NGAL, and MMP-8 in patients treated with SDX (Group A) with respect to untreated patients (Group B), with a time-dependent pattern (Table 4). BODY.RESULTS.MMPS AND NGAL TISSUE EXPRESSION: Western blot analysis showed a lower expression of MMP-2, MMP-9, NGAL, (P<0.01), and MMP-8 (P<0.05) in patients treated with SDX, with respect to untreated patients (Figure 1). BODY.RESULTS.QUALITY OF LIFE: Quality of life was significantly higher for Group A (treated group) patients than for those in Group B (control group) (Figures 2 and 3) in the following areas of investigation: pain/discomfort; anxiety/depression; mobility; self care; usual activities. BODY.DISCUSSION: In this study, we evaluated the effects of SDX on clinical and biomolecular parameters in patients with mixed arterial and venous chronic leg ulcers. Studies have shown that the majority of leg ulcers are associated with venous disease (estimates range from 40%–80%), since other risk factors, including immobility, obesity, trauma, arterial disease, vasculitis, diabetes, and neoplasia, may also be present.41,42 Mixed ulcers have the features of CVU in combination with signs of arterial impairment; therefore, diagnosis of mixed ulcers is crucial because CVU is best managed using multilayer graduated compression bandaging,43,44 while compression is not appropriate for mixed ulcers45 because it may cause deterioration of tissue vitality and limb loss.46 However, recent studies have shown that compression therapy from 20–30 mmHg can improve arterial perfusion and venous function in patients with ABPI between 0.5 and 0.8 and support the ulcer healing.13,47 As previously described,8,15,19–26 the pathophysiological processes that characterize chronic ulcer onset are the activation of immune system cells and the secretion of specific protease enzymes known as MMPs. Previously, we documented that MMPs are involved in several vascular diseases.48 In the present study, we have documented that MMP-2, MMP-8, MMP-9, and NGAL were strongly expressed in patients with mixed wound etiology with respect to the control Group C. Recently, several authors reported the involvement of MMPs in venous ulcers26,49,50 and their association with NGAL values.20,51 In the present study, we documented higher levels of NGAL in patients with mixed ulcer that could justify the severity of mixed vascular pathology observed in our study group. NGAL is a 25 kDa protein stored in the granules of human neutrophils and released during the activation of these cells. NGAL positively modulates the activity of MMP-9, forming the NGAL/MMP-9 complex and protecting, in this way, MMP-9 from proteolytic degradation. Another important result is the high concentration of MMP-8, the predominant collagenase present in normal healing wounds. Over-expression and activation of this collagenase may be involved in the pathogenesis of non-healing chronic leg ulcers.22,52 The current results confirm the chronic nature of mixed ulcers and their tendency to slow down the normal healing processes. Recently, we and others documented in CVU patients that treatment with doxycycline as well as with a new nutraceutical substance modifying plasma MMP values improved both clinical symptoms and healing of ulcers.51–53 Evaluating the inflammatory nature of mixed ulcers and the involvement of MMPs in the physiopathology of these ulcers, it has been suggested that some drugs mimicking the action of endogenous tissue inhibitors of MMPs may be used in the treatment of venous and arterial diseases, including mixed ulcers.28–31,54–56 In particular, Mannello et al32 documented that SDX is able to inhibit the MMP-9 gelatinase secretion and activity. SDX is a highly purified mixture of glycosaminoglycans composed of low-molecular-weight heparin (80%) and dermatan sulfate (20%).57 Due to the concomitant presence of both fast-moving heparin, with affinity for antithrombin III, and dermatan sulfate, with affinity for heparin cofactor II (HCII), SDX shows lipidemic (>10 LRU/mg), anticoagulant (<100 IU/mg), anti-Xa (70–100 IU/mg), HCII (<180 U/mg), Activated Partial Thromboplastin Time (APTT) (~50 U/mg), and antithrombotic effects.58 SDX, as well as heparin, may be able to inhibit leukocyte function and the release of elastase;59 moreover, SDX also shows in vitro and in vivo profibrinolytic actions.28–32 SDX is useful in the treatment and secondary prevention of ischemic arterial cardiovascular events,56,60 deep vein thrombosis,61 and systemic and local inflammations.62 Moreover, Andreozzi30 reported that SDX treatment is associated with significant improvements in the clinical signs and symptoms of venous ulcers, while Coccheri et al55 documented in patients with venous leg ulcers that SDX associated with local treatment induced an improvement in ulcer healing without the development of side effects. In our study, we documented that SDX is able to reduce plasma and tissue levels of MMPs and NGAL. These effects may be related with the action of SDX on proteases that possess cysteine residues; we did, in fact, find that SDX has early effects on gelatinase (MMP-2 and MMP-9). In this study, SDX treatment increased the healing of ulcers with an improvement in clinical symptoms and in quality of life of the enrolled patients. These effects showed a time-dependent pattern, with an initial improvement in the first month and with a complete remission within 3 months. During the follow-up at 6 months, we did not record any signs of wound disease and observed no side effects related to SDX treatment. BODY.CONCLUSION: SDX represents a safe and efficacious treatment for patients with ulcers of mixed etiology; however, further studies are necessary to validate the observations presented here.

TITLE: A four-drug combination therapy consisting of low-dose tacrolimus, low-dose mycophenolate mofetil, corticosteroids, and mizoribine in living donor renal transplantation: A randomized study ABSTRACT.OBJECTIVE:: We compared a three-drug combination therapy (control group) consisting of tacrolimus, mycophenolate mofetil, and corticosteroids in living donor renal transplantation with a four-drug combination therapy (study group), in which the doses of tacrolimus and mycophenolate mofetil were halved and the immunosuppressive drug mizoribine was added, in order to determine whether the incidence rates of acute rejection after transplantation between the study group and the control group are similar, whether the study group regimen prevents the occurrence of calcineurin inhibitor–induced renal damage, and whether the study group regimen prevents adverse effects such as diarrhea caused by mycophenolate mofetil. ABSTRACT.METHODS:: We investigated the incidence of acute rejection, serum creatinine levels, and estimated glomerular filtration rate and the incidence of adverse effects such as diarrhea. ABSTRACT.RESULTS:: There was no significant difference between the two groups in the incidence of acute rejection. Renal function (estimated glomerular filtration rate and serum creatinine) was maintained in the control group whereas in the study group renal function gradually improved, with a statistical difference observed at 12 months. The incidence of gastrointestinal symptoms including diarrhea was significantly higher in the control group than in the study group. There was no significant difference in the incidence of cytomegalovirus infection and other adverse effects. ABSTRACT.CONCLUSION:: These results suggest the study group therapy is an effective regimen in preventing acute rejection and the deterioration of renal function. These results also show this therapy can reduce the incidence of adverse effects such as gastrointestinal symptoms. BODY.INTRODUCTION: The renal transplantation center of Henan Provincial People's Hospital performs about 40 ABO compatible living donor renal transplants per year. ABO incompatible living donor renal transplants are not permitted by the Chinese government. The drug regimen used for such renal transplants is a three-drug combination therapy consisting of tacrolimus as the calcineurin inhibitor (CNI), mycophenolate mofetil (MMF) as the immunosuppressive drug, and corticosteroids. Tacrolimus is an effective drug widely used for the suppression of acute rejection in renal transplantation in China. However, as a CNI, its typical adverse effects include hypertension, hyperglycemia, and nephrotoxicity, with renal damage being an adverse effect that requires particular attention.1–4 MMF is an immunosuppressive drug widely used in China that is effective for the suppression of renal transplant rejection. However, gastrointestinal disorders (including diarrhea) and infections are common adverse effects with high rates of incidence.5–9 Mizoribine (MZR) is an immunosuppressant developed in Japan that inhibits DNA synthesis by selectively inhibiting inosine monophosphate dehydrogenase in the de novo pathway.10–12 The mechanism of action of MZR is similar to MMF and is used for the suppression of acute rejection in renal transplantation instead of MMF. However, MZR is known to possess antiviral activity against cytomegalovirus (CMV) and hepatitis C virus (HCV) and has few significant adverse effects such as infections.13–17 Suppression of nephrotoxicity, which is presumably caused by CNIs, is considered to be an important factor for the long-term engraftment of transplanted kidneys. We also have experienced difficulties with refractory diarrhea and infections during treatment with MMF. In this study, we thought that a decrease in the doses of tacrolimus and MMF would prevent deterioration of renal function caused by CNIs and reduce the incidence of adverse effects such as gastrointestinal symptoms caused by MMF. However, we were concerned about a decline in immunosuppression, so we added MZR, which has a mechanism of action similar to MMF and few significant adverse effects. We examined whether this new protocol could reduce the problematic adverse effects of tacrolimus and MMF while preserving their efficacy, to become, at least in China, a useful treatment method for living donor renal transplantation. BODY.METHODS.TRIAL DESIGN AND PARTICIPANTS: This study was a randomized, controlled, open-label, parallel group, single-site clinical trial. A total of 60 patients were screened for this study, with 56 patients finally enrolled and randomized in a parallel group design between a four-drug combination therapy group (study group) and a three-drug combination therapy group (control group) at the renal transplantation center of Henan Provincial People's Hospital (Figure 1). If the medical chart number of a patient to be enrolled in the study was odd, the patient was allocated to the study group. If the chart number was even, the patient was allocated to the control group. Patients were enrolled by the lead author. All patients included in this study had undergone an ABO-identical or compatible living donor renal transplantation. Study patients ranged in age from 17 to 60 years. Patients with serious infections or cardiac diseases, women who were pregnant or seeking to become pregnant during the study period, and patients with white blood cell counts of <3000/mm3 were excluded from the study. This study was conducted according to the Declaration of Helsinki. A review of the protocol was conducted by the hospital institutional review board (2011 Henan Provincial People's Hospital IRB Approval No. 19), and written informed consent was obtained from all the patients who participated in this study. Figure 1.CONSORT 2010 flow diagram for this study. BODY.METHODS.IMMUNOSUPPRESSIVE PROTOCOLS: The corticosteroid therapy in the control group and the study group was the same, with methylprednisolone 800 mg administered by intravenous infusion, followed by prednisone starting at 40–50 mg, the dose of which was tapered by 5 mg every 2 weeks. After 3 months, the prednisone dose was reduced to 10 mg, and this was further reduced to 5 mg while observing the patient's condition and maintained until 12 months. Tacrolimus in the control group was started at a dose of 0.1 mg/kg after transplantation and adjusted to reach target trough levels of 8–10 ng/mL after 1 week. This was adjusted to 6–8 ng/mL from 3 months after the transplantation and then further reduced and maintained at 5–7 ng/mL by 12 months. Tacrolimus in the study group was also started at a dose of 0.1 mg/kg after transplantation and adjusted to reach target trough levels of 5–7 ng/mL after 1 week. This was adjusted to 3–5 ng/mL from 3 months after transplantation and maintained at the same level until 12 months. The dose of MMF in the control group was started at 750–1000 mg twice daily, and after 3 months was maintained at 750 mg twice daily until 6 months. Afterward, it was reduced to 500–750 mg until 12 months. The dose of MMF in the study group was started at 500 mg twice daily and maintained until 6 months, after which it was reduced to 250–500 mg until 12 months. MZR was administered at a dose of 150 mg daily (100 mg, 50 mg) in patients whose body weight was less than 50 kg and at a dose of 100 mg twice daily in patients whose body weight was more than 50 kg. MZR was maintained at the same dose until 12 months. BODY.METHODS.STUDY PARAMETERS: The two groups showed no significant differences in patient background characteristics, such as underlying age, gender, dialysis period, disease in recipient, and number of human leukocyte antigen (HLA) mismatches (Table 1). At each follow-up visit (monthly for 12 months after enrollment), serum creatinine and trough levels of tacrolimus were determined by a biochemistry laboratory in our hospital. Estimated glomerular filtration rate (estimated GFR) was calculated using a version of the modification of diet in renal disease (MDRD) formula as follows: GFR (mL/min/1.73 m2) = 186 × (serum creatinine)−1.154 × (age)−0.203 × 0.742 (if female).18 Acute rejection was judged according to the Banff classification (1997) after conducting a renal biopsy. Table 1. Patient characteristics in this study. Study group (n = 28) Control group (n = 28) p value Age (years ±  SD ) 33.1 ± 6.4 33.6 ± 5.3 0.787 Gender (female/male) 6/22 9/19 0.531 Pretransplant dialysis 28 (100.0%) 28 (100.0%) – Duration of dialysis before transplantation (months ±  SD ) 0.82 ± 0.10 0.83 ± 0.19 0.742 Indication for transplantation  Chronic glomerulonephritis 8 (28.6%) 9 (32.1%) 1.000  Diabetic nephropathy 3 (10.7%) 4 (14.3%)  Focal glomerulosclerosis 9 (32.1%) 8 (28.6%)  Membranoproliferative glomerulonephritis 6 (21.4%) 6 (21.4%)  Unknown 2 (7.1%) 1 (3.6%) Donor age (years ±  SD ) 54.5 ± 5.9 55.7 ± 8.7 0.531 Donor type  Father 9 (32.1%) 10 (35.7%) 1.000  Mother 18 (64.3%) 17 (60.7%)  Sibling 1 (3.6%) 1 (3.6%) HLA-AB mismatches  0 3 (10.7%) 4 (14.3%) 0.156  1 3 (10.7%) 8 (28.6%)  2 21 (75.0%) 13 (46.4%)  3 1 (3.6%) 3 (10.7%)  4 0 (0.0%) 0 (0.0%) HLA-DR mismatches  0 7 (25.0%) 4 (14.3%) 0.337  1 18 (64.3%) 23 (82.1%)  2 3 (10.7%) 1 (3.6%) ABO blood type  Identical 27 (96.4%) 25 (89.3%) 1.000  Compatible 1 (3.6%) 3 (10.7%) SD: standard deviation; HLA: human leukocyte antigen. BODY.METHODS.ENDPOINTS: This study was an exploratory study designed to evaluate, as a drug regimen following renal transplantation, the incidence of acute rejection 1 year between the study group and the control group, whether the four-drug combination therapy prevents the occurrence of CNI-induced renal damage, and whether the four-drug combination therapy prevents gastrointestinal symptoms (in particular, diarrhea) caused by MMF. BODY.METHODS.SAMPLE SIZE: Among the three endpoints (i.e. the incidence of acute rejection, the occurrence of CNI-induced renal damage, and gastrointestinal symptoms (in particular, diarrhea)), the incidence of diarrhea was the primary focus for which the sample size was set. The previously reported incidence rates of diarrhea caused by MMF were 32.1%, 31.5%, and 31.4%.5,6,9 In direct comparisons of MZR with MMF, diarrhea incidence rates were not reported, but Y. Kawasaki reported a diarrhea incidence rate of 0.22% among 916 renal transplant cases treated with MZR.17 From these reports, we estimated that a sample group of approximately 23 subjects would be needed to reach a level of significance of 0.05 and a power of 0.8 using Fisher's exact test. Taking into account a subject attrition rate of 10%, we set the target number of subjects for this study to 60 patients. BODY.METHODS.STATISTICAL ANALYSES: The data are presented as mean ± standard deviation (SD) or percentages. Statistical analyses were performed with IBM SPSS Statistics (Version 19.0, Armonk, NY, USA). Nominal data were compared using the chi-square test or Fisher's exact test, and numeric means were compared using the unpaired t test. For serum creatinine levels and estimated GFR, repeated-measures analysis of variance (ANOVA) with Bonferroni corrections was used. All tests were two-sided, and a p value <0.05 was considered statistically significant. BODY.RESULTS: A total of 56 patients were enrolled and randomized between January 2012 and July 2013, with 28 patients allocated to the three-drug combination group (control group) and 28 patients allocated to the four-drug combination group (study group). The follow-up period was 1 year post-transplantation. Five patients allocated to the study group withheld consent, and one patient in the study group was lost to follow-up because the patient relocated 4 months post-transplantation to another city. One patient in the control group was lost to follow-up due to gastrointestinal bleeding (stomach ulcer) (at 6 months post-transplantation) and two patients due to diarrhea (at 8 and 9 months post-transplantation) (Figure 1). Baseline characteristics were similar between the two groups. Main indicators for transplantation were chronic glomerulonephritis and focal glomerulosclerosis in the majority of cases, with no difference between the two groups. The donor type was a parent (mother or father) in most cases in both groups (Table 1). Steroids and MMF were administered according to the protocol for each group. Target trough levels of tacrolimus were adjusted according to the protocol for each group (Figure 2). Figure 2.Comparison of profiles of tacrolimus trough levels between the two groups. At 1 year after transplantation, patient and graft survival rates were 100% in both groups. The results of biopsy for identification of acute rejection are shown in Table 2. The incidence of acute rejection was 4.3% in the study group and 7.1% in the control group, and there was no significant difference between the two groups (p = 1.000). Biopsy examination of the acute rejections revealed one patient with Banff grade 1A in the study group and one patient with Banff grade 1A and another with Banff grade 1B in the control group. Methylprednisolone was administered at a dose of 500 mg intravenously for three consecutive days in two patients as treatment for graft rejection. Anti-thymocyte globulin was administered at a dose of 1.5 mg/kg intravenously for seven consecutive days in one patient as treatment for graft rejection (Table 2). Table 2. Acute rejection in this study. Study group (n = 23) Control group (n = 28) p value Acute rejection 1 (4.3%) 2 (7.1%) 1.000  Banff 1A 1 1  Banff 1B 0 1  Treatment MP MP, ATG MP: methylprednisolone; ATG: anti-thymocyte globulin. When serum creatinine levels were compared between the two groups, no difference was found at baseline (0.5 month post-transplantation, Figure 3). While serum creatinine levels did not change for 12 months in the control group, they gradually decreased in the study group, and at 12 months a significant difference was found between the two groups (p = 0.011, Figure 3). Similarly, the estimated GFR (MDRD formula) did not change in the control group while it gradually increased in the study group, and at 12 months a significant difference was found between the two groups (p = 0.005, Figure 4). Figure 3.Comparison of serum creatinine levels between the two groups. Figure 4.Comparison of estimated GFR between the two groups. Adverse effect results are summarized in Table 3. CMV infection occurred in one patient in the study group (4.3%) and six patients in the control group (21.4%); however, there was no significant difference between the two groups (p = 0.112). Gastrointestinal disorders occurred in eight patients (28.6%) in the control group and in one patient (4.3%) in the study group, with a significant difference found between the two groups (p = 0.031, Table 3). Moreover, intravenous infusion of a proton pump inhibitor was given to one patient in the control group due to the development of gastrointestinal bleeding (stomach ulcer). While hyperuricemia presented in eight patients in the study group (34.8%) and four patients in the control group (14.3%), there was no significant difference between the two groups (p = 0.166). One patient in the study group presented with a serum uric acid level of 783 μmoL/L and one patient in the control group presented with a level of 639 μmoL/L. However, all patients with hyperuricemia were managed with a combined treatment of sodium hydrogen carbonate and allopurinol. The degree of hyperuricemia was similar between the two groups with the serum uric acid mean ± SD of study group and the control group, respectively, 575.8 ± 100.1 and 556.5 ± 75.3 μmoL/L (p = 0.743) (Table 3). Patients with hyperuricemia were managed with a combined treatment of sodium hydrogen carbonate and allopurinol. There was no significant difference in the incidence rates of other adverse effects (urinary tract infections, pneumonia, leukopenia, anemia, and alanine aminotransferase increased). Table 3. Adverse effects in this study. Study group (n = 23) Control group (n = 28) p value CMV infection 1 (4.3%) 6 (21.4%) 0.112 Urinary tract infections 3 (13.0%) 9 (32.1%) 0.184 Pneumonia 1 (4.3%) 2 (7.1%) 1.000 Leukopenia 2 (8.7%) 6 (21.4%) 0.269 Anemia 1 (4.3%) 4 (14.3%) 0.362 Gastrointestinal disorder 1 (4.3%) 8 (28.6%) 0.031 Appetite loss 1 (4.3%) 1 (3.6%) 0.056 Diarrhea 0 (0.0%) 5 (17.9%) Nausea 0 (0.0%) 1 (3.6%) Gastrointestinal bleeding 0 (0.0%) 1 (3.6%) ALT increased 1 (4.3%) 5 (17.9%) 0.204 Hyperuricemia 8 (34.8%) 4 (14.3%) 0.166 µmoL/L ±  SD 575.8 ± 100.2 556.5 ± 75.3 0.743 CMV: cytomegalovirus; ALT: alanine aminotransferase; SD: standard deviation. BODY.DISCUSSION: Five patients allocated to the study group (four-drug combination therapy) withheld consent due to concerns over being allocated to the study group regimen in which the doses of tacrolimus and MMF were half of that of conventional treatment and MZR was added. Three patients withdrew from the control group (three-drug combination therapy) due to adverse events, but no patient in the study group withdrew due to adverse events. One patient from the study group left the study due to a transfer to another hospital for a non-medical reason (Figure 1). The results showed that there was no difference in the rates of rejection between the two groups, and satisfactory outcomes were also achieved with the study group, in which the doses of tacrolimus and MMF were reduced by half and MZR was added to the regimen. These results suggest that the reduced immunosuppressive effects of tacrolimus and MMF were compensated for by MZR. When graft function was compared, serum creatinine and estimated GFR (MDRD formula) did not change after 1 year in the control group whereas these parameters gradually improved in the study group, with a significant difference observed between the two groups in serum creatinine and estimated GFR after 12 months. These results suggest that graft function in the control group was maintained because the follow-up period was short. We believe that renal function improved in the study group because of the lower dose (trough level) of tacrolimus in the study group compared to the control group. CNIs are powerful immunosuppressants, and adequate suppression of T-cell function definitely plays an important role in the suppression of acute rejection. However, it is well known that CNIs trigger renal damage.2–4 The chronic nephrotoxicity of CNIs (CNI toxicity) is considered to be one of the reasons why mid- and long-term survival rates are not improving, and protocols based on CNI avoidance or withdrawal have been considered.19–21 Various protocols based on CNI reduction have been explored, and there has been a report where at 12 months post-transplantation the estimated GFR was higher and the occurrence of acute rejection was lower with low-dose tacrolimus than with standard-dose cyclosporine.22 On the other hand, there is a report that CNI toxicity and the development of interstitial fibrosis (IF) and tubular atrophy (TA) without any specific etiology correlate.23 Outside of CNI dosing, acute rejection appears to play a strong role in the development of IF/TA in that if acute rejection occurs within 1 year from renal transplantation, the rate of IF/TA presentation increases significantly at 2 years after transplantation.24 This suggests that suppressing rejection itself is an important factor in reducing the development of IF/TA. Our results showed that a CNI dose reduction protocol may be able to prevent progress to IF/TA due to CNI nephrotoxicity better than the control group. Moreover, the fact that graft rejection was favorably suppressed in this study during the first year raises the possibility that the development of IF/TA can be halted through the suppression of rejection. A lower incidence of CMV infection in the study group was observed when the incidence rates of adverse effects were compared, but there was no significant difference between the two groups. In China, CMV prophylactic therapy has been widely used since 2008 for CMV infection prevention in at-risk renal transplant patients. However, while CMV prophylactic therapy can reduce the incidence rates of CMV infection when compared with preemptive therapy, it is now well-recognized that late-onset CMV infection can develop when the administration of ganciclovir is ceased.25,26 In this study, we expected the incidence of CMV infection in the two groups to be similar since the same prophylactic antiviral therapy was administered in both groups. However, we did note a CMV infection incidence rate that was slightly higher in the control group, though insignificantly, than in the study group (p = 0.112). MZR is known to possess antiviral activity against CMV in experimental studies.13,14 Other clinical reports have demonstrated a significantly lower incidence rate of CMV infection with MZR compared to MMF.15,27 Furthermore, MZR can reduce the dose of ganciclovir administered due to a mutually potentiating effect between MZR and ganciclovir.14 These reports may explain why the rate of incidence of CMV infection may have possibly have been lower in the study group. The incidence of gastrointestinal disorders was 28.6% in the control group and 4.3% in the study group, with a significant difference found between the two groups (p = 0.031). The incidence rate, however, of diarrhea in the control group was 17.9% and lower than the previous MMF reports that were used to determine the sample size of this study. The lower incidence rate may have been due to insufficient questioning by study investigators or patients may have had a lower level of awareness regarding any manifestation of diarrhea. Furthermore, the incidence rates of diarrhea reported with MMF may have been increased due to the use of questionnaires like the Gastrointestinal Symptom Rating Scale (GSRS).28 Diarrhea not only lowers the quality of life of transplant patients, but it can also decrease the absorption of immunosuppressive drugs from the digestive tract and possibly trigger graft rejection, and its prevention is therefore important. The development of diarrhea is a well-known and important adverse effect of MMF and has been reported to occur at a higher incidence than with MZR.15,27 In our study, serious gastrointestinal bleeding occurred in one patient in the control group. Adequate management is necessary when using MMF. Hyperuricemia occurred at a rate of 34.8% in the study group and 14.3% in the control group; however, there was no significant difference between the two groups (p = 0.166). Since hyperuricemia may be a cause of renal damage, lower the survival rate of the kidney, and be a cause of CNI toxicity and the development of IF/TA, attention is required. It has been reported that the incidence of hyperuricemia is higher with MZR than with MMF.15,27 Similar results were observed in this study with a slightly higher incidence of hyperuricemia in the study group. However, there are also reports that there is no difference in the incidence of hyperuricemia between MZR and MMF.29,30 With respect to degree of hyperuricemia, one patient in the study group had a serum uric acid level of 783 μmoL/L, but the serum uric acid levels of this patient were elevated prior to transplantation and further increased up to the fourth day post-transplantation. Thus, the serum uric acid levels of this patient may have increased due to unstable renal function. The mechanism of action of MZR is similar to MMF in that it inhibits DNA synthesis by selectively inhibiting inosine monophosphate dehydrogenase in the de novo pathway.10–12,31,32 However, why the incidence of hyperuricemia is higher with MZR than MMF is largely unknown. Unlike MMF, MZR is excreted in urine and this difference in excretion mechanism gives rise to hyperuricemia, but since gradual lowering of uric acid can be managed through the concomitant use of sodium hydrogen carbonate and allopurinol, adequate management is therefore considered to be possible. This study had a number of limitations. First, this study was an open-label study subject to bias since patients could elect to withhold consent if allocated to the study group. As described earlier, only patients allocated to the study group withheld consent due to concerns over the study group regimen. Second, as this study was conducted at a single hospital in a mid-sized city in Central China, bias may be present in patient characteristics. Third, the study sample size was small, and the study duration was short. Due to these limitations, further long-term studies with longer follow-up periods and larger numbers of patients drawn from a more diverse patient population are needed to assess any long-term benefits of the study group regimen to transplant patients. In conclusion, the four-drug combination therapy (study group) tested in this study reduced nephrotoxicity using a lower CNI dose and suppressed acute graft rejection as well as the control group. Furthermore, since gastrointestinal disorders due to MMF could also be reduced, the study group regimen can be considered a recommended protocol. Moreover, in China, we reduce the amount of drugs used and focus on preventing the occurrence of adverse effects because the physique of Chinese patients is comparatively smaller than Europeans and Americans. Given the foregoing, the study group regimen could at least be satisfactorily adopted for use in China.

TITLE: Dentascan an excellent tool for assessment of variations in the management of periodontal defects ABSTRACT.BACKGROUND:: The purpose of the present study was to envisage the effectiveness of demineralized freeze-dried bone allograft (DFDBA) and bovine bone graft (BBG) for promoting defect fill in periodontal intrabony defects using dentascan. ABSTRACT.MATERIALS AND METHODS:: A total of 13 subjects (15 intrabony defects) aged between 24 and 56 years affected by moderate to severe periodontitis were randomly divided into Control (CG) and Test groups (TG1 and TG2). In CG only debridement, TG1 debridement plus DFDBA, and TG2 debridement plus BBG were performed. The clinical parameters probing pocket depth (PPD), clinical attachment level (CAL) was used. The radiological analysis was done by dentascan, which is a single-slice spiral computed tomographic scanner. Six months after, regenerative treatment clinical measurements were recorded. The bone fill was assessed using Dentascan as previously mentioned. ABSTRACT.RESULTS:: PPD reduction and CAL gain were significant in all the groups after 6 months whereas, on intergroup comparisons, insignificant finding was observed both at baseline and after 6 months. Coronoapical bone status decreased significantly in all groups, buccolingual measurements decreased significantly in TG1 and TG2, but no such trend was seen in CG. Significant reduction in mesiodistal bone status was noticed only in TG1 whereas insignificant on intergroup comparisons. ABSTRACT.CONCLUSION:: Dentascan-based analysis attested that DFDBA was superior to BBG. BODY.INTRODUCTION: Recently, dental computed tomographic (CT) reformatting programs that use thin transverse images of the jaw to reformat multiple panoramic and cross-sectional views were developed. Since images are reformatted, streak artifacts that degrade bone visualization at direct coronal CT are projected over the crowns of the teeth, permitting optimal viewing of bone. As a result, these programs have been successfully used to evaluate implants, cysts, tumors, and surgical procedures. The developments of dental CT reformatting programs, however, have completely revolutionized and changed the fashion, in which we radiographically evaluate the jaw today.[1] The programs are useful because they provide accurate information about the height and width of the jaws as well as information about the location of vital structures. The technique of dental CT, also called Dentascan, was developed by[2] Schwarz et al. in 1987. They first used curved multiplaner reconstruction of the jaw. Periodontal therapy involves the elimination of bacterial plaque and the correction of anatomical defects produced by disease process. Both nonsurgical and surgical treatment modalities have been used to manage periodontal diseases. It is assumed that the application of bone grafts would potentially manipulate the biological response into a regenerative rather than a predominantly reparative pattern of periodontal healing.[3] The use of bone grafts for reconstruction of osseous defects produced by periodontal diseases dates back to 1923 by[4] Hegedus and was popularized by[5] Nabers and O'leary in 1965. Many osseous grafting materials have been used toward the goal of obtaining periodontal regeneration, for example, autografts, allografts, heterografts (or xenografts), and alloplastic materials. These various grafting materials may produce radiographic evidence of bone fill and clinical evidence of improvement in probing depth and clinical attachment level (CAL).[6] Schwartz et al. 1998 reported that variations in the amount of bone formation induced by demineralized freeze-dried bone allograft (DFDBA) were related to the source and processing of the bone. In addition to processing variations, it has been demonstrated that young donor bone results in significantly greater quantities of bone morphogenetic proteins (BMPs) retained in the bone allograft matrix compared with older donor bone. On the other hand, bovine bone grafts (BBGs) have long been used in the treatment of periodontal bone defects because of their potential for periodontal regeneration.[7] Due to a paucity of literature on the role of Dentascan in periodontics, the purpose of the present study was to envisage the effectiveness of DFDBA and BBG for promoting defect fill in periodontal intrabony defects using Dentascan. BODY.MATERIALS AND METHODS: A cross-sectional study was conducted in the Department of Periodontology, faculty of dental sciences, K. G. Medical University, Lucknow. A total sample size was 13 subjects (with 15 intrabony defects) out of 9 males and 4 females with age group 24–56 year (mean 40 years) affected by moderate to severe periodontitis were recruited from the outpatient. Inclusion criteria No contributory medical historyAt least ≥5 mm of probing pocket depth (PPD) of the test toothRadiographically detectable intrabony defects and presence of two wall intraosseous defects was confirmed during surgeryAbility to control oral hygieneGood cooperation. BODY.MATERIALS AND METHODS.EXCLUSION CRITERIA: Subjects on medications for the past 6 monthsThree walls or one wall intraosseous defectsSmokersTobacco chewersGrade three mobility of the test tooth with intrabony defects. BODY.MATERIALS AND METHODS.STUDY DESIGN: Fifteen intrabony defects were randomly divided into two groups; control and test group based on the assigned treatment. Control group (CG) (n = 5): Only debridement. Test groups: Group-I (TG1) (n = 5): assigned treatment with DFDBA, granules size: >1040 μ. Group-II (TG2) (n = 5): assigned treatment with BBG, granules size: 1000–2000 μ. BODY.MATERIALS AND METHODS.PRESURGICAL MANAGEMENT: For all subjects, general, oral, and full-mouth periodontal examination was carried out, and informed consent was obtained from the subjects after explanation of the procedure. Basic periodontal therapy was performed with detailed instructions in self-plaque control measures. Full mouth scaling and root planing and occlusal adjustments if necessary were done. The baseline examination was performed 4 weeks after the completion of initial therapy and achievement of low plaque index (15%).[8] Subjects evaluation was followed by impressions for fabrication of acrylic stent required for the measurements of clinical parameters in control and test groups during the study. BODY.MATERIALS AND METHODS.CLINICAL MEASUREMENTS.SOFT-TISSUE MEASUREMENTS: Included PPD and CAL using UNC-15 probe (Hu-friedy). All measurements were performed by PPD - Distance measured from free gingival margin to the base of the periodontal pocketCAL - When the gingival margin is coronal to the cementoenamel junction (CEJ), the CAL is calculated by subtracting the gingival margin level to CEJ from the probing depths. When recession is present, the CAL is calculated by adding the probing depth to the gingival margin level from CEJ. BODY.MATERIALS AND METHODS.CLINICAL MEASUREMENTS.RADIOLOGICAL EXAMINATION: Coronoapical, mesiodistal, and buccolingual measurements of intrabony defect were measured using Dentascan, which is a single-slice spiral CT scanner. Dentascan produces 1 mm thick slice interval, and all axial images acquired 12.5 cm field of view. Dentascan was operated at 120–140 kV and 100–200 mA which varied from subject to subject. BODY.MATERIALS AND METHODS.CLINICAL MEASUREMENTS.ANALYSIS: The acquired images were transferred to workstation and reformatted into axial sections, sagittal sections, and CT orthopantomographs. The buccolingual and mesiodistal measurements were taken using computer software placing caliper at widest point of bone loss. The coronoapical measurements were taken manually using CT orthopantomographs and sagittal reformatted images of individual tooth, using measurement scales provided along with the printed images. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: It was performed using two-way analysis of variance (ANOVA) which is an extension to the one-way ANOVA. There are two independent variables (hence the name two-way). After ANOVA, Newman–Keuls test was used to calculate difference between the means of all groups. P < 0.05 was considered as a statistically significant. BODY.MATERIALS AND METHODS.SURGICAL PROCEDURE: All instruments to be used in the surgery were sterilized by autoclaving (temperature 121°C at 15 psi for 15 min). The facial skin all around the oral cavity was scrubbed with povidone-iodine solution and subjects were asked to rinse with 0.2% chlorhexidine. After obtaining the local anesthesia (2% xylocaine with 1:80,000 adrenaline), crevicular incision with blade number 15 was given at the test site. Full thickness mucoperiosteal flap was reflected on facial/buccal and lingual/palatal exposing crestal bone using periosteal elevator to expose the test area. The vertical releasing incision was given when needed for better access. Papilla preservation flap was performed where interproximal spaces were available to ensure maximum closure and graft coverage postsurgically. The exposed intraosseous defect was debrided of granulation tissue using hand curettes. Adjacent tooth surfaces were planed with area specific curettes. The defect area was irrigated with sterilized saline to get rid off the residual debris. Osseous grafts (DFDBA or BBG) were placed in the defects as assigned. Flaps were repositioned, and complete closure was achieved. Interrupted or horizontal mattress sutures (3-0) were used and periodontal dressing was applied over the surgical site. After surgery, subjects were prescribed amoxicillin with clavulanic acid (625 mg) TDS, anti-inflammatory (Ibugesic 400 mg) three times per day and B-complex 1 capsule daily for 5 days. Subjects were instructed to rinse twice daily with 0.2% chlorhexidine and not to brush in the treated area for the first 2 weeks. One week after surgery sutures and periodontal dressing were removed. The site was cleaned, and the periodontal dressing was replaced if needed. The subjects were recalled every 4 weeks for 6 months for oral hygiene evaluation and prophylaxis. After 6 months PPD and CAL were measured, and bone fill was assessed using Dentascan as previously mentioned. BODY.RESULTS: The results obtained through the study are summarized in Tables 1–4 and Figures 1–7. The maximum reduction in PPD was 3.80 mm (45.2%) in the TG1 and minimum reduction 2.80 mm (32.6%) in CG. The maximum gain of CAL 3.80 mm (50%) was observed in TG1 followed by 3.00 mm (38.5%) in TG2 and 2.80 mm (35.9%) in CG[Table 1]. On intergroup comparison[Table 2] PPD and CAL at baseline and after 6 months showed nonsignificant difference (P > 0.05). Figure 1Pretreatment radiological measurement of coronoapical bone loss Figure 2Pretreatment radiological measurement of mesiodistal and buccolingual bone loss Figure 3Radiological measurement of coronoapical bone loss at 6 months after treatment Figure 4Radiological measurement of mesiodistal and buccolingual bone loss at 6 months after treatment Figure 5Placement of bone graft in the defect Figure 6Clinical measurement of coronoapical bone loss at the time of surgery Figure 7Clinical measurement of coronoapical bone loss at 6 months after treatment without exposure of the defect Table 1 Change in probing pocket depth and clinical attachment level (mm) at 6 month interval in control and test groups Table 2 Comparison of mean probing pocket depth (mm) and clinical attachment level (mm) between the groups Table 3 Change in coronoapical, mesiodistal, and buccolingual bone status (mm) at 6 month interval in control and test groups Table 4 Comparisons of mean coronoapical, mesiodistal and buccolingual bone status (mm) between the groups Alterations in hard tissue (bone status) using Dentascan[Table 3] depicts reduction in coronoapical bone status 3.00 mm (50%) in TG1 followed by 2.40 mm (41.4%) in TG2 and least 0.80 mm (14.3%) reduction was seen in CG. Reduction in buccolingual bone status in CG, TG1, TG2 was 0.40 mm (5.7%), 2.20 mm (30.6%) and1.40 mm (18.9%) respectively. The mesiodistal bone status was 0.80 mm (26.7%) in TG1 followed by 0.60 mm (21.4%) in TG2 and 0.40 mm (14.3%) in CG. On intergroup comparison at baseline coronoapical bone status did not differ significantly (P > 0.05)[Table 4]. On the contrary at 6 months significant reduction (P < 0.01) in coronoapical bone status was seen in CG versus TG1; (P < 0.05) in CG versus TG2 and nonsignificant difference (P > 0.05) in TG1 versus TG2. Although the reduction of mesiodistal bone status between the CG versus TG1 is twice, i.e., 0.40 mm[Table 3] but difference was statistically nonsignificant (P > 0.05). The Same trend of the nonsignificance was observed in CG versus TG2 and in between the two test groups. On intergroup comparison insignificant (P > 0.05) reduction of buccolingual bone status was observed. BODY.DISCUSSION: The present study was designed to compare the role of DFDBA and BBG for the treatment of intrabony periodontal defects. Test and control intrabony defect groups were homogeneous at baseline, and each subject participating in the study showed good oral hygiene level and a healthy gingival status. Among all the bone graft materials being developed, DFDBA has been shown in clinical trials, controlled studies, and human histological evaluations to be a highly efficacious materials for the reconstruction of periodontal osseous defects and the regeneration of the periodontium.[910] BBG has recently been shown to have the potential for periodontal regeneration.[7] Although, there is no denying fact that the most reliable method to assess the amount of bone fill is surgical reentry, yet in this study second surgical procedure was not performed. Trejo et al. 1998[11] emphasized that second surgical trauma to the subjects add to the disruption of the attachment apparatus and may account for the resultant significant loss of attachment and bone fill. Clinical examination and periapical radiographs are generally sufficient in the pre- and postoperative hard tissue measurements. Usually, these are affected by common errors such as angulations and distortion. Even with the best-standardized technique, the radiograph does not show the entire topography of the area before or after treatment. A comparative study of pretreatment bone levels and posttherapy bone gains shows that linear radiographic analysis significantly underestimates pretreatment bone loss and posttreatment bone fill.[12] Therefore, in the present study Dentascan, a unique new computer software program which provides CT imaging was used. It offers significant potential for identifying mineralized structures and enhances the usefulness of radiographic evaluation by measuring coronoapical, mesiodistal and buccolingual bone loss/fill. The particle size of DFDBA and BBG used in this study was >1040 μ and 1000–2000 μ, respectively. Fucini et al. 1993[13] however concluded that there is no statistic significant difference between defects grafted with different particle sizes of DFDBA when used in humans. At 6 months postsurgically, the percentage reduction of PPD in TG1 was 45.2%, 40.9% in TG2, and 32.6% in the CG[Table 1]. Highly significant (P < 0.001) reduction of PPD was obtained both with TG1 and TG2 when compared with CG. Comparable PPD reduction was reported by[14] Scheyer et al. 2002. Percentage gain in CAL after 6 months was 50% in TG1 whereas very little differences of 38.5% were observed in TG2 and 35.9% in the CG[Table 1]. In contrast to our study,[15] Richardson et al. 1999 reported a gain of 2.6 ± 1.6 mm and 3.6 ± 1.9 mm with DFDBA and BBG, respectively. Rummelhart et al. 1989 documented a gain of 1.7 mm in CAL with DFDBA. After 6 months, posttreatment coronoapiocal bone status decreased in all groups. The improvement was highest in TG1 (50%) followed byTG2 (41.4%) and least for CG (14.3%)[Table 3]. On intragroup comparisons, significant (P < 0.01 or P < 0.001) reduction in coronoapical bone status was noted in all the groups. When compared to control significant coronoapical bone level reduction of 2.20 mm (3.5 fold) and 1.60 mm (2.9 fold) was noted in TG1 and TG2, respectively. Masters et al. 1996[16] also reported similar results of 2.20 mm bone fill with DFDBA. According to the results, DFDBA (TG1) appeared to be a favorable graft material. Dissimilar results between our study and other studies can be attributed to the variation in the degree of DFDBA demineralization between the tissue banks, which might have an effect on bone fill. With the use of Dentascan in the present study, it was possible to record two more measurements buccolingual and mesiodistal. The findings of our study demonstrated that TG1 and TG2 led to substantial improvement buccolingually when compared to nongrafted CG. Reduction in buccolingual bone status with TG1 (30.6%) was 5.3 and with TG2 (18.9%) 3.3 fold higher than the CG (5.7%). While in buccolingual measurements, no significant difference (P > 0.05) was present when TG1, TG2, and CG were compared at 6 months from baseline. Bone status mesiodistally showed significant reduction (P < 0.05) only in TG1 at 6 months when compared to baseline. Nonsignificant difference (P > 0.05) was noted in TG2 and CG at 6 months period [Table 4]. It was interesting to note that TG1 (DFDBA) maintained consistency in reducing all the three bony parameters, thus proving a better treatment choice for the management of two wall intrabony defects. Appreciable results of DFDBA (TG1) might be attributed to the exposure of BMPs on demineralization which subsequently stimulated new bone formation. Within the limits of the present study, it can be concluded that at 6 months after surgery both the grafts resulted in significant PPD reduction and CAL gain. Although significant improvement was noted in bone fill and percentage gain with both the materials DFDBA (TG1) and BBG (TG2), there was no significant difference between the two. However, DFDBA was found to be superior to BBG. Further longitudinal studies with larger sample size and use of barrier membranes should be undertaken to substantiate the osteogenic potential of DFDBA and BBG. BODY.CONCLUSION: DFDBA is better treatment choice in compare to BBG for the management of two wall intrabony defects. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Effect of Radial Extracorporeal Shock Wave Therapy on Hemiplegic Shoulder Pain Syndrome ABSTRACT.OBJECTIVE: To investigate the effect of radial extracorporeal shock wave therapy (rESWT) on hemiplegic shoulder pain (HSP) syndrome. ABSTRACT.METHODS: In this monocentric, randomized, patient-assessor blinded, placebo-controlled trial, patients with HSP were randomly divided into the rESWT (n=17) and control (n=17) groups. Treatment was administered four times a week for 2 weeks. The visual analogue scale (VAS) score and Constant-Murley score (CS) were assessed before and after treatment, and at 2 and 4 weeks. The Modified Ashworth Scale and Fugl-Meyer Assessment scores and range of motion of the shoulder were also assessed. ABSTRACT.RESULTS: VAS scores improved post-intervention and at the 2-week and 4-week follow-up in the intervention group (p<0.05). Respective differences in VAS scores between baseline and post-intervention in the intervention and control groups were –1.69±1.90 and –0.45±0.79, respectively (p<0.05), between baseline and 2-week follow-up in the intervention and control groups were –1.60±1.74 and –0.34±0.70, respectively (p<0.05), and between baseline and 4-week follow-up in the intervention and control groups were –1.61±1.73 and –0.33±0.71, respectively (p<0.05). Baseline CS improved from 19.12±11.02 to 20.88±10.37 post-intervention and to 20.41±10.82 at the 2-week follow-up only in the intervention group (p<0.05). ABSTRACT.CONCLUSION: rESWT consisting of eight sessions could be one of the effective and safe modalities for pain management in people with HSP. Further studies are needed to generalize and support these results in patients with HSP and a variety conditions, and to understand the mechanism of rESWT for treating HSP. BODY.INTRODUCTION: Hemiplegic shoulder pain (HSP) is one of the most common problems after stroke with a prevalence of 34%–84%, and it can inhibit recovery and reduce quality of life [12]. One pathology alone cannot account for shoulder pain after stroke. In general, the causes of HSP are thought to be subluxation of the shoulder, rotator cuff problems, adhesive capsulitis, and complex regional pain syndrome. In a patient with HSP, more than two types of shoulder pathology exist [3]. Because HSP can have multiple causes and patients have a variety of medical conditions, it is necessary to consider targeted treatments and the patient's status. Well-known treatments for HSP are oral medications including non-steroidal anti-inflammatory drugs (NSAIDs), a subacromial-subdeltoid bursa steroid injection or fluoroscopically guided anterior approach intra-articular steroid injection, transcutaneous electrical nerve stimulation (TENS), and range of motion exercises. However, treatments that are currently being used for HSP cause several complications such as mechanical injury to joint structures during fluoroscopically guided anterior approach intra-articular injection or blind intra-articular injection, and there are limitations on the use of steroids in patients who have underlying diseases and unstable medical conditions such as diabetes mellitus, cardiac problems, etc. Extracorporeal shock wave therapy (ESWT) has been suggested as a non-invasive and alternative treatment for shoulder pain, and the effects of ESWT on various musculoskeletal disorders have been reported [456]. ESWT is a sequence of single sonic pulses, which have a high peak pressure (100 MPa), fast pressure rise (<10 ns), and short duration (10 μs). During ESWT, shock waves are transmitted by an appropriate generator to a specific target area, with an energy flux density ranging from 0.003–0.890 mJ/mm2. Compared to conventional focused ESWT, radial extracorporeal shock-wave therapy (rESWT) does not focus shock waves on a target zone. Because the waves of rESWT disperse eccentrically from the applicator tip without concentrating the shock wave field on the targeted tissue, ultrasound or fluoroscopy is not required. The therapeutic effect of rESWT occurs 0–3.5 cm deep within the skin's surface [78]. It has shown promising results in patients with musculoskeletal problems [9] and in those with various causes of HSP, including spasticity, rotator cuff problems, adhesive capsulitis, and complex regional pain syndrome [10111213141516]. Several studies on the effect of rESWT (or ESWT) in post-stroke patients have focused on the spasticity of muscles, and although research on shoulder pain after stroke has been conducted, to the best of our knowledge, none of the studies had a control group or assessed shoulder pain as a primary outcome [10]. Therefore, the aim of our study was to investigate the beneficial effects of rESWT on HSP after stroke. We hypothesized that eight sessions of rESWT on the greater and lesser tuberosities as the insertion sites of subscapularis and supraspinatus would be safe and effective in reducing HSP symptoms. BODY.MATERIALS AND METHODS.SUBJECTS: In our monocentric, randomized, patient-assessor blinded, placebo-controlled trial, we recruited patients who were hospitalized from March 2015 to July 2015. The study was approved by the appropriate ethical committees. Patients who (1) understood the study process and signed the informed consent form, (2) had a brain lesion confirmed by brain imaging, (3) had the first stroke more than 3 months ago, (4) were hemiplegic due to stroke, and (5) had shoulder pain and limited range of motion (ROM) or loss of motion in the proximal arm on the hemiplegic side were included. Patients who (1) could not express their own pain intensity, (2) had a history of trauma to the shoulder on the affected side or a history of surgery on the shoulder on the affected side, (3) had a history of prior use of oral NSAIDs 3 days before this study, (4) had a history of shoulder pain before the stroke, (5) took warfarin medication with an international normalized ratio above 4.0, (6) had received a previous shoulder intra-articular injection or other interventions on the affected shoulder within 1 month before rESWT, (7) had received a cardiac pacemaker, (8) had osteoporosis, (9) refused the procedure before or during the study, and (10) had psychological problems were excluded. A researcher who was not involved in processing or analyzing the data used a computerized randomization program, and patients were randomly assigned to the rESWT group or the control group. BODY.MATERIALS AND METHODS.INTERVENTIONS: Each patient sat in a wheelchair or firm chair with the affected side of the shoulder kept open. HSP has various causes, including rotator cuff tendinopathies, adhesive capsulitis, spasticity, complex regional pain syndrome; therefore, to determine the effect of rESWT on the causes of HSP, we referred to previous studies in which rESWT was used to treat shoulder problems [10111213]. In the studies on the effect of ESWT on shoulder pain, Galasso et al. [11] performed stimulation around the supraspinatus tendon, Kim et al. [10] used the subscapularis muscle belly as the target site, Seo et al. [12] performed stimulation around the rotator cuff tendons in their study of rotator cuff tendinopathy, and Chen et al. [13] used three target sites around the shoulder in their study of adhesive capsulitis. By referencing these previous studies, we arbitrarily chose the greater and lesser tuberosities of the humeral head as the stimulation sites to stimulate rotator cuff tendons and the shoulder capsule. These stimulation sites were the subscapularis and supraspinatus insertion sites, and the stimulations were guided by ultrasonography. The ultrasonography-guided interventions were performed by a single rehabilitation physician who did not assess the outcome measures. The shoulder was externally rotated, and the elbow was flexed at 90° to stimulate the subscapularis insertion site. To stimulate the supraspinatus insertion site, the shoulder was internally rotated, and the elbow was slightly extended. The Masterpuls MP200 (Storz Medical AG, Tagerwilen, Switzerland) was used to perform rESWT (Fig. 1A). In a previous study by Kim et al. [10], treatment was performed 5 times for 2 weeks. In our study, treatment was administered four times a week for 2 weeks (a total of eight sessions), which was more intensive than the treatment in the study by Kim et al. [10]. Patients received 3,000 pulses, 1,500 pulses per site at a frequency of 12 Hz per session with the submaximal pressure between 0.39 and 1.95 mJ/mm2 (1.0 and 5.0 bar), depending on the level which the patient could tolerate without local anesthetics. In the control group, the same protocol was used. Sham stimulation was performed as in previous studies with a control group. In these studies, sham treatments were performed by using the same sound from a compact disc player without stimulation [11] or minimal stimulation [17] to create a similar situation, except without shock wave stimulation. In our study, stimulation was not delivered as the transmitter head was removed; hence, the patients received the same frequency of air pressure and sound (Fig. 1B). BODY.MATERIALS AND METHODS.OUTCOME MEASURES: Information on the general characteristics was collected from patients' medical records. Abduction muscle strength of the shoulder on the affected side was assessed using the manual muscle test, along with the Medical Research Council scale and Brunnstrom stage, and outcome measures were assessed by a single rehabilitation physician who was blinded to randomization. Outcome measures were evaluated before (baseline) and after 1 day of the 2-week intervention (post-intervention), and follow-up evaluations were performed at 2 weeks and 4 weeks after the intervention (Fig. 2). As primary outcome measures, the visual analogue scale (VAS) score of the affected shoulder and the Constant-Murley score (CS) were used. Secondary outcome measures were the Modified Ashworth Scale (MAS) score, ROM of the glenohumeral joint of the affected side (flexion, abduction, internal rotation, and external rotation), and Fugl-Meyer Upper Extremity Assessment (FMA-UE) score. The subjective intensity of shoulder pain was measured using a 10-cm VAS. VAS is the most commonly used scale for the quantification of pain. There is evidence showing that it has superior metrical characteristics than discrete scales; thus a wider range of statistical methods can be applied to the measurements [18]. The CS is a functional evaluation method for the shoulder. The CS is composed of four domains: pain intensity, limitation of activities of daily living, mobility of the shoulder joint (ROMs for flexion, abduction, internal rotation, and external rotation), and muscle power of the shoulder. The CS is based on a 100-point scale, composed of subjective and objective parameters. Subjective measurements yield 35 points. Objective measurements are awarded a maximum of 65 points, with 40 points for full ROM and 25 points for normal muscle strength [19]. The MAS score is the most commonly used to manually assess spasticity. For the convenience of performing statistical analysis, a MAS grade 1+ was matched to 2 points; and grades 2, 3, and 4 were matched to 3, 4, and 5 points, respectively [20]. ROM was measured passively with a goniometer for forward flexion, abduction, and internal and external rotation of the shoulder. The measurement was taken with the patient in a sitting position. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Categorical variables were analyzed using the chi-square test, and continuous variables were analyzed by the Mann-Whitney U test to determine comparisons between the rESWT and control groups at baseline. To analyze the effects of the intervention within groups, the Wilcoxon signed-rank test was used. Differences between post-intervention and baseline, between 2-week follow-up and baseline, and between 4-week follow-up and baseline were calculated in both groups. To determine the intra-individual difference between the rESWT and control groups, the Mann-Whitney U test was used. The values reported post-intervention minus those at baseline in the rESWT and control groups were compared using the Mann-Whitney U test. Similarly, the 2-week follow-up data minus the baseline data, and the 4-week follow-up data minus the baseline data between the rESWT and control groups were analyzed. To investigate the predictive value of the effect of rESWT, multiple linear regression analyses using the backward elimination technique were applied to the data from the rESWT group to determine the correlation between baseline variables and the degree of pain reduction after 2 weeks of rESWT intervention. A p-value <0.05 was considered statistically significant. All data are presented as mean±standard deviation. All statistical analyses were performed using SPSS software ver. 18.0 for Windows (SPSS Inc., Chicago, IL, USA). BODY.RESULTS: Of the 91 stroke patients screened, 40 were enrolled in the study according to the inclusion and exclusion criteria. The flow chart of the study patients and a description of the missing data are presented in Fig. 3. BODY.RESULTS.GENERAL CHARACTERISTICS: Thirty-four patients included 17 men and 17 women. Of the 17 subjects in the rESWT group, 7 were men and 10 were women, whereas the control group was composed of 10 men and 7 women. The average age was 65.88±8.27 years and 66.11±15.79 years in the rESWT and control groups, respectively. After performing the chi-square test and Mann-Whitney U test to examine the preliminary homogeneity of baseline variables between the two groups, both groups were found to be homogeneous as there was no significant difference (p>0.05) (Table 1). BODY.RESULTS.PRIMARY OUTCOME MEASURES.PAIN INTENSITY (VAS): The VAS score significantly improved post-intervention and at the 2-week and 4-week follow-up compared to baseline in the intervention group. The VAS scores at baseline before treatment, post-intervention, 2-week follow-up, and 4-week follow-up were 5.01±1.21, 3.32±1.50, 3.41±1.54, and 3.40±1.52, respectively (p<0.05). In the control group, the VAS scores significantly improved post-intervention from 5.05±1.34 to 4.60±1.20 (p<0.05). Respective differences in the VAS score between baseline and post-intervention in the intervention and control groups were –1.69±1.90 and –0.45±0.79 (p=0.038), between baseline and 2-week follow-up in the intervention and control groups were –1.60±1.74 and –0.34±0.70 (p=0.009), and between baseline and 4-week follow-up in the intervention and control groups were –1.61±1.73 and –0.33±0.71 (p=0.016), which were statistically significant (Table 2). BODY.RESULTS.CONSTANT-MURLEY SCORE: The CS significantly improved from 19.12±11.02 at baseline to 20.88±10.37 post-intervention and to 20.41±10.82 at the 2-week follow-up only in the intervention group (p<0.05). The CS score at the 4-week follow-up was 20.05±11.34 (p=0.064), and when compared to the baseline value, it was not statistically significant. Differences in the CS between baseline and post-intervention, 2-week follow-up, and 4-week follow-up in the intervention and control groups were slightly increased without statistical significance (Table 2). BODY.RESULTS.SECONDARY OUTCOME MEASURES: ROMs of the shoulder joint, including internal rotation, external rotation, abduction, and flexion, were significantly improved post-intervention, and at the 2-week and 4-week follow-up compared to those at baseline only in the intervention group (Table 3). The MAS and FMA-UE scores in the rESWT group showed minimal improvement post-intervention, and at the 2-week and 4-week follow-up after the intervention compared to those at baseline, but this difference was not statistically significant (p=0.157, 0.083, and 0.102, respectively; p=0.068, 0.066, and 0.109, respectively) (Table 3). BODY.RESULTS.PREDICTIVE VALUE FOR THE EFFECT OF RESWT: Among the baseline variables (age; pain duration; baseline VAS score, CS, MAS score, and FMA-UE score; and ROMs of the shoulder), only the baseline VAS score was correlated with the degree of pain reduction by rESWT with R2=37.4%, β=–0.962, and p=0.009. BODY.RESULTS.ADVERSE EVENTS: After the eight sessions of rESWT, four adverse events were noted after the final intervention. Three patients had petechiae at the treatment site, which resolved spontaneously, and a small bulla was noted in 1 patient, which completely healed after a few days with a simple dressing. BODY.DISCUSSION: To the best of our knowledge, this is the first randomized controlled study that focused on the effects of rESWT on pain and function in people with HSP. The results of the study demonstrated that eight sessions of rESWT on the subscapularis and supraspinatus insertion sites of a hemiplegic shoulder reduced the pain and its effects lasted for at least 4 weeks. Our results on the effectiveness of rESWT in HSP are similar to those of a previous study by Kim et al. [10], in which the pain was reduced and the effect was maintained for 4 weeks. Although the mechanisms of the pain improvement effect with rESWT (or ESWT) are unclear, it has been proposed that rESWT generates oscillations in tissue that leads to improved microcirculation and metabolic activities [21]. The immediate pain reduction effect after rESWT (or ESWT) can be explained by the result of a hyperstimulation analgesic effect [22]. Other mechanisms underlying the various causes of HSP, including rotator cuff problems, adhesive capsulitis, and complex regional pain syndrome, were suggested to lead to the anti-fibrotic effect [23], anti-inflammatory effects [11], and modulation of pain [14]. In the present study, the control group also showed pain improvement post-intervention, but the degree of pain improvement was negligible compared with that in the rESWT group, in which the difference was statistically significant. The placebo effects of ESWT have been suggested in various musculoskeletal conditions, including shoulder pain and soft-tissue lesions [242526]. The placebo and time effect can influence the results of pain improvement in a placebo group [27], but in the present study, the placebo effect seemed minimal. In our study, the amount of pain reduction after intervention was 1.69 (from 5.01 to 3.32) and 0.45 (from 5.05 to 4.60) in the real rESWT group and the control group, respectively. The amount of pain reduction in the real rESWT group was considered to be too small to ensure patient satisfaction and the difference between the results of the two groups did not seem to be large enough. However, the clinical meaning that we derived from these results was that pain reduction to a VAS score of less than 4 was seen only in the real rESWT group. In terms of functional improvement in the shoulder after rESWT (or ESWT), there have been conflicting results [1728]. Our study focused on patients with HSP, and improvement in the CS occurred and its effect lasted for only 2 weeks after the intervention with statistical significance in the rESWT group; however, the effect did not reach statistical significance in the between group analyses. Improvement of the CS score was not considered to have a clinical significance. The shoulder muscle strength in the enrolled patients with HSP was too weak to show a significant functional improvement. Further future studies are needed to determine functional improvement after rESWT in patients with HSP who have a minimal loss of shoulder muscle strength. The results of the current study correspond with those of previous studies that demonstrated a positive effect on the ROM of the shoulder [2930]. HSP is associated with limited ROM of the shoulder [3132]. This is thought to be due to a combination of synovial inflammation and capsular fibrosis [333435], and they can aggravate the conditions of decreased active shoulder movement because of paralysis, synergistic pattern movements, and spasticity. The histologic features of adhesive capsulitis are capsular thickening and contracture of the ligaments around the shoulder, which is accompanied by an increased level of several inflammatory cytokines [333435]. The anti-inflammatory effects and anti-fibrotic effects of ESWT have been proposed as the mechanism of therapeutic effects of rESWT in several studies [233637], and this seems to be a possible explanation for the positive effect on ROM in the current study. In our study, spasticity assessed by the MAS score improved but the result did not reach statistical significance. Unlike our study, Vidal et al. [29] showed a significant decrease in spasticity measured by the Ashworth scale in spastic cerebral palsy patients after rESWT. In studies on stroke patients, spasticity was also improved [15163038]. The mechanisms of ESWT on spasticity after stroke are still unknown. Variable mechanisms have been proposed, including induction of nitric oxide synthesis, decrease in spinal excitability, and induction of mechanical vibrations [39]. There are several reasons for the absence of change in the MAS score in the present study. In many studies that showed decreased spasticity after ESWT, the stimulation sites included the muscle belly or musculotendinous junction, but in our study, the muscle insertion sites were stimulated. The initial lower MAS score and flaccidity of the hemiplegic shoulders in our study may have occurred due to another reason. Among various predictive values, higher pain scores were associated with better treatment outcomes following rESWT treatment. This is an interesting result, as it conflicts with the result of a previous study [40]. In our study, about half of the patients in the rESWT group had a VAS score of less than 5. The small amount of pain reduction in the real rESWT might be considered to be due to the proportion of patients with initial lower VAS scores in the rESWT group. In order to verify the actual pain relief effect, further study assessing the patient's satisfaction after treatment is needed. The limitations of our study include the small number of subjects; in this situation, we used nonparametric methods so that statistical power was reduced. Therefore, the study sample was too small to generalize the results of our study. Another limitation was the relatively short follow-up duration. Since our follow-up period was 4 weeks after the last rESWT, long-term follow-up studies are needed to precisely assess the duration of the effect of rESWT. Comparative studies on HSP treatments, such as steroid intra-articular injections, oral medications, and TENS, and on the treatment protocol of rESWT are needed in the future. The current study demonstrated that eight sessions of rESWT at the subscapularis and supraspinatus insertion sites improved the VAS score in patients with HSP. Therefore, rESWT could be one of the effective and safe modalities for pain management in people with HSP. Further studies with various treatment protocols are needed to generalize and support these results in patients with HSP and a variety conditions, and to understand the mechanism of rESWT for treating HSP.

TITLE: Effect of Antihelminthic Treatment on Vaccine Immunogenicity to a Seasonal Influenza Vaccine in Primary School Children in Gabon: A Randomized Placebo-Controlled Trial ABSTRACT.BACKGROUND: Helminth infections are a major public health problem, especially in the tropics. Infected individuals have an altered immune response with evidence that antibody response to vaccination is impaired. Hence, treatment of helminth infections before vaccination may be a simple intervention to improve vaccine immunogenicity. In the present study we investigated whether a single-dose antihelminthic treatment influences antibody responses to a seasonal influenza vaccine in primary school children living in Gabon, Central Africa. ABSTRACT.METHODS: In this placebo-controlled double-blind trial conducted in Gabon the effect of a single-dose antihelminthic treatment with 400 mg albendazole versus a placebo one month prior to immunization with a seasonal influenza vaccine was investigated. Antiviral antibody titers against all three vaccine strains were assessed by haemagglutination inhibition (HI) test at baseline (Day 0; vaccination) and four weeks (Day 28) as well as 12 weeks (Day 84) following vaccination. Vaccine-specific memory B-cell response was measured at Day 0 and Day 84 by vaccine-specific Enzyme-linked Immunospot (ELISpot) assay. The trial is registered with the Pan African Clinical Trials Registry (PACTR) (PACTR201303000434188). ABSTRACT.RESULTS: 98 school children aged 6–10 years were randomly allocated to receive either antihelminthic treatment or placebo and were vaccinated one month after the treatment. The prevalence of helminths at baseline was 21%. Vaccine-specific HI titers against at least one of the three vaccine strains increased at Day 28 and Day 84 in all participants. HI titers against both influenza A strains as well as memory B-cell response were modestly higher in the antihelminthic treated group compared to the placebo group but the difference was not statistically significant. Total but not specific IgA was elevated in the antihelminthic treated group compared to the control group at Day 28. ABSTRACT.CONCLUSION: In our setting antihelminthic treatment had no significant effect on influenza vaccine immunogenicity. A trend towards better antiviral and vaccine immunogenicity in the antihelminthic treated group encourages studies to be conducted with alternative treatment schedules or in populations with a higher helminth burden. BODY.INTRODUCTION: Infection with geohelminths, mainly Ascaris (A.) lumbricoides, Trichuris (T.) trichiura and hookworm, is a major public health problem affecting 20% of the world's population, mainly for those living in Sub-Saharan Africa (SSA). As access to public health programs is widely lacking, geohelminthiasis is considered by the World Health Organization (WHO) as one of the most neglected tropical diseases with serious health, nutritional and social outcomes for the affected individuals[1–3]. Vulnerable groups are children[2] and pregnant women[3]. Chronic infection with geohelminths has an impact on health as well as on cognitive skills[4–8] and it is known that infection with helminths leads to immune response alterations. Usually, T-helper type 2 (Th2) immune responses[9–12] are predominant and a general suppression of innate and adaptive T- and B-cell responses via the activation of regulatory T-cells (Treg) and/or induction of anti-inflammatory cytokines[10,13,14] may lead to general hyporesponsiveness of the immune system[14,15]. Vaccination is one of the most effective tools to prevent infectious diseases. Nonetheless seroconversion and therefore efficacy are variable in vaccinated individuals depending on age, environment and genetic host factors[16–18]. In addition, acute and chronic infections have an influence on vaccine outcome[19,20]. The interaction of geohelminth infection and vaccination is not well investigated although immunization programs for infants are well implemented in areas where geohelminths are highly endemic. Until now it has been shown that A. lumbricoides has an impact on the immune response induced by an oral cholera vaccine[21] and that intestinal parasites influence the outcome of a Bacillus-Calmette-Guérin (BCG) vaccination[22]. There is evidence that the presence of geohelminths, especially T. trichiura negatively influences immune responses against GMZ2, a malaria vaccine candidate[23]. Past studies from Gabon showed that Gabonese school children are heavily infected with intestinal parasites (infection rates of A. lumbricoides 46%, or T. trichiura 71%) and 74% of examined children were at least positive for one of the investigated helminths[24–26]. Regular antihelminthic treatment in high-risk groups like school children is considered as an effective tool for controlling the burden of geohelminth infection but is not widely implemented in Gabon. The WHO promotes helminth control by periodic deworming once or twice a year, depending on prevalence, as a cost-effective intervention[27,28]. However regular deworming is not yet implemented in all endemic countries[29]. Antihelminthic treatment would be a cost-effective and easy tool to reduce worm burden[30] and may simultaneously increase vaccine immunogenicity. Therefore in the present study we investigated the effect on vaccine immunogenicity of pre-treatment with a single-dose of albendazole four weeks prior to a scheduled seasonal influenza vaccination in Gabonese primary school children. BODY.MATERIALS AND METHODS.TRIAL DESIGN AND SETTING: For this double-blinded randomized trial healthy primary school children from Lambaréné and surroundings were randomized to receive either antihelminthic treatment (albendazole 400 mg) (Micro Lab ltd, India) or placebo (Laboratories Sterop, Belgium) four weeks (Day -28) prior to vaccination with either a seasonal influenza vaccination (VAXIGRIP, Sanofi Pasteur, season 2011/2012) intra muscularly (i.m.) (n = 98), Polysaccharide Meningococcal A+C vaccine (Sanofi Pasteur) sub cutaneously (s.c.) (n = 104) or an oral cholera vaccine (Dukoral, Sanofi Pasteur) (n = 106) administered at Day 0. Vaccinations were given in three subsequent time slots. The first cohort of primary school children was vaccinated with the influenza vaccine, the second with meningococcal vaccine and the third cohort received two times the cholera vaccine. Inclusion criteria were age from 6 to 10 years (primary school children), a signed informed consent form (ICF), good general health upon clinical examination and no acute symptoms of geohelminths infection. Furthermore the participants and their legal representative was asked if he/she will be resident in the area until the end of the study. Exclusion criteria were the participation in another clinical trial, known contraindication to antihelminthic treatment or to the administration of one of the chosen vaccines including i.m. or s.c. administration, known immunization against the vaccine antigens, known infection with pathogens of one of the vaccine antigens in the past except for influenza (because the influenza vaccine strain composition is different each year), any confirmed or suspected immunosuppressive or immunodeficient condition resulting from disease (e.g., malignancy, HIV infection) or immunosuppressive/cytotoxic therapy as well as acute disease at the beginning of the study and before vaccination. If a child was febrile at the scheduled time of vaccination, injections were postponed until the infection was cured. If a child had a known acute or chronic disease like malaria, AIDS or tuberculosis as well as a haemoglobin level < 7 g/dl or signs of haematuria and/or proteinuria tested by urine sticks (Combur 9 test) the child was not included and referred to the Albert Schweitzer Hospital (ASH) for treatment. If menarche was reported a pregnancy test was be performed at Day -28 and Day 0 prior to antihelmintic treatment and prior to vaccination. Here, we report results of the first part of the study, where the children were vaccinated with the seasonal influenza vaccine. Children, who were infected with Schistosoma (S.) haematobium, as well as children with any other symptomatic infection were excluded from the study and treated accordingly. All parasite positive participants (including those without symptoms) received appropriate treatment after study termination. BODY.MATERIALS AND METHODS.IMMUNOLOGICAL INVESTIGATIONS: Blood was taken on the day of vaccine injection (Day 0), Day 28 (four weeks after vaccination) and Day 84 (12 weeks after vaccination). The primary immunological endpoint of the study was functional antibody level measured as haemagglutination inhibition (HI) testing. To assess memory B-cell response (secondary immunological endpoint) against the vaccine antigens, an Enzyme-linked Immunospot (ELISpot) assay was performed. BODY.MATERIALS AND METHODS.ASSESSMENT OF ANTI-INFLUENZA ANTIBODY TITERS: Pre- and post-vaccination samples were analyzed by a validated microtiter haemagglutination inhibition (HI) test at the German National Reference Center for Influenza (NRZ Influenza, Robert Koch Institute (RKI), Berlin) as previously described[31]. Prior to testing, each serum was treated with receptor-degrading enzyme to inactivate non-specific inhibitors at a final serum dilution of 1:10. Sera were then diluted serially two fold into microtiter plates. Each virus strain was adjusted to 4 HA units/25 μl which was verified by back titration and 25 μl of this virus suspension was added to each of the 96 wells. After incubation at room temperature (RT) for 30 min freshly prepared 0.5% turkey red blood cells (RBCs) were added, the plates were mixed, followed by a further incubation at RT for 30 min. Human sera serving as positive controls and negative controls were included on each plate. HI titers were reported as the reciprocal of the last serum dilution that contained non-agglutinated RBCs. BODY.MATERIALS AND METHODS.ASSESSMENT OF TOTAL IMMUNOGLOBULINE (IG) ISOTYPES AND SUBCLASSES: Pre- and post-vaccination samples (Day 0, Day 28 and Day 84) were analyzed by using a Multiplex assay (Biorad, Germany) for detection of multiple antibodies, like IgG subclasses (IgG1; IgG2, IgG3 and IgG4), total IgE, total IgM and total IgA at baseline, Day 28 and Day 84 post vaccination. The assay was conducted according to the manufacturer's specifications. BODY.MATERIALS AND METHODS.ASSESSMENT OF VACCINE-SPECIFIC IGA BY ENZYME-LINKED IMMUNOSORBANT ASSAY (ELISA): To assess vaccine-specific IgA concentrations plates (Nunc, Germany) were coated with 0.5 μg/ml of the vaccine antigen, incubated over night at 4°C and blocked with blocking buffer (0.3% milkpowder (Roth, Germany), 0.1% Tween-20 (Sigma, Germany) and PBS (Life technologies, USA) for 1 h at RT. Samples were plated in serial dilutions for 2 h at RT. As secondary antibody a polyclonal rabbit anti-human IgA/HRP (Dako, Germany) was used. For visualization TMB one (Kem En Tec, Denmark) and H2SO4 (Merck, Germany) was used. OD was measured using a Photometer (Phomo, Anthos, Germany) at wave length 450 nm and 620 nm as reference. BODY.MATERIALS AND METHODS.VACCINE-SPECIFIC MEMORY B-CELL ELISPOT: As previously described, peripheral blood mononuclear cells (PBMCs) were frozen on Day 0 and Day 84 and later used for memory B-cell ELISpot[32,33]. In brief, PBMC were separated from heparinized full blood by gradient centrifugation (Ficoll-Plaque PLUS, GE Healthcare, Sweden), counted and frozen in 90% fetal calf serum (FCS Gold PAA, Germany) and 10% DMSO (Sigma, Germany) at -150°C. Before ELISpot, cells were thawed, counted and seeded at a density of 1*106 cells per ml in RPMI 1640 (Sigma, Germany), complemented with sodium pyruvate, non-essential amino acids, L-glutamine, penicillin, streptomycin (all supplemented from Life Technologies, USA) and 10% heat inactivated FCS (FCS Gold, PAA, Germany). Maturation of circulating memory B-cells into antibody-secreting cells (ASC) was performed by in vitro stimulation with 2.5 μg/ml CpG-2006 (TIB-MOLBIOL, Germany) and 10 ng/ml recombinant human IL-15 (R&D systems, USA) in 24 wells cell culture plates (Corning Costar) for 6 days at 37°C, 5% CO2 as described[34]. Following maturation, 2*105 cells were serially diluted on Ag-specific 96 well plates (Millipore, Germany). The vaccine (VAXIGRIP) was coated over night at a concentration of 5μg/ml in PBS. Plates were washed and blocked for 1 hour (h) with complete medium. Meanwhile cells were washed and counted. After seeding cells were incubated at 37°C, 5% CO2 for 2 h allowing them to secrete antibodies. Secreted antigen (Ag)-specific antibodies were detected using biotin labeled anti-human IgG antibody (Sigma, Germany, 1:500 in PBS, 3% BSA) and ExtrAvidin peroxidase (SIGMA, Germany, 1:600 in PBS, 5% BSA). AEC substrate (SIGMA, Germany) was added for 10 min at RT. After staining the plates were dried over night. Spots were counted by the CTL ImmunoSpot (CTL, USA). BODY.MATERIALS AND METHODS.ASSESSMENT OF PARASITE BURDEN: Stool samples were collected at Day -28, Day 0 and Day 84 and analyzed by using the qualitative Merthiolate-Iodine-Formaldehyde (MIF)-technique[35]. In brief, 10 ml of Merthiolate-Formaldehyde-Solution (5% Formaldehyd, 1% Glycerine (Merck, Germany)) and Lugol's Solution (10% potassium iodine, 5% iodine (Merck, Germany)) were added to each walnut size stool sample (approx. 5 g) filtered through a metal wire, centrifuged for 5 min 1500 rounds per minute (rpm) and analyzed by microscopy at the end of the study. Urine was examined for the presence of S. haematobium by urine filtration method[36] using 10 ml of well mixed urine passed through a filter (12 μm pore size, Millipore, Germany). The filter was transferred to a glass slide, stained with methylene blue and analyzed by microscopy. At Day 0 and Day 84 thick blood smears were performed to assess malaria parasites retrospectively. If a child presented with fever or any other symptom suggestive of malaria, a rapid test (Paracheck Pf) was performed and the volunteer was treated with appropriate treatment. BODY.MATERIALS AND METHODS.SAMPLE SIZE CALCULATION: We assumed that a 30% difference of immune responses between antihelminthic treatment and placebo groups is clinically relevant. In a pilot trial with the malaria vaccine candidate GMZ2 we observed a difference in antibody response greater than 30% with a standard deviation of 0.5[23]. A sample size of 45 individuals (n = 45) per group is required to detect such a difference with a power of 80% at a significance level of 0.05. To allow for 15% of loss to follow-up a total of 52 (n = 52) schoolchildren per group were required. Sample size calculation was done using R v2.9.0[37]. BODY.MATERIALS AND METHODS.RANDOMIZATION, DATA ENTRY AND STATISTICAL ANALYSIS: The randomization and data management was performed on the "Koordobas" database system (Institute for Clinical Epidemiology and applied Biometry, University of Tübingen) The medical personal treating participants were not involved in the outcome evaluation. Group differences at follow-up visits (Day 28, Day 84) were determined with a rank based ANCOVA (Analysis of Covariance) corrected for baseline titer and gender. Differences in ELISpot counts were assessed by a Wilcoxon test. Geometric mean titers were calculated according to 10mean(log10(1+HI-titer)). A two sided alpha of 0.05 was used as significance level. BODY.MATERIALS AND METHODS.ETHICAL APPROVAL: The study was approved by the Comité d'Ethique Régional Indépendant de Lambaréné and the Comité National d ́Ethique pour la Recherche du Gabon. The trial is registered with PACTR (PACTR201303000434188) and the study was conducted in accordance to the Declaration of Helsinki and followed the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) guidelines. The child's parents or their legally accepted representatives provided written informed consent before study participation. BODY.RESULTS: From December 2011 until September 2012 out of 113 screened primary school children from Lambaréné and surroundings 98 were randomized to receive either antihelminthic treatment (n = 50) or placebo (n = 48). 92 participants were vaccinated with a seasonal influenza vaccine and 82 terminated the study with visit 4 at Day 84 post vaccination (Fig 1). Baseline characteristics were similar between the two groups except that the fraction of females was higher in the antihelminthic treated group (57% versus 34% in the placebo group) (Table 1). A possible effect of gender was taken into account in exploratory and sensitivity analyses. 10.1371/journal.pntd.0003768.g001Fig 1Study profile. +Patients were excluded, because of infection with S. haematobium. *Participants not terminating the study are summarized as lost to follow up (n = 16). 10.1371/journal.pntd.0003768.t001 Table 1 Baseline characteristics and helminth infection at day -28. Antihelminthic treatment Placebo Gender Male 22 (22.4%) 32 (32.7%) Female 28 (29.6%) 16 (16.3%) Age 6 years 15 (15.3%) 16 (16.3%) 7 years 9 (9.2%) 8 (8.2%) 8 years 14 (14.3%) 12 (12.2%) 9 years 10 (10.2%) 12 (12.2%) 10 years 2 (2%) 0 Mean age 7.43 Helminth Single infection A . lumbricoides 2 (2%) 2 (2%) F . hepatica 0 2 (2%) T . trichiura 4 (4%) 3 (3%) Multiple infection T . trichiura/ A . lumbricoides 2 (2%) 4 (4%) T . trichiura/ A . lumbricoides/ A . duodenale 0 1 (1%) Negative 41 (41.84%) 33 (33.3%) NA 1 (1%) 3 (3%) BODY.RESULTS.ANTI-INFLUENZA ANTIBODY TITERS: Antibodies against the three influenza vaccine strains A/California/7/09 (A(H1N1)pdm09), A/Perth/16/09 (A(H3N2)) and B/Brisbane/60/08 were determined by HI testing. HI titers increased against at least one vaccine strain after vaccination at Day 28 and Day 84 in all participants (Fig 2). The increase of HI values for A(H1N1)pdm09 and A(H3N2) tended to be higher in the antihelminthic treated group compared to the control group but the difference was not statistically significant (Figs 2 and 3). 10.1371/journal.pntd.0003768.g002Fig 2Antibody titers against the three vaccine strains at baseline (day 0), day 28 and day 84.Red lines indicate the mean of all volunteers of the antihelminthic treated group (AT) and blue lines indicate the mean of all participants of the placebo group. Dashed lines indicate antibody titers of each participant. 10.1371/journal.pntd.0003768.g003Fig 3Differences of HI titers between the respective visits (day 28, day 84) and day 0 (baseline).Red and blue colors represent the pre-treated (AT) and control group. Thirty-four participants already had detectable antibodies at baseline for the A(H1N1)pdm09 strain. The baseline titers ranged between 15 and 640 with a median of 120 in the antihelminthic treated group and 80 in the placebo group and the GMT was 28 in the antihelminthic treated group and 15 in the control group; 19 participants in the antihelminthic treated group and 17 participants in the placebo group were without a detectable baseline HI titer. At Day 28 the HI titers increased up to 5000 (median: 640 in both groups and a GMT of 134 in the antihelminthic treated group and 84 in the control group,) and decreased until Day 84 (median: 320 in both groups and GMT of 138 in the antihelminthic treated group and 94 in the control group) (Tables 2 and 3). 10.1371/journal.pntd.0003768.t002 Table 2 Median, 25% and 75% quartile of vaccine strain specific HI titers. Day 0 Day 28 Day 84 A(H1N1)pdm09 Antihelminthic treatment 120 (0,160) 640 (0,1280) 320 (35,960) Placebo 80 (0,60) 640 (0,960) 320 (20,480) A(H3N2) Antihelminthic treatment 120 (20,240) 640 (320,960) 320 (280,640) Placebo 80 (40,160) 640 (480,1280) 320 (240,600) Influenza B Antihelminthic treatment 0 (0,40) 320 (120,320) 160 (80,320) Placebo 0 (0,20) 320 (120,520) 160 (80,800) 10.1371/journal.pntd.0003768.t003 Table 3 GMT of vaccine strain specific antibodies. Day 0 Day 28 Day 84 A(H1N1)pdm09 Antihelminthic treatment 28 134 138 Placebo 15 84 94 A(H3N2) Antihelminthic treatment 73 554 344 Placebo 72 516 258 Influenza B Antihelminthic treatment 12 142 120 Placebo 10 164 97 Antibody titers against the strain A(H3N2) ranged from 15 to 160 (median of 120 in the antihelminthic treated group and 80 in the control group), whereas the GMT was 73 for the antihelminthic treated and 72 for the placebo group at Day 0. For this strain also an increase of antibodies at Day 28 with a median of 640 in both groups and a GMT of 554 in the antihelminthic treated group and 516 in the placebo group as well as decreasing values at Day 84 with an median of 320 in both groups and a GMT of 344 in the antihelminthic treated group and 258 in the control group were observed (Tables 2 and 3). Antibodies against the influenza B vaccine strain had a median of 320 at Day 28 in both groups and declined until Day 84 with a median of 160 in both groups. GMT at Day 28 was 142 in the antihelminthic treated group and 164 in the placebo group and at Day 84 120 in the antihelminthic treated group and 97 in the control group. At baseline the median was 0 in both groups and the GMT was 12 for the antihelminthic treated group and 10 for the placebo group (Tables 2 and 3). BODY.RESULTS.ASSESSMENT OF TOTAL IG ISOTYPES AND SUBCLASSES: Ig isotypes and subclasses (IgG1-4, IgA, IgE and IgM) pre- and post-vaccination were assessed by a multiplex system. Total IgA was elevated in the antihelminthic treated group compared to the control group at Day 28 (p-value 0.006) (Fig 4). 10.1371/journal.pntd.0003768.g004Fig 4Total IgA at day 0, day 28 and day 84 in antihelminthic treated (AT) (red blots) and placebo group (blue blots). The total concentration of the subclasses IgG1 and IgG3 were slightly elevated in the antihelminthic treated group (for IgG1 p-value was 0.042 and for IgG3 p-value was 0.03 in the model-based analysis, but was not significant using the Wilcoxon-test (p-value 0.347 and 0.160)). To evaluate whether the elevation of total IgA indicates a higher amount of vaccine-specific IgA we performed a vaccine-specific ELISA. Here, no difference of vaccine-specific IgA was detected between the antihelminthic treated and control group (Fig 5). 10.1371/journal.pntd.0003768.g005Fig 5Vaccine specific IgA at day 0, day 28 and day 84 in antihelminthic treated (AT) (red) and placebo group (blue). BODY.RESULTS.ASSESSMENT OF ASC: B-cell ELISpot assay to measure vaccine-specific memory B-cells was performed with samples from 75 individuals. Antigen-specific memory B-cells were detectable in all vaccinated subjects at Day 84 (Fig 6). Median number of vaccine-specific ASC per 100,000 PBMC was 13 at Day 84 (range: 1 to 144) in all participants. 10.1371/journal.pntd.0003768.g006Fig 6Vaccine-specific IgG ASCs determined by B-cell ELISpot at day 0 and day 84. In 10 participants of the antihelminthic treated group and 12 participants of the placebo group a low number of ASCs (range: 0 to 47) was already detected at Day 0 (Fig 6). The number of detectable ASCs in the antihelminthic treated group at Day 84 ranged from 0 to 240 (median 19) and from 0 to 140 (median 3) in the placebo group (Fig 7). Nonetheless this difference was not statistically significant. 10.1371/journal.pntd.0003768.g007Fig 7Vaccine-specific IgG ASCs at day 0 and day 84, in antihelminthic treated (AT) and placebo group.Red and blue represent antihelminthic treated (AT) and control group. BODY.RESULTS.ASSESSMENT OF PARASITE BURDEN: The percentage of helminth burden in our setting was 21%. From these 21%, infection with A. lumbricoides and T. trichiura was 6% for each species. The burden did not differ between the visits (Tables 1 and 4). 10.1371/journal.pntd.0003768.t004 Table 4 Distribution of the worm burden in the two groups at day 0 and day 84. Day 0 Day 84 Antihelminthic treatment Placebo Antihelminthic treatment Placebo Single infection A . lumbricoides 2 (2.2%) 2 (2.2%) 5 (6.1%) 4 (4.9%) T . trichiura 2 (2.2%) 3 (3.3%) 3 (3.6%) 1 (1.2%) S . haematobium 0 0 2 (2.4%) 1 (1.2%) Taenia 0 0 0 1 (1.2%) Tapeworm 1 (1.1) 0 0 0 Multiple infection T . trichiura/ A . lumbricoides 1 (1.1%) 2 (2.2%) 0 3 (3.7%) S . haematobium/ T . trichiura 0 0 0 1 (1.2%) S . haematobium/ A . lumbricoides 0 0 1 (1.2%) 0 T . trichiura/ A . duodenale 1 (1.1%) 0 0 0 T . trichiura/A . lumbricoides/ A . deodenale 0 1 (1.1%) 1 (1.2%) 0 Neg 39 (42.4%) 32 (34.7%) 25 (30.5%) 24 (29.3%) NA 2 (2.2%) 4 (4.4%) 7 (8.5%) 3 (3.7%) At Day 0 and Day 84 9 and 10 volunteers were positive in the thick blood smear, respectively. BODY.DISCUSSION: Recent studies suggest that infection with helminths influences the immunological outcome of vaccination. A study recently conducted in rural Gabon showed that children infected with helminths had an impaired antibody response against an influenza vaccine compared to those free of infection[14]. During a phase Ib trial investigating immunogenicity of the malaria vaccine candidate GMZ2 in Gabonese children those infected with T. trichiura exhibited a lower antibody response against the vaccine antigens compared to those who were not infected with the parasite[23]. Since these and other studies in animal models and humans [21,38,39] show that helminths and other intestinal infections negatively influence vaccine immunogenicity we conducted a study to test the hypothesis that antihelminthic treatment prior to vaccination will increase immune responses to vaccine antigens. Because antibody response towards vaccine antigens is a surrogate marker for protection in areas where infectious diseases are highly endemic an effective immune response to vaccinated antigens is very important[40–42]. In the present study anti-viral antibody response was analyzed by HI test for each of the three influenza strains administered with a seasonal vaccine. The influenza vaccine was selected because a single-dose is sufficient to raise antibody responses towards a protective titer and the vaccine is not part of the Expanded Program on Immunization (EPI) in Gabon. Therefore, no or only low baseline titers would presumably be present in the study population. As expected there was an increase of HI titers against each vaccine strain after vaccination at Day 28 and Day 84 in all participants. This was observed mainly for the antibody concentration against the influenza A strains A(H1N1)pdm09 and A(H3N2). The low but frequently seen baseline HI titers against the influenza A (H1N1) and A(H3N2) in some participants suggest that these children were already in contact with circulating virus strains or that they have cross-reactive antibodies from recent circulating influenza strains or from other cross-reactive pathogens. In the present study we assessed that total IgA concentrations were higher in individuals of the antihelminthic treated group compared to the control group four weeks following vaccination. Because IgA is crucial for the control of influenza[43–45] and vaccine-specific IgA can be detected in mice following influenza vaccination[46] we also measured vaccine-specific IgA but saw no difference between the groups. Since total IgA was not different at baseline we do not know if the difference of total IgA at Day 28 is an effect of vaccination or a late effect of the antihelminthic treatment. This effect should be further investigated in more detail in particular because IgA has an important function during the defense of airborne and gastrointestinal infections. However in our setting we have not examined the role of this finding and we have not determined secretory IgA. Besides we could show that the number of vaccine-specific ASCs representing memory B-cells was elevated in the antihelminthic treated group compared to the control group but this difference did also not reach statistical significance. We assume that the already existing ASCs at baseline in samples of some participants are due to previous influenza infections. Since the antibody titers as well as the number of ASCs were not significantly elevated in the antihelminthic treated group the question arises if a better or more effective antihelminthic treatment would have had clearer effects on the vaccine immunogenicity. The fact that the overall helminth burden did not differ between the visits implies that single-dose antihelminthic treatment is not sufficient to cure or to prevent relevant helminth infection or that reinfection occurs rapidly. This becomes more evident since Adegnika et al. very recently showed that short antihelminthic treatment regimens are efficacious to cure A. lumbricoides but not T. trichiura infection. To eliminate T. trichiura infection at least a double treatment seems to be necessary[30]. Therefore in our setting the treatment regimen was not adequate to eliminate helminths sufficiently and to reconstitute the immune system. Cooper et al. investigated the effect of a double-dose (2x200mg) albendazole treatment prior to an oral cholera vaccination leading to a highly significant increase in anti-vibriocidal antibodies[21,47]. In contrast to the present study, the investigators only included individuals carrying A. lumbricoides[21,30]. In our setting we retrospectively analyzed the helminth burden of the volunteers at the end of the study and noticed that the number of infections amongst primary school children was not as high as originally suggested from recent studies, which reduces the power of our analyses. In a study performed 2004 the overall prevalence of helminth infection was 74%[24], whereas in our study population the worm burden was only 21%. This could be due to different reasons. First, in the study conducted by van den Biggelaar et al. the Kato Katz method to detect the egg load in fresh stool samples was used[24] whereas we used the MIF technique to detect the worm burden in preserved stool samples at the end of the study to ensure all investigators were blind to the infection status of the participants. The Kato Katz method has a higher sensitivity[48] compared to the MIF-technique[49] and this may explain partially the unexpected low prevalence of helminth infection although other studies using the same methodology gave higher prevalence rates. Besides the possibility that it is a chance finding the reduction of worm burden could be due to better public health facilities and better education of the population as well as to mass drug administration (MDA) even when it is administered on an irregular basis or by uncontrolled self-mediation. Gabon is a highly endemic country for STH according to the WHO, therefore MDA is recommended but in the region where the study was performed it is not regularly administered[29]. Private use is difficult to assess since individuals do not need prescription to buy antihelminthic treatment over-the-counter[50,51] and often the individuals do not report the use of antihelminthic treatment. Furthermore it could be that a member of a household was recently treated and therefore also infection of other family members decreased[52]. Taken all this into account our in our study population the helminth burden was lower as expected for reasons which were not elicited in this study and our sample size was not powered for such a low helminth prevalence. In conclusion, we showed that in our setting there was non-significant difference in virus-specific HI titers and ASCs against the vaccine antigens between the antihelminthic treated and the placebo group. Furthermore at Day 28 post vaccination total IgA was higher in the antihelminthic treated group compared to the control group. But there was no difference comparing vaccine-specific IgA titers. We can only speculate if a single dose antihelminthic treatment is sufficient to increase vaccine immunogenicity in a setting with higher helminth prevalence or if an appropriate more powerful treatment could contribute to a better immune response to vaccination. This has to be investigated in more detail and with different antihelminthic regiments. BODY.SUPPORTING INFORMATION: S1 ChecklistConsort checklist.(PDF)Click here for additional data file.

TITLE: Exploratory studies on the therapeutic effects of ABSTRACT: The effect of an Ayurvedic poly-herbo-mineral formulation Kumarabharana Rasa (KR) in the management of chronic tonsillitis (Tundikeri) in children has been assessed in this study. This clinical study was a double-arm study with a pre- and post-test design at the outpatient level in a tertiary Ayurveda hospital attached to a teaching institute located in district headquarters in Southern India. Patients (n = 40) with chronic tonsillitis satisfying diagnostic criteria and aged between 5 and 10 years were selected from the outpatient Department of Kaumarbhritya, SDM College of Ayurveda and Hospital, Hassan. Among them, 20 patients were treated with Kumarabharana rasa (tablet form) at a dose of 500 mg once daily for 30 days (Group A). The other 20 patients were treated with Godhuma Vati (placebo) at a dose of 500 mg once daily for 30 days (Group B). In both groups, Madhu was the Anupana advised. After completion of 30 days of treatment, the patients were assessed on the following day and another investigation took place 15 days later. Statistically significant effects (p < 0.05) in the reduction of all signs and symptoms of chronic tonsillitis after KR treatment were observed. These results indicate that Kumarabharana Rasa has an ameliorative effect in reducing the signs and symptoms of chronic tonsillitis. BODY.INTRODUCTION: 1 Chronic tonsillitis (CHT) is one of the most common otolaryngologic diseases.1 CHT is rare in infants and older people. In young children, tonsillitis is one of the recurrent upper respiratory tract infections. CHT is a highly prevalent disease in the pediatric age group, and it peaks between 3 and 10 years of age and then declines.2, 3 In general practice children frequently visit with recurrent throat problems4 and the incidence of this disease accounts for about 7% of all visits to the pediatrician.3 Children with CHT experience discomfort, and the disease also impacts on social, emotional, and financial aspects for family members.5 Tonsilitis is an infection of the tonsils.6, 7, 8 Despite its high prevalence, the etiology of CHT has remained indistinct. The surface and deep bacterial flora of chronic inflamed tonsils consist of an abundance of probable pathogenic aerobic and anaerobic bacteria, primarily of streptococcal origin.9, 10, 11, 12, 13 Tonsils are part of the immune system. Therefore, due to the decrease in immunity and the tonsils' incompetence in helping the immune system, they actually become a source of recurrent infections.3 The current treatment option for CHT is tonsillectomy, but it is not the ultimate solution. The generally accepted criteria for tonsillectomy are at least three to seven episodes of tonsillitis per year in spite of medical therapy, but there is no international consensus.14 In Ayurvedic thought, tonsillitis can be correlated to Tundikeri, which is one of the Urdhvajatrugata Roga (diseases of the head and neck); it is mentioned in Talugata Roga15 (diseases of the palate) as well as Kanthagata Roga (diseases of the throat).16 Ayurveda explains that it is caused by the vitiation and imbalance of Doshas (bodily humors), i.e., Vata, Pitta, and Kapha. Mainly derangement of Kapha and Rakta (blood) is preceded by impaired digestive capacity (Mandagni/Vishamagni) and obstruction of channels (Sroto Avarodha) namely Annavaha Srotas (gastrointestinal tract) and Pranavaha Srotas (respiratory tract) which is manifested as difficulty in swallowing, mouth breathing, choking spells at night, etc.15, 16 The present study was conducted to explore the efficacy of Kumarabharana Rasa (KR) in the management of chronic tonsillitis in children. KR has a combined action over vitiated Doshas due to its anti-inflammatory, antimicrobial, immunomodulatory, and rejuvinative effects. BODY.MATERIALS AND METHODS: 2 BODY.MATERIALS AND METHODS.DESIGN: 2.1 This study was an open-labeled double-arm setting with a pre- and post-test design. BODY.MATERIALS AND METHODS.PARTICIPANTS: 2.2 Children presenting with any of the symptoms of chronic tonsillitis (Tundikeri), i.e., Kathina Shotha (enlargement of tonsils), Ragatwa (hyperemia), Galoparodha (dysphagia), Mukha Daurgandhya (halitosis), Lasikagranthi Vriddhi (enlargement of lymph nodes), and Jwara (fever) were selected and registered from Kaumarabhritya (Ayurvedic Pediatrics) outpatient department of SDM College of Ayurveda and Hospital, Hassan between June 2012 and December 2013. Before initiating the study ethical clearance was obtained from institutional ethics committee of SDM College of Ayurveda and Hospital, Hassan (IEC No. SDMAH/IEC/57/11-12 dated 01-04-2012). Written informed consent was taken from the parents of the study participants before any study-related procedures were performed. Inclusion criteria were: children of both sexes between 5 and 10 years of age and who had repeated attacks of tonsillitis (chronic infections) in the past year. Exclusion criteria were: patients with acute tonsillitis, peritonsillar abscess, tonsillar cyst, tonsillolith, or any other systemic disorders; patients who had taken systemic steroids and/or antibiotics in the past 4 weeks. BODY.MATERIALS AND METHODS.STUDY DRUGS: 2.3 BODY.MATERIALS AND METHODS.STUDY DRUGS.KUMARABHARANA RASA: 2.3.1 This is a compound drug comprising Bhasmas (purified calx) of Swarna (gold), Rajata (silver), Pravala (coral) and Churna (powder) of Yastimadhu (Glycyrrhiza glabra Linn.), Amlaki (Emblica officinalis Gaertn.), Ashwagandha (Withania somnifera Dunal.), Sunthi (Zingiber officinale Rosc.), Pippali (Piper longum Linn.), Haritaki (Terminalia chebula Retz.), Vacha (Acorus calamus Linn.). All these drugs were processed with Swarasa (extract juice) of Guduchi (Tinospora cordifolia Miers ex Hook. F. & Thoms), Brahmi (Centella asiatica Linn.), and Tulsi (Ocimum tenuiflorum Linn.) separately then prepared in tablet form.17 BODY.MATERIALS AND METHODS.STUDY DRUGS.GODHUMA VATI (PLACEBO): 2.3.2 Wheat powder was processed and prepared in tablet form. Raw drugs were obtained from SDM Pharmacy, Udupi and authenticated in the Department of Dravyaguna, SDM College of Ayurveda and Hospital, Hassan. The medicine was prepared in the Teaching Pharmacy, SDM College of Ayurveda and Hospital, Hassan. Tablets of 500 mg were prepared and preserved in air-tight, properly labeled plastic bottles containing 30 tablets in each. BODY.MATERIALS AND METHODS.INTERVENTION: 2.4 A total of 53 patients were screened for chronic tonsillitis. Among them 40 patients were enrolled into the study fulfilling the inclusion and exclusion criteria. A convenient sampling technique was adopted, with 20 patients each in study group (KR) and the control group (Godhuma Vati). The patients in the study group were treated with KR (tablet form) at a dose of 500 mg once daily for 30 days. The patients in the control group were treated with Godhuma Vati (tablet form) at a dose of 500 mg once daily for 30 days. Parents were advised to crush the tablet to a powder and to give it to the child using honey as Anupana (vehicle for drug administration) before food, in the morning, for both groups. BODY.MATERIALS AND METHODS.OBSERVATION-BASED ASSESSMENT CRITERIA: 2.5 BODY.MATERIALS AND METHODS.OBSERVATION-BASED ASSESSMENT CRITERIA.SUBJECTIVE PARAMETERS: 2.5.1 The assessment of the signs and symptoms were done on the Day 0 and Day 31. Severity was assessed by grading 1–5 (absence to severe) for each symptom. Kathina Shotha (enlargement of tonsils) – no enlargement, enlarged within anterior pillars, enlarged within posterior pillars, enlarged beyond pillars, kissing tonsils with sleep apnea. Ragatwa (hyperemia) – no hyperemia, hyperemia of tonsil surface, pinkish appearance of pillars, reddish appearance of surroundings, reddish appearance of surroundings and pharynx. Galoparodha (dysphagia) – no pain while swallowing, pain during swallowing solid food substances, pain during swallowing semi-solid food substances, pain during swallowing liquid food substances, continuous pain/unable to swallow. Mukha Daurgandhya (halitosis) – no halitosis, foul breath experienced by the patient only, foul breath experienced by the patient and friends/parents, foul breath is experienced by a group of surrounding people, foul breath is experienced as soon as the patient opens the mouth. Lasikagranthi Vriddhi (enlargement of lymph nodes) – no palpable lymph nodes, palpable lymph nodes unilateral/warm, palpable lymph nodes bilateral/soft/fluctuant, palpable lymph nodes bilateral which are hard, palpable lymph nodes bilateral with tenderness. Jwara (fever) was measured according in degrees Fahrenheit (normal was 98.6° Fahrenheit). BODY.MATERIALS AND METHODS.OBSERVATION-BASED ASSESSMENT CRITERIA.OBJECTIVE PARAMETERS: 2.5.2 Assessments were based on routine laboratory blood investigations – hemoglobin % (Hb%), total leukocyte count (TLC), neutrophils, lymphocytes, eosinophils, and erythrocyte sedimentation rate (ESR) were performed on Day 0 and Day 31. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: 2.6 For the statistical analysis, the Statistical Package for Social Sciences (SPSS) version 16 (SSPS Inc., Chicago. IL, USA) was used. The independent samples t test and Mann-Whitney U test (for between-subjects designs) and paired samples t test and the Wilcoxon test (for within-subjects designs) were done. BODY.RESULTS: 3 In present study 40 patients were registered, of which only 37 participants completed the study. The sociodemographic profile of the participants shows that 57.5% (23) were male and the rest 42.5% (17) were females. The age groups of 5–6, 7–8, and 9–10 years consisted of 18 (45%), 27.5% (11), 27.5% (11) participants, respectively. The majority (90%, 36) of the participants belonged to the Hindu religion. Socioeconomic assessment revealed that 37.5% belonged to the lower-middle class followed by 30% in the upper-middle class strata. Prakriti (genetic phenotype) yielded the majority 87.5% (35) were having Pitta-Kapha Prakriti. A mixed diet was consumed by 80% (32) of the participants. Sleep patterns showed the 60% (24 participants) slept for around 10 hours/day but 62.5% (25) had disturbed sleep, while 37.5% (15) had normal sleep. The family history of the participants highlighted that 25% (10) had a positive family history of chronic tonsillitis. Duration of tonsillitis revealed that 27.5% (11) participants had a duration of tonsillitis for a period of 3 years or more, 17.5% (7) had a duration of tonsillitis for a period of 2 years or more and 55% (22) had a duration of onset of tonsillitis for a period of 1 year or less. Poor oral hygiene was observed among 67.5% (27) participants and only 10% (4) had good oral hygiene. Chief complaints reported were throat pain among 10% (4) patients, difficulty in deglutition in 17.5% (7) patients, halitosis in 12.5% (5) patients, fever in 10% (4) patients, breathing difficulty among 20% (8) patients (16.66%) with complaints of mouth breathing in 22.5% (9) patients. Only 5% (2) reported having Jugulo-digastric lymphadenopathy and 2.5% (1) having enlarged tonsils. Clinical diagnosis revealed 62.5% (25) patients with chronic parenchymatous tonsillitis, 32.5% (13) patients with chronic follicular tonsillitis and only 5% (2) were diagnosed as chronic fibroid tonsillitis. Within the group (Wilcoxon signed ranks test) statistical significance (p < 0.05) on signs and symptoms of chronic tonsillitis was found for KR intervention, but nothing significant was observed with Godhuma Vati intervention (Table 1). However, between groups (Mann Whitney U test), the effects of KR on signs and symptoms of chronic tonsillitis was found to be significant (p < 0.05; Table 2). Routine laboratory blood investigations assessed within the KR (study group) showed statistical significance (p < 0.05) on Hb%, TLC, lymphocytes and ESR, whereas Godhuma Vati (placebo) were nonsignificant (Table 3). The comparisons on the effect of KR (study group) and Godhuma Vati (placebo group) on laboratory investigations and fever were nonsignificant (p > 0.05) as detailed in Table 4. BODY.DISCUSSION: 4 CHT is an inflammation of the tonsils caused by a microbial infection. The symptoms are usually mild and often related to the common cold. In streptococcal infection the tonsils often swell and become coated and the throat is sore. The patient has a temperature, foul-smelling breath and may feel quite ill. Therefore drugs with anti-inflammatory and immunomodulatory effects may be useful for treatment of CHT. Ayurveda, as well as recent experimental results indicate the ingredients of a poly-herbo-mineral formulation KR as having anti-inflammatory, antimicrobial, and immunomodulatory effects. It is substantiated by the clinical observations among tonsillitis patients. The key ingredients being the metallic Bhasmas (purified calx), like Swarna Bhasma, is prepared by incinerating gold processed with herbal preparations.18, 19 Swarna Bhasma promotes immunity through phagocytosis.20 Gold poly-herbal formulations are found to increases vitality and immunity.21 The clinical applications of Swarna Bhasma and gold salts in Ayurveda are known for rejuvenation and immunomodulation for some chronic diseases.22 Gold nanoparticles have significant applications in targeted drug delivery.23, 24 Swarna Bhasma contains gold particles in the size range of nanoparticles.25 Recent studies shows that Swarna Bhasma contained not only gold, but also several microelements (Fe, Al, Cu, Zn, Co, Mg, Ca, As, Pb, etc.). Animal model studies show Swarna Bhasma-treated animals significantly increased superoxide dismutase and catalase activity, which are responsible for reducing free radical concentrations in the body.19 The antioxidant effect of Swarna Bhasma is considered to be one of the mechanisms for the immunomodulatory mode of action of the formulation. Rajata Bhasma (silver calx) is a nanoparticulate complex of silver along with Sn, Cd, Mg, K, Na, S, Ca, P, Si, Al, Cl, Ar, In, Fe, Cu, Ba, Hg, or Cr.26 Recent scientific trends indicate emergence of metallic silver in the form of silver nanoparticles as a potential antimicrobial agent, specially important, as several pathogenic bacteria have developed resistance against various antibiotics.27 Herbal components like Amalaki (Phylanthus embellica)28, 29 and Guduchi (Tinospora cordifolia)30 are having nourishing and rejuvinative property. Experimental studies reveal their immunostimulant activity which combats sepsis in animals.31, 32, 33, 34, 35, 36 Vacha, Sunthi, Pippali, Ashwagandha, Yastimadhu, Amalaki, and Haritaki all have immunomodulatory properties.37, 38, 39, 40 Pippali is an important drug for the treatment of cough, common cold, and is useful as a digestive. The active principle piperine is known for its bio-enhancing and anti-inflammatory effects.41 Yasthimadhu is a prominent Rasayana known for its immunomodulatory, anti-inflammatory, anti-allergic and antiviral activities. It also enhances permeability in drug delivery systems.42 Yastimadhu (Glycerrhiza glabra) also has cytoprotective and demulcent effects and is a popular home remedy for minor throat infections. Biologically active substances in liquorice root include glycyrrhizic acid (GL) and its aglycone (GA), phenolic compounds, oligosaccharides and polysaccharides, lipids, sterine, etc. Many researchers have suggested that the effects on the production of interferon (IFN) and Th2 cytokines might be one of the mechanisms involved in the anti-infective process. Recently, glycyrrhizin has been found to be active in inhibiting replication of the severe acute respiratory syndrome (SARS)-associated virus (FFM-1 and FFM-2) and also H5N1 influenza A virus-infected cells. GL is also reported to have modulatory effects on the complement system. Reports indicate that GL blocks C5 or a more distal stage of the complement cascade, suggesting that it might have a role in preventing tissue injury not only in chronic hepatitis, but also in autoimmune and inflammatory diseases. Chemical modification of GL and GA has been tried, and a significant improvement in anti-inflammatory, antiallergic, and antiulcer activities was observed. These observations indicate immune-modulating and biological response modifier activities associated with GL. Tinospora cordifolia (TC) is known for its immunomodulatory and cytoprotective activities Quaternary alkaloids and biotherapeutic diterpene glucosides of TC (syringin, cordiol, cordioside, and coriofolioside) showed an immunopotentiating activity. Research work has been conducted on berberine, jatrorrhizine, tinosporaside, and columbin, which shows a possible mechanism of immunomodulatory activity as an activation of macrophages, leading to increases in granulocyte–macrophage colony-stimulating factor (GM-CSF), leukocytosis, and improved neutrophil function. TC also inhibits C3 convertase of the classical complement pathway. Research on polysaccharide (α-d-glucan) derived from TC shows the activation of nuclear killer (NK) cells, complement system, and Th1 pathway cytokines, coupled with low nitric oxide synthesis.43 Aswagandha has shown promising immunomodulatory effect in inflammatory experimental models.44 Sunthi (Zingiber officinalis) has anti-inflammatory property which inhibits LPS-induced NF-κB activation by preventing degradation of the IκB-α, as well as the phosphorylation of ERK1/2, SAPK/JNK, and p38 MAPKs which were associated with a decrease in the expression of inducible nitric oxide synthase (iNOS) and cyclooxyenase-2 (COX-2).45 Vacha (Acorus calamus) is reported for its inhibitory effect on mast cell-dependent anaphylactic reactions in allergic reactions of the respiratory tract.46 Amla (Phyllanthus emblica) has been reported to suppress the expression of LPS-induced pro-inflammatory genes (COX-2, iNOS, TNF-α, IL-16, and IL-6) in RAW 264.7 murine macrophage cells in a dose-dependent manner.47 Honey is known to enhance the action of these medicines, and has a potential immunomodulatory effect.48, 49, 50, 51 BODY.CONCLUSION: 5 Thus the poly-herbo-mineral formulation KR has a combined mechanism of action on tonsillitis by reducing the number of attacks. From the Ayurvedic perspective it controls the imbalance of Doshas and thus effectively reduces the signs and symptoms of chronic tonsillitis. However, following the reverse pharmacological perspective we find scientific evidence of its components against inflammation and microbial invasion via multiple mechanisms mostly as immunomodulators, as discussed above. Future studies on KR formulation along the lines of reverse pharmacology may produce more conclusive information about its combined mechanism of action at the molecular level. BODY.CONFLICTS OF INTEREST: All authors have no conflicts of interest to declare.

TITLE: Effect of an Educational and Psychological Intervention on Knowledge and Quality of Life among Patients with Psoriasis ABSTRACT.BACKGROUND:: Psoriasis is one of the most common skin disorders with a prevalence rate of 0.1%–3%. Chronic nature, frequent relapses, absence of permanent cure, and the cosmetic disfigurement of psoriasis have a negative impact on quality of life (QoL) by causing psychological stress. Patients with psoriasis often have unambiguous ideas about the causes, controllability, consequences, and expected time-course of their disease. ABSTRACT.AIM:: The aim of this study is to assess the effectiveness of a video-assisted teaching program regarding psoriasis on the level of knowledge and relaxation therapy on QoL among patients with psoriasis. ABSTRACT.MATERIALS AND METHODS:: Experimental design was adapted. One hundred and four participants diagnosed with psoriasis were randomly allocated either to an experimental or to a control group. Fifty-two participants were included in each group by simple random sampling. A video-assisted teaching program on psoriasis and relaxation exercises was taught to the participants over a period of 3 months. The tools used were: Psoriasis Knowledge Assessment Questionnaire, modified psoriasis disability index, and modified psoriasis life stress inventory. ABSTRACT.RESULTS:: In the experimental group, the knowledge score was increased significantly from 9 ± 2.2 at baseline to 23.6 ± 1.5 after the intervention. The disability score was decreased from 15.6 to 9.9 and the stress score related to the illness was decreased from 22.8 to 16.9 after the intervention. ABSTRACT.CONCLUSION:: Educational intervention about disease process and relaxation exercises was effective in improving the knowledge and QoL of patients with psoriasis. BODY.INTRODUCTION: Psoriasis is a chronic inflammatory hyperproliferative disease of the skin and currently affects approximately 0.6% to 4.8% of the world's population.[1] In India, the overall incidence of psoriasis has been reported to be 1.02%.[2] In addition to the skin and joint manifestations, psoriasis impairs many aspects of individual well-being, including emotional, physical, sexual, and financial status.[3] Further, many of the difficulties experienced by patients with psoriasis make demands that outstrip the coping measures of patient and their family or social network. They need sufficient education and support from the health-care providers to manage their condition effectively.[4] This study aimed to assess the effectiveness of a video-assisted teaching program regarding psoriasis on the level of knowledge and relaxation therapy on quality of life (QoL) among patients with psoriasis. BODY.MATERIALS AND METHODS: Before commencing the study, approval was obtained from Institute Ethics Committee for human studies of the institute, where the study was conducted. Participants signed the written informed consent after being explained about the study. Privacy was provided to the participants and confidentiality was maintained throughout the study. An experimental research design was used to select the sample of 104 participants diagnosed with psoriasis who attended the inpatient and outpatient units of the department of dermatology at a tertiary care hospital. Sample size was estimated using the statistical formula for comparing two related means. The expected change in the mean score of psoriasis life stress inventory (PLSI) as an impact of intervention was 4 with a standard deviation (SD) of 10, and the sample size was estimated at 5% level of significance and 80% power. The samples were selected using simple random sampling. The list of patients registered in the psoriasis clinic was considered. A total of more than 200 patients were undergoing treatment in the psoriasis clinic. The estimated sample size for the study was 51 in each group. The samples were selected using random numbers generated through computer. Patients of both sexes, 18–65 years of age, and with the body surface area involvement of >10% were included in the study. Irrespective of patient's disease status and treatment status, patients were recruited. Participants with acute illness and those who had the diagnosis of any psychiatric disorder were excluded from the study. No participant was found to have psychiatric illness as per clinical evaluation. BODY.MATERIALS AND METHODS.PSORIASIS KNOWLEDGE ASSESSMENT QUESTIONNAIRE: A closed-ended questionnaire was developed to assess the knowledge regarding psoriasis. It consisted of 25 statements pertaining to basic facts on psoriasis (nine items), triggering factors (five items), treatment aspects (four items), and knowledge related to disease process (seven items). The study participants were requested to give "true, false, or do not know" responses. The score ranged from 0 to 25. Reliability coefficient (Cronbach's alpha) was ranged from 0.80 to 0.86. Content validity and face validity were established by the experts from the field of dermatology, psychiatry, and psychology (documented in Appendix I). BODY.MATERIALS AND METHODS.MODIFIED PSORIASIS DISABILITY INDEX: The original psoriasis disability index (PDI)[5] is a 15-item validated self-administered questionnaire designed to quantify the functional disability in the aspects of daily activities, employment, personal relationships, leisure, and treatment effects in participants with psoriasis. The score ranged from 0 to 45. The higher the score, the more the QoL is impaired. In the modified PDI, two questions were added since it would be appropriate for the patients with psoriasis in our specific cultural context: (1) "How is your work in kitchen affected by this condition?" and (2) "How much did the patient experience financial burden due to the treatment of psoriasis?" (Under the subdivisions of work or school and treatment-related questions, respectively). The resulting score thus ranged from 0 to 51 instead of 0–45. The necessary permission was obtained from the authors to use the questionnaire. The modified version of PDI was adapted for the present study from the published literature. BODY.MATERIALS AND METHODS.MODIFIED PSORIASIS LIFE STRESS INVENTORY: The original 15-item version of PLSI[6] was developed to assess the stress associated with potential psoriasis-related psychosocial problems. The score ranged from 0 to 45. The higher the score the more the QoL is impaired. In the present study, the stress experienced by the participants was assessed by the modified PLSI which was an 18-item questionnaire. It was found one question – "sunbathing in the company of others"– inappropriate, when applied to our patients and was deleted. Further, four questions were added – feeling less sexually attractive, concerned about children developing psoriasis, fear of relapse, and difficulty in coping if no family support. Thus, the resulting score ranged from 0 to 54 instead of 0–45. Modified version of PLSI was adapted from the literature that was already available. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The data were analyzed using both descriptive and inferential statistics using SPSS version 19.0 (IBM Corp. Released 2010. Armonk, NY). The data for the categorical variables on the sociodemographic factors were expressed as frequency and percentage. To calculate the responses in the individual items in knowledge, PLSI, and PDI questionnaire, frequency and percentage were used. The overall scores in knowledge, PLSI, and PDI were expressed as mean with SD. The comparisons of the categorical data between the groups were carried out using Chi-square test. The changes in the level of knowledge, PLSI, and PDI scores over time in each group were analyzed using two-way repeated measures of analysis of variance with repeated contrast test. All statistical analyses were carried out at 5% level of significance, and P < 0.05 was considered as statistically significant. BODY.MATERIALS AND METHODS.DATA COLLECTION PROCEDURE: A pretest was conducted by the principal investigator for all the participants in the experimental group as well as in the control group to assess their knowledge on psoriasis and their QoL using modified PDI and modified PLSI. All the participants in the control group received routine dermatological treatment for 12 weeks. All the participants in the experimental group received routine dermatological treatment along with video-assisted teaching program on psoriasis and progressive muscle relaxation exercises along with deep breathing exercises and were followed every week for over 12 weeks. Three posttests were conducted for both the groups with the same questionnaires which were used for pretest at different time points (at the end of 4th, 8th, and 12th week). BODY.MATERIALS AND METHODS.VIDEO-ASSISTED TEACHING PROGRAM ON PSORIASIS: A video-teaching program was prepared with the details regarding the nature of psoriasis, clinical types, triggering factors, and the diagnostic methods. It also included the description on medications and ointments that are used for the treatment of psoriasis. The side effects of the drugs, precautions to be followed while using ointments, and the signs and symptoms to be reported while taking the drugs were discussed with the participants. The participants were explained about phototherapy and photochemotherapy, and orientation was given to the phototherapy room. Skin care measures and the coping skills were also discussed. Sessions were conducted for four consecutive days and then continued for 12 weeks for reinforcement. An educational pamphlet was provided at the end of the session to reinforce their knowledge. BODY.MATERIALS AND METHODS.JACOBSON'S PROGRESSIVE MUSCLE RELAXATION TECHNIQUE: The participants in the experimental group were divided into small groups with five in each group and were trained to learn breathing techniques as well as the relaxation techniques. After gaining mastery of relaxation, they were given a regular practice for the period of 12 weeks and the practice was observed by the researcher. BODY.RESULTS: The results of Chi-square analysis showed that there was no significant difference between the groups with respect to demographic variables and the groups were comparable. Comparison of the participants' knowledge and QoL scores before intervention between the groups is given in Table 1. The change in the mean knowledge score at different time points as an impact of intervention in the experimental group is depicted in Figure 1. The corresponding change of knowledge score in the control group is also depicted in the same figure by line diagram. In the experimental group, the knowledge level was found to be 9.0 before intervention. It was increased to 18.5, 23.2, and 23.6, respectively, at the 1st, 2nd, and 3rd month after the intervention. Similarly, in the control group, the knowledge level was found to be 9.7 before intervention. It was increased to 10.9, 11.3, and 11.4, respectively, at the 1st, 2nd, and 3rd month. Table 1 Comparison of the participants’ knowledge and quality of life before intervention ( n =104) Figure 1Mean knowledge scores at different time points between the groups (n = 104) BODY.RESULTS.EFFECTIVENESS OF INTERVENTION ON DISABILITY RELATED TO VARIOUS DOMAINS OF PSORIASIS DISABILITY INDEX IN THE EXPERIMENTAL GROUP: PDI measures illness-related disability in five aspects, i.e. daily activities, work, personal relations, leisure, and treatment. The mean PDI score in the experimental group was significantly (P < 0.001) decreased from 15.6 (±6.9) at baseline to 9.9 (±5.1) after 3 months of intervention [Figure 2]. The mean disability score related to daily activities was significantly (P < 0.001) decreased from 5.5 (±3.1) at baseline to 3.6 (±1.7) after 3 months of intervention. The mean disability score related to work was significantly (P < 0.001) decreased from 3.9 (±2.8) at baseline to 2.4 (±1.4); the mean disability score related to personal relations was significantly (P < 0.001) decreased from 1.8 (±1.7) at baseline to 1.3 (±1.3); the mean disability score related to leisure time activities was significantly (P < 0.001) decreased from 2.4 (±1.9) at baseline to 1.4 (±1.4); the mean disability score related to treatment was significantly (P < 0.001) decreased from 2.1 (±1.4) at baseline to 1.4 (±0.8) after the intervention. Figure 2Mean disability (psoriasis disability index) scores at different time points between the groups (n = 104) The mean PLSI scores in the experimental group were significantly (P < 0.001) decreased from 22.8 (±9.8) at baseline to 16.9 (±6.7) after 3 months of intervention [Figure 3]. Thus, the results of the study suggested that the scores were significant at P < 0.001 level when compared with the control group. Figure 3Mean stress scores (psoriasis life stress inventory) at different time points between the groups (n = 104) BODY.DISCUSSION: Many researchers have found that patient's attitudes, knowledge, and behavior toward their disease have a profound effect on its course and severity. For example, a patient with psoriasis who knows about the Koebner phenomenon will be able to avoid injury to the skin. Furthermore, patients with a good understanding of their disease are more self-motivated by increasing their sense of control. Many research findings indicate that it is important to educate patients with psoriasis regarding their condition.[47] The results of the present study revealed that the knowledge level was significantly increased in the experimental group due to the impact of the educational program. Similarly, research studies[891011] had reported that education and demonstration of topical therapies by specialist dermatology nurses were found to be effective in reducing the severity of clinical symptoms in patients with psoriasis. Other studies which support our study findings are given below: Wahl et al. investigated patients' knowledge regarding psoriasis before and after patient education in the context of climate therapy. The authors found significant improvement in the knowledge score immediately after the intervention and 3 months after the intervention when compared to the baseline scores.[12] Balato et al. evaluated the use of mobile phone-based interventions in improving treatment adherence among twenty patients with psoriasis. The intervention group reported a significantly better improvement of disease severity as well as adherence to therapy in a statistically significant way whereas the control group remained stable.[11] Bostoen et al. assessed the effect of an educational program on disease severity and QoL in patients with psoriasis or atopic dermatitis. It was found that disease severity and QoL improved significantly for patients with psoriasis after the intervention.[13] Pagliarello et al. evaluated the effect of a single 2-h empowerment-based educational program in a spa setting at Italy. The experimental group reported higher levels of empowerment with improved knowledge about the disease as compared to the control group.[14] Lora et al. investigated the efficacy of an educational intervention for patients with psoriasis in improving disease knowledge and attitude toward physicians and systemic treatments. The intervention consisted of information on psoriasis and its treatment. After 6 months, a better knowledge about the disease and a better attitude to treatment were retained. It is clearly evident that a single educational intervention may be helpful in improving psoriasis knowledge and give psychological relief to patients.[15] Werfel et al. conducted an educational program for the management of psoriasis. Five 2-h classes focused on topics relevant for the patients and the management of the disease by dermatologists, psychotherapists/psychologists, and dieticians. This program was accepted very well and seen as helpful by the concerned.[16] It has long been acknowledged that the clients affected with psoriasis may benefit greatly from psychotherapeutic interventions. It is possible that stress management through relaxation may result in improvement in these patients. Stress may worsen psoriasis severity and may even lengthen the time to disease clearance. According to Zachariae et al.,[17] 66% of their patients reported that their psoriasis was exacerbated by stress and 35% of them reported that the onset of their psoriasis occurred during a time of worry and stress. Rakhesh et al.[18] reported that psoriasis sufferers were most likely to feel self-conscious, disturbed/inconvenienced by the shedding of the skin, lived in a constant fear of relapse, and avoided social interactions. The findings of the present study showed that the stress score related to the illness was found to be 22.8 before the intervention in the experimental group. It was decreased to 20.4, 18.4, and 16.9, respectively, at the 1st, 2nd, and 3rd month after the intervention due to the impact of educational and relaxation therapy. Similarly, Kabat-Zinn et al.[19] found that meditation-based stress reduction intervention increased the rate of resolution of psoriatic lesions in patients undergoing phototherapy or photochemotherapy treatment. Price et al.[20] reported that patients with psoriasis who were taught specific relaxation techniques had a significant reduction in their anxiety level and showed physical improvement. The present study findings were also supported by the findings of Gaston et al.[9] and Malhotra and Mehta.[21] Fortune et al. investigated the effects of a cognitive-behavioral psoriasis symptom management program (PSMP) on patient-held perceptions about their condition. Results suggested that at 6-month follow-up, patients who had undergone PSMP showed significant reductions in illness severity.[22] Ersser et al.[23] searched the literature to assess the effectiveness of psychological and educational interventions in changing outcomes for children with atopic eczema. Four studies focused on intervention directed toward the parents and examined the effect of relaxation techniques (hypnotherapy and biofeedback) on severity. Three educational studies identified significant improvements in disease severity between intervention groups. One study found that video-based education was found to be more effective in improving severity than direct education and the control (P < 0.001). The single psychological study found relaxation techniques resulted in reducing the clinical severity as compared to the control at 20 weeks. Price et al.[20] investigated whether psychotherapy helps patients with psoriasis. Participants who were taught specific relaxation techniques for use whenever they felt under stress had significantly reduced anxiety level at the end of the study with the modest trend toward physical improvement. No such changes were seen in a matched control group. BODY.CONCLUSION: The central and peripheral nervous systems, as well as the endocrine system, play an important role in the pathogenesis of psoriasis. Endogenous factors, such as stress, and exogenous factors, such as trauma, cause an increased release of neuropeptides such as substance P. Thus, as a result of stress playing such a prominent role in psoriasis, it seems evident that stress reduction programs should be considered when trying to treat the condition. The results of the present study also suggest that an educational intervention may be helpful in improving the knowledge and give psychological relief to patients with psoriasis. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Ultrasound Guided Needle Aspiration versus Surgical Drainage in the management of breast abscesses: a Ugandan experience ABSTRACT.BACKGROUND: Despite breast abscess becoming less common in developed countries, it has remained one of the leading causes of morbidity in women in developing countries. A randomized controlled trial was conducted at Mulago hospital complex in Kampala Uganda to establish whether ultrasound guided needle aspiration is a feasible alternative treatment option for breast abscesses. ABSTRACT.RESULTS: A total of 65 females with breast abscess were analyzed, of these 33 patients were randomized into the ultrasound guided needle aspiration and 32 patients in the Incision and drainage arm. The mean age was 23.12, most of them were lactating (66.2%), primipararous (44.6%) with peripheral abscesses (73.8%) located in the upper lateral quadrant (56%).The mean breast size was 3.49 cm. The two groups were comparably in demographic characteristic and breast abscess size. Survival analysis showed no difference in breast abscess healing rate between the two groups (Log rank 0.24 df 1 and P = 0.63). Incision and drainage was found to be more costly than ultrasound guided aspiration (cost effective ratio of 2.85). ABSTRACT.CONCLUSION: Ultrasound guided needle aspiration is therefore a feasible and cost effective treatment option for both lactating and non lactating breast abscesses with a diameter up to 5 cm by ultrasound in an immune competent patient BODY.BACKGROUND: Breast abscess is a common cause of morbidity in women. While they are less common in developed countries as a result of improved maternal hygiene, nutrition, standard of living and early administration of antibiotics, breast abscess remain a problem among women in developing countries [1]. The treatment of breast abscesses poses a difficult clinical problem [2]. Traditionally, management of breast abscess involves incision and drainage; however this is associated with need for general anesthesia, prolonged healing time, regular dressing, difficulty in breast feeding, and possible unsatisfactory cosmetic outcome [3]. Even with the aggressive approach of incision and drainage combined with use of antibiotics, breast abscess recurrence rate is reported to be between 10 and 38% [2]. Breast abscesses can be treated by repeated needle aspiration with or without ultrasound guidance [4-6]. Ultrasound has been shown to be useful in diagnosis of breast abscesses, guiding needle placement during aspiration and also enables visualization of multiple abscess loculation and thus useful in needle aspiration of breast abscesses [7]. This procedure has been used successful and is associated with less recurrence, excellent cosmetic result and has less costs [8]. Incision and drainage is still the most common mode of treatment for breast abscesses in Uganda. There is no data to compare the outcome of breast abscess treatment when using ultrasound guided needle aspiration versus surgical incision and drainage. The aim of this study was to establish whether ultrasound guided needle aspiration is a feasible alternative treatment option for breast abscesses in Mulago hospital. BODY.METHODS.STUDY DESIGN AND SETTING: This was a randomized controlled clinical trial with no blinding done between October 2006 and March 2007. The study was a hospital based which was conducted in Mulago hospital complex which is in Kampala city with a population of about 1.2 M people. Mulago is a National referral and teaching hospital in Uganda, it has bed capacity 1500.The study was conducted in the Accident and Emergency (A & E) department and breast outpatient clinic. BODY.METHODS.STUDY SUBJECTS: The study included all female patients aged 14 and above who presented to A&E department and Breast Clinic with breast abscess with a diameter of up to a maximum of 5 cm by ultrasound. Patients with recurrent or chronic breast abscess and those with necrotic skin overlying the abscess or abscess already draining were excluded from the study. Patients with clinical features of immune suppression (WHO clinical stage III and IV) and those known to be allergic to penicillin antibiotics were also excluded. Recruitment of patients was carried out in the Accident and Emergency department, and Breast Outpatient Clinic. Patients who met the inclusion criteria were enrolled into the study. Clinical diagnosis was made basing on the presence of breast pain, swelling, ± fever and presence of a fluctuant tender breast swelling. The patients diagnosed clinically were subjected to ultrasound scan (high frequency linear transducer of 7.5 MHZ) in the radiology department. The diagnosis was confirmed sonographically by the presence of a thick walled echo complex mass, predominantly cystic with internal echoes and septations. The size of the abscess was estimated. In this study, healing was defined as achieving breast abscess resolution. Breast abscesses resolution was defined as clinically no breast tenderness, swelling or wound at the previous site of the abscess and sonographically complete absence of fluid collection, normal breast glandular and fibro fat tissue with no edema BODY.METHODS.RANDOMIZATION: Patients were randomized to either incision and drainage or needle aspiration arm using computer-generated numbers. A computer program (random generator number, Microsoft excel version 5:0) was used to generate random number list. Patients were assigned to either needle aspiration (A) or incision and drainage (B).The principal investigator randomized patients to either A or B as they presented at the Accident and Emergency department. There was no blinding. BODY.TREATMENT PROCEDURE AND FOLLOW UP.INCISION AND DRAINAGE: Patients in the incision and drainage arm were admitted in the Emergency ward and prepared for surgery under general anesthesia in casualty theatre by the principal investigator. In the operation theatre with the patient positioned supine, the breast was swabbed using Chlorhexidine- Cetrimide (Cetrimide 15% w/v, Chlorhexine 1.5%w/v Isopropylalcohol4%w/v) 35 mls in 1 L of water. A skin depth incision was made at the area of maximum fluctuation along skin lines and a sinus forceps used to reach the abscess cavity. Initial pus was swabbed with a sterile pus swab which was transported for Culture and sensitivity. The pus was then evacuated and loculi broken down digitally, the wound was packed with sterile gauze. After recovery, the patient was taken back to emergency ward. Post operatively the patient was put on analgesics and antibiotics, Diclofenac 75 mg i/m stat, then 50 mg orally for 3 days and Cloxacillin 500 mg 8hry for 10 days respectively. The patient was discharged home the next morning to undergo daily wound dressing at a nearby clinic until the wound heals. Patients whose culture and sensitivity results showed resistance to Cloxacillin were excluded from the study and the antibiotic treatment changed accordingly. BODY.TREATMENT PROCEDURE AND FOLLOW UP.ULTRASOUND GUIDED NEEDLE ASPIRATION: Patients under the needle aspiration arm were managed in the department of Radiology Ultrasound room as outpatient cases. Under aseptic condition, a small area of skin adjacent to the abscess was anaesthetized by 1% Lignocaine through a 23 G needle. Aspiration was done under ultrasound guidance using a 16 G needle and a 20 mls syringe. Initial aspirated pus was sent for culture and sensitivity. Aspiration was done until there was no significant residual pus. After the procedure the patient was discharged on antibiotics and analgesics, Cloxacillin 500 mg orally 8hry for 10 days and Diclofenac 75 mg i/m stat then 50 mg orally 8hry for 3 days respectively. Similarly patients whose culture and sensitivity results showed resistance to Cloxacillin were excluded from the study and the antibiotic treatment changed accordingly. In order to minimize non- compliance to treatment in both arms, drugs were provided by the principal investigator to the patients who could not afford buying the drugs. Patients were required to come back with the packs of drugs during follow up visits to countercheck whether the patients had taken the drugs. In both arms, lactating patients were advised to resume breast-feeding on both breasts as soon as possible as they could tolerate the pain as the baby breast feed. The patient's follow up was done at the OPD by the principal investigator on day 7, day 14 and 30 days. At every follow up, clinical assessment of symptoms and signs was done to assess resolution of the abscess. Ultrasound scan was done to assess radiological resolution of the abscess which was defined as complete absence of fluid collection, normal breast glandular and fibro fatty tissues without edema. In situation where the abscess persisted in case of ultrasound guided needle aspiration, re-aspiration was to be done on day 7, if it still persisted on day 14 it was considered treatment failure and hence converted to the traditional incision and drainage. Breast abscess recurrence and acceptance were assessed at the last visit (day 30). Patients who had not achieved complete resolution of the breast abscess at the end of the study period were referred to the Breast outpatient clinic for further follow up. BODY.TREATMENT PROCEDURE AND FOLLOW UP.COST ESTIMATION: All costs were done in Ugandan shillings. Costs incurred by the patients and they included; cost for antibiotics, analgesics, syringes (20 cc) and cannulas (FG 16) used during U.S.S guided aspiration. These were estimated basing on open market price obtained in the local pharmacies. Costs for lodging, professional fee, surgery, anesthesia, amenities, sundries, doctor in care fee and health care fee were estimated basing on the Mulago hospital private patients' charges for the year 2007. Cost for ultrasound guided aspiration was valued basing on the charges as per radiology department for interventional ultrasound guided procedures. Costs for daily dressing for patients in the incision and drainage group was obtained from patient basing on how much she was charged every time she would go for wound dressing at the nearby clinic. BODY.TREATMENT PROCEDURE AND FOLLOW UP.DATA COLLECTION AND STATISTICAL ANALYSIS: Data was collected using a structured and coded interviewer administered questionnaire. Administered in the questionnaire were; Age, Parity, Social economic status, Smoking, Time of presentation from onset of symptoms and Size of breast abscess. Outcome variables included; Time to breast abscess resolution, Breast abscess recurrence, Acceptance of ultrasound guided needle aspiration procedure and Cost of the procedures. Statistical analysis was done using SPSS computer software version 11.5. Categorical data was summarized into proportions, percentages and rates. Continuous data was summarized into mean, median, mode, range and standard deviation. Tables were used to present data. Chi-square was used to compare the differences between the two groups where the outcome was categorical and if continuous, t-test was used. Statistical significance was defined as a P value of less than 0.05. Survival Analysis using Kaplan-Meier and Cox Regression was used to compare the healing rates between the two groups. For the cost data, costs in each intervention arm were summed up to give the total expenditure per intervention. The cost effectiveness ratio was determined by dividing the total cost of each intervention group by the number of patients successfully treated. BODY.TREATMENT PROCEDURE AND FOLLOW UP.ETHICAL ISSUES: Approval to carry out research was obtained from; Faculty of Medicine Research Committee, National Science and Research Council, Mulago Hospital Complex and the department of surgery, Mulago hospital before the commencement of the study. BODY.RESULTS: A total of 71 patients with breast abscesses were seen during the study period, of which 65 patients met the inclusion criteria and consented for the study. Six patients were excluded due to their abscesses being already draining pus and others having clinical features of immune suppression. Of the 65 patients, 33 patients were randomized into Ultrasound guided needle aspiration group and 32 patients into incision and drainage group. During the follow up period, Ultrasound guided aspiration had a success rate of 93.1% (27/33) single aspiration, with 6.9% (2/33) re aspirated on the 1st visit due to persistence of the abscess. There was no abscess converted to I & D in the U.S guided needle aspiration group (Figures 1 &2). Figure 1A sonogram of a 22 yrs old female showing a right breast abscess. Note the oval shape of the abscess measuring 2.64 cm by 1.54 cm before aspiration under U.S.S guidance. Figure 2U.S.S guided needle aspiration of the Right breast abscess of the above patient. Note the needle in the breast abscess cavity during the aspiration process (see the arrows).Incision and drainage group had a recurrence rate of 3.1% (1/32) during the follow up period. 5 (7.7%) women of the 65 were lost to follow up, 4 patients were from the Ultrasound guided aspiration group and 1 patient from Incision and drainage group. Of the 4 patients in the aspiration group, 2 missed the 2nd visit and the other 2 patients missed the 3rd visit. The patient in the Incision and drainage group lost to follow up in the 3rd visit. Figure 3Right breast after U.S.S guided needle Aspiration of the Abscess of the above patient. Note the disappearance of the abscess cavity leaving behind inflamed tissues as indicated by arrows. During the study period, no breast abscess recurrence was observed in the U.S aspiration group, and all the patient (100%) treated by Ultrasound aspiration highly accepted the procedure (Figure 3). In both groups majority of patients were healed by the 3rd visit that is 65.5% (19) for Ultrasound guided aspiration group and 58.1% (18) for the Incision and drainage group. There was no difference in healing rate between the two study arms at all the three visits (Table 1). Table 1 Healing rates per group Study group A B Odds ratio 95%CI P-value Visit 1( Day 7 ) 3% (1) 9.4% (3) 0.30 0.03-3.07 0.29 Visit 2 ( Day 14 ) 29% (9) 21.9% (7) 1.46 0.47-4.58 0.51 Visit 3( Day 30 ) 65.5% (19) 58.1% (18) 1.37 0.48-3.91 0.55 Demographic characteristics, clinical presentation of patients, size, shape and location of the abscess had no effect on the healing rate between the two study groups (P > 0.001) (Table 2). Table 2 Mean Healing time of the two study arms at different variables Variable Mean healing time in weeks Mean Difference 95%CI P-Value US Aspiration I&D Age in yrs  ≤ 20 27.5 30.0 -2.46 -6.2-1.3 0.19  > 20 21.5 19.9 1.61 -4.8-8.1 0.61 Symptoms  ≤ 7 days 20.6 22.0 -1.42 -11.8-8.9 0.77  > 7 days 28.7 24.0 4.70 -0.7-10.2 0.09 B/feeding  Yes 26.1 25.8 0.25 -4.9-5.3 0.92  No 22.5 15.8 6.70 -2.5-15.9 0.15 Parity  Primiparous 26.0 26.4 -0.45 -7.1-6.2 0.88  Multiparous 21.3 21.6 -0.35 -8.1-7.4 0.93  Nulliparous 24.7 30.0 -5.33 -28.3-17.6 0.58 Const.symp2  Yes 26.8 26.0 0.80 -8.4-9.9 0.83  No 23.7 22.9 0.80 -4.6-6.2 0.62 B/affected 3  Right 23.3 24.1 -0.73 -7.8-6.3 0.83  Left 24.9 23.3 1.55 -4.9-7.9 0.62 Q/affected 4  LUQ 5 23.6 20.3 3.26 -3.3-9.9 0.32  MUQ 6 18.0 27.3 -9.33 -19.9-1.3 0.08  LLQ 7 24.7 30.0 -5.33 -51.2-40 0.67  MLQ 8 25.7 31.0 -4.0 -49.2-39 0.62 SizeB/Absc 9  ≤ 3 cm 15.3 13.5 1.74 -5.0-8.5 0.59  > 3 cm 27.1 30.0 -2.90 -5.93-0.11 0.06 Shape  Oval 22.0 30.0 -8.0 -18-3.0 0.11  Irregular 24.8 23.1 -3.7 -3.7-71 0.52  Multiloculated 24.6 14.0 10.7 -35.2-56 0.42 Location  Subareolar 17.5 25.4 -7.8 -20.2-4.5 0.18  Periphery 26.8 23.1 3.7 -1.2-8.6 0.14  Indeterminate 22.0 30.0 -8.0 -184-168 0.67 BODY.RESULTS.SURVIVAL ANALYSIS: The duration of healing for study group A was 24.24 days and for group B was 24.16 days (Table 3). The probability of not healing was equal in the first week in both study arms. In the second week, the probability of not healing was slightly higher in group A than B. While in the third week, the probability of not healing was slightly higher in group B than A. Statistically there is no difference in the probabilities of not healing between patients in group A and B (Log rank: 0.24, df 1 P 0.63) (Figure 4). The Hazard rate of patients in Arm A was 0.93 times less the hazard rate of group B and this was not statistically significant (P > 0.001) (Figure 5). Table 3 Descriptive analysis Study Arm Mean duration of survival (95% CI) Median duration of survival (95% CI) A 24.24 (21.27-27.22) 30.00 B 24.16 (21.06-27.27) 30.00 (27.74-32.26) Figure 4Survival Function. Figure 5Hazard Function. BODY.RESULTS.COST-EFFECTIVENESS ANALYSIS: The number of patients effectively treated by Ultrasound guided Needle aspiration (excluding loss to follow up) were 29 and for incision and drainage were 31 patients (Table 4). The cost effectiveness ratio for incision and drainage arm was 2.85 times that of Ultrasound guided needle aspiration. Table 4 Cost-Effectiveness analysis  Category Intervention U.S. Guided Needle aspiration Incision & Drainage  Admission 620000  Professional fee 2900000 3100000  Preoperative fee 620000  Surgery fee 6100000  Anesthetic fee 3200000  Amenities 580000 620000  Sundries 290000 310000  Lodging 930000  Intervention U.S.S 870000  Cannula (FG 16) 43500  Syringe 20cc 29000  Drugs 246500 259600  Dr. in charge care fee 290000 310000  Health care fee 290000 310000  Wound dressing cost 489500  Total 5539000 16869100  Number of patients 29 31  Cost-effectiveness ratio 191000 544164.5 BODY.DISCUSSION: The breast is one of the sex organs of a female, in case of breast disease care should be taken to insure that its beauty is minimally compromised in order to preserve its value and function. Despite of breast abscess becoming less in developed countries due to improved maternal hygiene, nutrition, standard of living and early use of antibiotics, breast abscess remain a problem among women in developing countries [1]. Treatment of breast abscess traditionally has been incision and drainage however this has been found to be associated with possible unsatisfactory cosmetic outcome, difficult in breast feeding and needs general anesthesia, prolonged healing time, and regular dressing [3]. Repeated aspiration with or without ultrasound guidance has been found to be another treatment option for breast abscess and this has been reported to be associated with less recurrence, excellent cosmetic result and has less costs [4-6,8]. This study was conducted to establish whether ultrasound guided needle aspiration is a feasible alternative treatment option for the breast abscess in Mulago hospital. This discussion is based on 65 patients with breast abscesses randomized into 33 for Ultrasound guided aspiration and 32 for incision and drainage intervention. The healing rate, recurrence, cost effectiveness were compared between the two groups and acceptance of Ultrasound guided needle aspiration was assessed. Healing rate of the two groups had no statistically significant difference both overall and at each visit (Log rank: 0.24 df 1 P-0.63), this was similar with what was found elsewhere [4].This similarity in the healing rate between the two treatment option could be explained by the fact that regardless of the way pus is removed from the cavity (that is incision and drainage, needle aspiration or spontaneous rupture onto the skin surface) the healing process is the same which is by collapse of the cavity wall and adherence to one another by fibrin, later by granulation tissue. The remaining bacteria destroyed by polymorphs [9]. There was no recurrence of breast abscesses observed in the Ultrasound guided needle aspiration group during the study period. There was 3.1% (1/32) recurrence rate observed in the incision and incision group. However this recurrence rate was far less than 31% recurrence in the incision and drainage group which has been reported in another study [8].This small recurrence rate observed may have been resulted from a short follow up period and it was not possible to compare the recurrence rate of the two study groups. All the patients treated with Ultrasound guided needle aspiration highly accepted this modality (100%).This was consistence with what other studied found [8,10-12].This high acceptance rate may have been resulted from the convenience of the procedure which was an outpatient one, having no wound to nurse and absence of scar after healing. The cost effectiveness ratio of Ultrasound guided aspiration was found to be much less than that of Incision and Drainage, thus indicating that Ultrasound guided aspiration provides savings to the hospital and the patient, hence more cost effective than Incision and Drainage. This was consistence with what was found elsewhere [11,13]. Since Ultrasound guided aspiration is an outpatient procedure as opposed to the Incision and Drainage which is inpatient procedure. Studies done to compare outpatient versus inpatient surgical procedures showed that outpatient procedures were cost effective [14,15]. BODY.CONCLUSION: There is no difference in terms of healing rate of breast abscess between Ultrasound guided aspiration and surgical incision and drainage, Ultrasound guided needle aspiration is highly accepted by women with breast abscesses in Mulago hospital. Ultrasound guided aspiration is more cost effective than Incision and Drainage in management of breast abscess, therefore Ultrasound guided needle aspiration is an effective treatment option for both lactating and non lactating breast abscess. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: ABC conceived and designed the study and did the literature search, coordinated the write-up, editing and submission of the article. AMG & KEM coordinated the write-up, editing and supervised the study. PLC participated in the literature search, writing of the manuscript and editing. All the authors read and approved the final manuscript.

TITLE: Clinical and Angiographic Outcomes of the First Korean-made Sirolimus-Eluting Coronary Stent with Abluminal Bioresorbable Polymer ABSTRACT.BACKGROUND AND OBJECTIVES: This trial evaluated the safety and efficacy of the Genoss drug-eluting coronary stent. ABSTRACT.METHODS: This study was a prospective, multicenter, randomized trial with a 1:1 ratio of Genoss drug-eluting stent (DES)TM and Promus ElementTM. Inclusion criteria were the presence of stable angina, unstable angina, or silent ischemia. Angiographic inclusion criteria were de novo coronary stenotic lesion with diameter stenosis >50%, reference vessel diameter of 2.5–4.0 mm, and lesion length ≤40 mm. The primary endpoint was in-stent late lumen loss at 9-month quantitative coronary angiography follow-up. Secondary endpoints were in-segment late lumen loss, binary restenosis rate, death, myocardial infarction (MI), target lesion revascularization (TLR), target vessel revascularization (TVR), and stent thrombosis during 9 months of follow-up. ABSTRACT.RESULTS: We enrolled 38 patients for the Genoss DESTM group and 39 patients for the Promus ElementTM group. In-stent late lumen loss at 9 months was not significantly different between the 2 groups (0.11±0.25 vs. 0.16±0.43 mm, p=0.567). There was no MI or stent thrombosis in either group. The rates of death (2.6% vs. 0%, p=0.494), TLR (2.6% vs. 2.6%, p=1.000), and TVR (7.9% vs. 2.6%, p=0.358) at 9 months were not significantly different. ABSTRACT.CONCLUSION: This first-in-patient study of the Genoss DESTM stent showed excellent angiographic outcomes for in-stent late lumen loss and major adverse cardiac events over a 9-month follow-up. BODY.INTRODUCTION: Percutaneous coronary intervention (PCI) using a drug-eluting stent (DES) is a standard treatment for significant coronary artery occlusive disease. First-generation DESs have shown significantly lower restenosis rates compared to bare metal stents.1)2)3)4) Although DES reduced the rate of restenosis and target lesion revascularization (TLR), the risk of stent thrombosis increased.5)6)7) Stent thrombosis is a clinically serious complication because of its high mortality rate. It is related to early discontinuation of antiplatelet agents, resistance to antiplatelet agents, delayed endothelization, hypersensitivity reaction to polymers, and several clinical or procedural factors.6)7)8) As one possible cause of stent thrombosis is a hypersensitivity reaction to a durable polymer, the NoboriTM stent (Terumo Corporation, Tokyo, Japan) and BioMatrixTM (Biosensors International, Singapore) used a bioresorbable polymer that did not remain in the stent after a certain time period and showed good clinical outcomes compared to a durable polymer DES.9)10) The Genoss DESTM stent (Genoss Company Limited, Suwon, Korea) is the first sirolimus-eluting stent with biodegradable polymers made in Korea. The objectives of this first-in-patient prospective randomized study were to evaluate the efficacy and safety of the Genoss DESTM stent in comparison to the Promus ElementTM (Boston Scientific, Natick, MA, USA). BODY.METHODS.PATIENT POPULATION: This study was a prospective, multicenter, single blind (patient blind), randomized study with 1:1 ratio of patients using the Genoss DESTM stent and Promus ElementTM, conducted from March 2013 to April 2015. The study enrolled 80 patients at 4 Korean centers. Inclusion criteria were the presence of stable angina, unstable angina, or silent ischemia in patients between 20 and 80 years of age. Angiographic inclusion criteria were de novo coronary stenotic lesion with diameter stenosis (DS) >50%, reference vessel diameter (RVD) of 2.5 to 4.0 mm, and a lesion length ≤40 mm. Exclusion criteria were the presence of acute myocardial infarction (MI), cardiogenic shock, low ejection fraction (<40%), contraindication to antiplatelet agents, and chronic total occlusion lesion, restenosis, left main disease, and stenosis of the graft vessel. The protocol was approved by the Institutional Review Board of the Ajou University Hospital, Yonsei University Wonju Christian Hospital, Seoul National University Hospital, and Seoul St. Mary's Hospital, and all patients provided written informed consent. BODY.METHODS.THE GENOSS DES™ SIROLIMUS-ELUTING STENT: The Genoss DESTM is a sirolimus-eluting cobalt-chromium coronary stent system for the treatment of coronary artery diseases. The stent platform features open-cell 2 links with a uniform architecture that permits a high level of flexibility with good radial force, shortening, and conformability. Two kinds of strut thickness (70 and 78 μm) are adopted, depending on the stent diameter (2.25–2.50 and 2.75–4.00 mm, respectively). To achieve favorable clinical outcomes, the initial burst release of drug from DES is needed to suppress vascular injury and inflammation induced by catheter manipulation and stent implantation. Accordingly, the Genoss DESTM is designed to release approximately 70% of its initial drug payload (1.15 μg/mm2) within the first 30 days following implantation. To achieve the desired drug release profile and minimize the amount of polymer, a 4 μm ultra-thin coating is applied to only the abluminal side of the stent. This bioresorbable coating consists of a proprietary blend of poly-DL-lactide-co-glycolide (PLGA) and poly-DL-lactide (PLA), which are almost fully resorbed within 9 months. BODY.METHODS.STUDY PROCEDURE: PCI was performed using standard interventional techniques. Intravenous heparin was administered to maintain an activated clotting time of more than 250 seconds, and antiplatelet therapy was used before the procedure with aspirin (100–325 mg) and clopidogrel (loading dose of 300–600 mg) or ticlopidine (500 mg/day). Dual antiplatelet therapy was maintained during the 9 months of follow-up. Glycoprotein IIb/IIIa receptor antagonists were used at the operator's discretion. After predilation, Genoss DESTM, or Promus ElementTM was implanted under rated burst pressure, and post-dilatation was performed if needed. This study permitted 2 stents to overlap in cases of long lesions. BODY.METHODS.FOLLOW-UP: Clinical follow-up was performed at the time of discharge, and following a period of 1, 5, and 9 months. All patients were planned to undergo a follow-up angiography and intravascular ultrasound (IVUS) at 9±1 months. BODY.METHODS.QUANTITATIVE CORONARY ANGIOGRAPHY AND INTRAVASCULAR ULTRASOUND: Quantitative coronary angiography (QCA) analyses were performed by 2 independent observers blinded to treatment group using the Cardiovascular Angiography Analysis System II (CAAS II; Pie Medical, Maastricht, Netherlands). The percent DS, minimal luminal diameter, RVD, and lesion length were measured and calculated before the intervention, at the end of the procedure, and at the 9-month follow-up. IVUS was performed at the end of the index procedure and at follow-up, and analyzed by 2 independent observers blinded to clinical and procedural information according to the American College of Cardiology clinical expert consensus document on standards for acquisition, measurement, and reporting of IVUS studies.11) All IVUS analyses were performed using computerized planimetry software (EchoPlaque 3.0; Indec Systems, Santa Clara, CA, USA). BODY.METHODS.STUDY ENDPOINTS: The primary endpoint was in-stent late lumen loss at 9-month angiography follow-up determined by QCA. Secondary endpoints were in-segment late lumen loss, binary restenosis rate, death, MI, TLR, target vessel revascularization (TVR), and stent thrombosis during 9-month follow-up. BODY.METHODS.DEFINITION: MI was defined as chest pain and increased creatine kinase-myocardial band level to more than twice the upper limit of the normal range with or without a Q-wave on electrocardiography. Binary restenosis was defined as luminal narrowing of more than 50%, and TLR and TVR were defined as repeated PCI or bypass surgery of a previously stented site or vessel, respectively. Stent thrombosis was defined using the Academic Research Consortium definition.12) BODY.METHODS.STATISTICAL ANALYSIS: This study was a non-inferiority trial of the Genoss DESTM stent for in-stent late lumen loss at 9-month angiography follow-up compared to the Promus ElementTM stent. We assumed an in-stent late lumen loss of 0.20±0.28 mm in the Promus ElementTM stent group based on prior study.13) We calculated 35 patients per group, using a one side α level 0.025 and non-inferiority margin of 0.19 mm with a statistical power of 80%. Assuming approximately 10% of patients would drop out, we calculated 40 patients for each group. Data are presented as mean±standard deviations for continuous variables, and frequency (percentage) for categorical variables. Comparison of continuous variables was performed using Student's t-test, and χ2 or Fisher's exact tests were used for categorical variables. A p value <0.025 was considered statistically significant for the primary endpoint, and <0.050 for the secondary endpoints. Statistical analyses were performed using PASW software (version 18.0; SPSS Inc., Chicago, IL, USA). BODY.RESULTS: Between March 2013 and April 2015, 85 patients were screened for enrollment in the study. Five patients did not meet inclusion criteria and a total of 80 patients were randomly assigned to the Genoss DESTM or Promus ElementTM group at a 1:1 ratio. In the Genoss DESTM group, 2 patients were excluded as one patient did not receive stent implantation and the other patient had a protocol violation. In the Promus ElementTM group, 1 patient was excluded owing to the patient's withdrawal from the study (Figure 1). Figure 1Flow chart of patient enrollment.DES = drug-eluting stent. Baseline clinical and angiographic characteristics are shown in Tables 1 and 2, respectively. There were no significant differences in clinical characteristics including age and clinical diagnosis between the 2 groups (Table 1). Target vessels were not significantly different, but the left anterior descending artery was targeted more often in the Promus ElementTM group. Stent overlap was performed in 8 (21.1%) patients in the Genoss DESTM group and in 3 (7.7%) patients in the Promus ElementTM group (Table 2). Table 1 Baseline clinical characteristics Genoss DES™ (n=38) Promus Element™ (n=39) p Age (years) 64±8 63±8 0.591 Male 31 (81.6) 31 (79.5) 1.000 Diabetes 12 (31.6) 12 (30.8) 1.000 Hypertension 24 (63.2) 29 (74.4) 0.332 Hyperlipidemia 15 (39.5) 13 (33.3) 0.640 Current smoker 9 (23.7) 13 (33.3) 0.451 Diagnosis 0.549 Stable angina 17 (44.7) 19 (48.7) Unstable angina 21 (55.3) 19 (48.7) Silent ischemia 0 1 (2.6) Data are shown as mean±standard deviation or number (%). DES = drug-eluting stent. Table 2 Angiographic and procedural characteristics Genoss DES™ (n=38) Promus Element™ (n=39) p Target vessel 0.068 Left anterior descending 18 (47.3) 26 (66.7) Left circumflex 4 (10.5) 6 (15.4) Right coronary 16 (42.1) 7 (17.9) AHA/ACC classification 0.089 A 4 (10.5) 7 (17.9) B1 6 (15.8) 14 (35.9) B2 10 (26.3) 8 (20.5) C 18 (47.4) 10 (25.6) Stent diameter (mm) 3.14±0.26 3.15±0.30 0.970 Stent length (mm) 25.5±8.6 24.1±5.0 0.395 Mean stent number 1.2 1.1 0.099 Stent overlap 8 (21.1) 3 (7.7) 0.114 Data are shown as mean±standard deviation or number (%). ACC = American College of Cardiology; AHA = American Heart Association; DES = drug-eluting stent. Angiographic follow-up at 9 months was performed for all patients, and IVUS follow-up was performed in 35 (92%) patients in the Genoss DESTM group and in 37 (95%) patients in the Promus ElementTM group. QCA results are shown in Table 3. There were no significant differences in RVD. For the primary endpoint, in-stent late lumen loss at 9 months was not significantly different between the groups (0.11±0.25 mm for Genoss DESTM vs. 0.16±0.43 mm for Promus ElementTM, p=0.567) nor was in-segment late lumen loss (0.11±0.26 mm for Genoss DESTM vs. 0.15±0.43 mm for Promus ElementTM, p=0.558). Angiographic restenosis at 9 months was observed in 1 (2.6%) patient in the Genoss DESTM group and in 2 (5.1%) patients in the Promus ElementTM group (p=1.000). IVUS minimal lumen area after stenting during the index procedure (6.95±1.98 vs. 7.47±2.47 mm2, p=0.272) and after 9 months (6.95±1.98 vs. 7.29±2.34 mm2, p=0.508) were not significantly different (Table 4). For clinical outcomes, there were no MI or stent thrombosis events during 9-month follow-up. One death occurred in the Genoss DESTM group due to aggravated renal function 6 months after the index procedure. However, the rate of death, MI, TLR, and TVR at 9 months were not significantly different (Table 5). Table 3 QCA analysis Genoss DES™ (n=38) Promus Element™ (n=39) p RVD (mm) Pre-procedure 3.24±0.41 3.12±0.43 0.199 Post-procedure 3.30±0.30 3.33±0.30 0.652 9-month follow-up 3.17±0.35 3.16±0.39 0.932 Minimal lumen diameter (mm) In-stent Pre-procedure NA NA NA Post-procedure 2.95±0.35 3.03±0.30 0.269 9-month follow-up 2.84±0.42 2.87±0.56 0.743 In-segment Pre-procedure 0.83±0.29 0.88±0.45 0.572 Post-procedure 2.90±0.27 3.00±0.36 0.159 9-month follow-up 2.79±0.40 2.85±0.56 0.615 DS % In-stent Pre-procedure NA NA NA Post-procedure 10.6±3.48 9.16±4.85 0.140 9-month follow-up 14.18±9.31 13.73±14.30 0.871 In-segment Pre-procedure 74.41±8.62 72.65±12.14 0.465 Post-procedure 12.10±3.93 10.12±4.94 0.056 9-month follow-up 15.46±9.39 14.54±14.08 0.738 Acute gain (mm) In-stent 1.79±0.40 1.82±0.36 0.742 In-segment 1.74±0.39 1.78±0.38 0.579 Late loss (mm) In-stent 0.11±0.25 0.16±0.43 0.567 In-segment 0.11±0.26 0.15±0.43 0.558 Lesion length (mm) 23.8±8.1 22.8±4.9 0.531 Restenosis 1 (2.6) 2 (5.1) 1.000 Data are shown as mean±standard deviation or number (%). DES = drug-eluting stent; DS = diameter stenosis; NA = not available; QCA = quantitative coronary angiography; RVD = reference vessel diameter. Table 4 IVUS analysis results Genoss DES™ (n=35) Promus Element™ (n=37) p Minimal lumen area (mm 2 ) Post-procedure 6.71±1.74 7.28±2.41 0.255 9-month follow-up 6.30±1.78 6.72±2.28 0.387 Minimal stent area (mm 2 ) Post-procedure 6.90±1.77 7.47±2.47 0.272 9-month follow-up 6.95±1.98 7.29±2.34 0.508 EEM Post-procedure 14.87±3.23 14.91±4.40 0.963 9-month follow-up 15.42±3.27 14.74±3.90 0.423 Data are shown as mean±standard deviation. DES = drug-eluting stent; EEM = external elastic membrane; IVUS = intravascular ultrasound. Table 5 Clinical outcomes Genoss DES™ (n=38) Promus Element™ (n=39) p Death 1 (2.6) 0 0.494 Cardiac 0 0 1.000 Non-cardiac 1 (2.6) 0 0.494 MI 0 0 1.000 TLR 1 (2.6) 1 (2.6) 1.000 TVR 3 (7.9) 1 (2.6) 0.358 Stent thrombosis 0 0 1.000 Data are shown as number (%). DES = drug-eluting stent; MI = myocardial infarction; TLR = target lesion revascularization; TVR = target vessel revascularization. BODY.DISCUSSION: This prospective, multicenter, randomized first-in-patient study showed that in-stent late lumen loss of Genoss DESTM stents was not inferior to Promus ElementTM stents as determined by QCA at 9-month angiography follow-up. Moreover, there were no differences in clinical outcomes and no stent thrombosis during 9 months of follow-up. The Genoss DESTM stent is the first DES made in Korea. It consists of a cobalt-chromium alloy stent platform, an abluminal coating of bioresorbable polymers, and a drug-eluting coating with sirolimus. Sirolimus is an anti-proliferative and anti-inflammatory agent that inhibits progression from the G1 to the S phase of the cell cycle. Coronary DES using sirolimus has demonstrated good clinical outcomes.4)14) For the Genoss DESTM stent, animal studies using pigs were performed to determine the optimal dose of the drug, comparing high and low doses of sirolimus and paclitaxel (unpublished data). Consequently, sirolimus was loaded on the stent at a concentration of 1.15 μg/mm2. The stent was designed to release approximately 70% of the total drug amount within 30 days after implantation. To control the drug release profile, it possesses an ultra-thin (4 μm) bioresorbable layer applied to an abluminal stent surface. This bioresorbable layer consists of a proprietary blend of PLGA and PLA, which are completely resorbable within 6 months, to reduce the possibility of stent thrombosis due to polymer hypersensitivity. In addition, the cobalt-chromium alloy stent platform reduces strut thickness and maximizes flexibility. In-stent late luminal loss at 9 months with the Genoss DESTM stent was 0.11±0.25 mm, comparable with previous DES trials (Table 6). Moreover, this study included a number of patients at high risk of unstable angina or complex lesions. The proportion of patients with unstable angina was around 50% in both groups and that of American Heart Association (AHA)/American College of Cardiology (ACC) type C lesions was 47.4% for the Genoss DESTM group and 25.6% for the Promus ElementTM group. Angiographic in-stent restenosis was observed in only 2.6% of patients in the Genoss DESTM group, even though stent overlap was permitted such that mean lesion length was very high (25.5 mm for the Genoss DESTM group) compared with previous studies (Table 6). Considering patient and angiographic characteristics, in-stent late lumen loss of the Genoss DESTM stent at 9-month follow-up was acceptable in real clinical practice. Table 6 Comparison of angiographic outcomes with previous studies In-stent late loss (mm) Lesion length (mm) Platinum QCA 13) 0.17±0.25 15.4±7.0 Nobori 1 15) 0.11±0.30 10.56  SPIRIT I 16) 0.10±0.21 10.1±2.6 RESOLUTE 17) 0.22±0.27 15.61±6.13 Genoss DES™ (current study) 0.11±0.25 25.5±8.6 Data are shown as mean±standard deviation. DES = drug-eluting stent; QCA = quantitative coronary angiography. For clinical outcomes, there were no differences in death, MI, TLR, or TVR between the 2 groups. TLR occurred in only 2.6% of patients in both groups at 9-month angiography follow-up. In the Genoss DESTM group, 2 cases of TVR without TLR occurred, which were new lesions in the proximal portions of the right coronary artery independent of previously stented sites. Although not statistically significant, the stent overlap rate at the target lesion was higher in the Genoss DESTM group than in the Promus ElementTM group (21.1% vs. 7.7%). However, the clinical event rate of the Genoss DESTM group was very low. The Genoss DESTM stent did not develop stent thrombosis during the 9-month follow-up despite complex lesion characteristics. This study has several limitations. For assumptions about sample size, the non-inferiority margin was relatively high and the number of patients was small. This made the statistical power weak. However, the Genoss DESTM stent showed excellent in-stent late lumen loss compared to current second-generation DESs. In addition, as this is a first-in-patient trial, more studies with larger populations should be conducted in real clinical practice. In conclusion, this first-in-patient study of the Genoss DESTM stent showed excellent angiographic outcomes for in-stent late lumen loss and major adverse cardiac events during the 9-month follow-up.

TITLE: Comparison of Clinical Efficacy of Newfactan® versus Surfacten® for the Treatment of Respiratory Distress Syndrome in the Newborn Infants ABSTRACT: Newfactan® is a domestically developed, bovine lung-derived, semi-synthetic surfactant. The aim of this study was to compare the clinical efficacy of Newfactan® with that of Surfacten® in the treatment of respiratory distress syndrome (RDS). Newfactan® or Surfacten® was randomly allocated to 492 newborn infants who were diagnosed as RDS and required surfactant instillation in four participating hospitals. The comparisons were made individually in two subsets of infants by birth weight (<1,500 g group [n=253] and ≥1,500 g group [n=239]). Short-term responses to surfactant and acute complications, such as the total doses of surfactant instilled, response type, extubation rate, ventilator settings, changes in respiratory parameters, air leak, patent ductus arteriosus, pulmonary hemorrhage, and intraventricular hemorrhage, and mortality during the 96 hr after surfactant instillation were measured. Long-term outcome and complications, such as total duration of intubation, bronchopulmonary dysplasia and periventricular leukomalacia, and ultimate mortality were measured. There were no significant differences in demographic and perinatal variables, short-term responses to surfactant and acute complications, and long-term outcome and complications between Newfactan® and Surfacten® in both birth weight groups. We concluded that Newfactan® was comparable to Surfacten® in the clinical efficacy in the treatment of RDS in both birth weight groups. BODY.INTRODUCTION: Pulmonary surfactant is the cornerstone of the treatment of respiratory distress syndrome (RDS) in the newborn infants. A number of pulmonary surfactants have been developed, but only several agents are used in the present days in the world (1). In Korea, Surfacten® and Exosurf® came into the market in 1990, and Curosurf® in 1996. Surfacten® and Curosurf® are modified natural surfactants derived from bovine and porcine lung, respectively. While on the other, Exosurf® is a purely synthetic surfactant. However, Exosurf® has dropped out of the market before long, and Surfacten® imported from Japan has been the most commonly prescribed pulmonary surfactant for the treatment of RDS in the newborn infants in Korea since the first clinical report by Namgung et al. (2) and the following multi-center clinical trial by Bae et al. (3, 4). However, because of the high cost of Surfacten®, the need for less expensive domestic pulmonary surfactant agent has emerged. Thereupon, Newfactan®, a semi-synthetic pulmonary surfactant derived from bovine lung, was co-developed by a domestic pharmaceutical company and a medical college. Both in vitro and in vivo studies confirmed the efficacy of Newfactan® (5, 6). However, the clinical data on the efficacy of Newfactan® are scant (7-9), especially in extremely low birth weight infants whose survival rate has improved remarkably recently. The initial clinical report on its efficacy in RDS was not a comparative study, but a single agent study (7). A single comparative study of Newfactan® with Surfacten® was done so far (8). In that study, a total of 60 newborn infants were enrolled with 30 infants in each surfactant group. There were no differences in the efficacy in the treatment of RDS, the incidence of complications, and mortality between Surfacten® and Newfactan® groups. However, this study has some limitations as the authors mentioned in the discussion. Firstly, the number of the newborn infants enrolled was small because only two centers participated in the study. Secondly, the study lacks the data on the extremely low birth weight infants, especially the infants with birth weight of less than 800 g, whose survival rate has improved dramatically recently. Therefore, further comparative studies with the larger number of newborn infants, especially the extremely low birth weight infants whose birth weight was less than 1,000 g are required to verify the comparability of Newfactan® to Surfacten® in clinical efficacy in the treatment of RDS. Therefore, we did a multi-center clinical comparative study enrolled a fairly large number of the newborn infants, especially the extremely low birth weight infants. A total of 112 extremely low birth weight infants were included in our subjects. BODY.MATERIALS AND METHODS.SUBJECTS: A total of 492 newborn infants who were admitted to the neonatal intensive care units in four hospitals: Samsung Seoul Hospital; Samsung Cheil Hospital; Kangnam Cha Hospital; and Soonchunhyang University Bucheon Hospital and diagnosed as RDS, and judged to require pulmonary surfactant instillation therapy between May 2002 and July 2004 were enrolled. The diagnosis of RDS and the judgment of the requirement for surfactant instillation therapy were made by the staff neonatologists at each participating hospital. Newborn infants who had a major congenital anomaly were excluded. Institutional review board (Samsung Seoul Hospital, Seoul, Korea) approved the study protocol and informed consent was obtained from a parent before study participation. BODY.MATERIALS AND METHODS.STUDY PROTOCOL: Newfactan® or Surfacten® was randomly allocated and instilled to the newborn infants who were newly enrolled to the study during the study period. The order of allocation was individualized by each participating hospital. The two surfactants were alternatively allocated in two stratified blocks by birth weight, which are <1,500 g and ≥1,500 g block. However, in the hospitals where the infants with a birth weight of less than 1,500 g were in the minority, the allocation was done without stratification. The method of surfactant instillation was the same among the four participating hospitals. Surfactant instillation was used as a rescue therapy. Newfactan® or Surfacten® was dissolved in 4 mL of 0.9% saline and 4 mL/kg of the dissolved solution was instilled into the trachea via endotracheal tube by using orogastric tube. To distribute the surfactant to the whole lung evenly, four-position method was used. Surfactant was instilled to a maximum of 3 doses according to the infant's condition. Second or third dose was given after 6 hr when the infant became worse clinically and radiologically at the staff neonatologist's discretion. BODY.MATERIALS AND METHODS.OUTCOME MEASURES: The comparisons of outcome were also done by stratifying the subject infants into two subgroups by birth weight, which are <1,500 g group and ≥1,500 g group. Short-term responses to surfactant and acute complications include the total doses of surfactant instilled, response type, extubation rate, changes in respiratory parameters, air leak, patent ductus arteriosus, pulmonary hemorrhage, intraventricular hemorrhage, and short-term mortality during the 96 hr after surfactant instillation. Long-term outcome and complications include the total duration of intubation, bronchopulmonary dysplasia (BPD) and periventricular leukomalacia, and ultimate mortality. We chose arterial-alveolar oxygen ratio (a/APO2=PaO2/[713 ×FiO2-PaCO2/0.8]) and ventilatory index (VI=mean airway pressure×FiO2/PaO2) (10) as respiratory parameters. The response type to surfactant was defined into three types: 'Favorable' when VI dropped below 0.047 within 6 hr after surfactant instillation; 'Relapse' when VI rebounded above 0.047 between 6 and 96 hr after surfactant instillation among 'favorable' cases; and 'Poor' when VI did not drop below 0.047 within 6 hr after surfactant instillation (11). When the infant was already extubated, the response type was assigned to ''Favorable'', FiO2 to 21%, and mean airway pressure (MAP) to 0 cmH2O. Extubation was defined as a successful extubation that does not require re-intubation within 7 days after the extubation. Air leak includes pneumothrax, pneumomediastinum, and pulmonary interstitial emphysema. Patent ductus arteriosus was diagnosed by echocardiography and only symptomatic cases were identified. Intraventricular hemorrhage and periventricular leukomalacia were diagnosed by brain ultrasonography, and only high-grade (≥grade III) intraventricular hemorrhage was identified. Periventricular leukomalacia was identified only in infants who survived more than 28 days after birth. Pulmonary hemorrhage was diagnosed when the fresh blood gushed out of the endotracheal tube in the presence of typical chest radiography finding. The diagnosis of BPD was made in the infants who needed supplemental oxygen at 36 wk postmenstrual age (gestational age <32 wk) or for more than 28 days after birth (gestational age ≥32 wk) with consistent radiographic change (persistent hazy opacification or cystlike pattern of density and lucency) (11). BPD was also identified only in the infants who survived more than 28 days after birth. BODY.MATERIALS AND METHODS.STATISTICAL METHODS: Statistical analyses were done by using SPSS version 11.5 (SPSS Inc. Chicago, IL, U.S.A.). All categorical variables were expressed as percent (%) and continuous variables were expressed as mean±standard deviation. Categorical variables between Newfactan® group and Surfacten® group were compared by chi-square test or Fisher's exact test. Continuous variables were compared by Student t-test or Mann-Whitney U test. Repeatedly measured variables like respiratory parameters were compared by using repeated measures ANOVA. P value of less than 0.05 was considered significant. The statistical analyses were supervised by a biomedical statistician at Samsung Seoul Hospital. BODY.RESULTS.DEMOGRAPHIC VARIABLES: Of a total of 492 newborn infants, 224 infants were given Newfactan® (Newfactan® group) and 268 infants were given Surfacten® (Surfacten® group), and 253 infants had the birth weight of less than 1,500 g (<1,500 g group) and 239 infants had the birth weight of more than 1,500 g (≥1,500 g group). The followings are the distribution of enrolled infants according to the participating hospital: 180 infants in Samsung Seoul Hospital; 156 infants in Samsung Cheil Hospital; 124 infants in Kangnam Cha Hospital; 32 infants in Soonchunhyang University Bucheon Hospital (Fig. 1). A total of 112 extremely low birth weight infants were included in <1,500 g group, which consisted of 13 infants with a birth weight of <600 g, 41 infants with a birth weight of 600-799 g, and 58 infants with a birth weight of 800-1,000 g (Fig. 1). There were no significant differences in birth weight (1,074±255 g for Newfactan® group versus 1,031±276 g for Surfacten® group) and gestational age (28.1±2.3 vs. 28.4±2.6 wk) between Newfactan® group and Surfacten® group in <1,500 g group as well as in ≥1,500 g (birth weight 2,496±714 vs. 2,346±596 g, gestational age 35.2±3.0 vs. 34.6±3.0 wk). There were no significant differences in sex ratio between Newfactan® group and Surfacten® group in both birth weight groups (Table 1). BODY.RESULTS.PERINATAL VARIABLES: There were no significant differences in perinatal variables between Newfactan® group and Surfacten® group in both <1,500 g group and ≥1,500 g group (Table 1). The respiratory parameters just before the surfactant instillation indicating the severity of RDS, such as FiO2, MAP, VI, and a/APO2 were not significantly different between Newfactan® group and Surfacten® group in both birth weight groups (pretreatment values in Fig. 2, 3). BODY.RESULTS.SHORT-TERM RESPONSES TO SURFACTANT AND ACUTE COMPLICATIONS: In <1,500 g group, the total doses of surfactant instilled were not significantly different between Newfactan® group and Surfacten® group. 75% of the infants in Newfactan® group and 77% of the infants in Surfacten® group received only one dose of surfactant, and 23% of the infants in both Newfactan® group and Surfacten® group received two doses. Only two infants in Newfactan® group received three doses of surfactant. There was no significant difference in the response type to surfactant between Newfactan® group and Surfacten® group. The 'Favorable' type was observed in 84% of the infants in Newfactan® group and in 87% of the infants in Surfacten® group. The 'Poor' type was observed in 9% of the infants in Newfactan® group and 10% of the infants in Surfacten® group. The 'Relapse' type was observed in 7% of the infants in Newfactan® group, and in 3% of the infants in Surfacten® group. The extubation rate by the 96 hr after surfactant instillation was not significantly different between Newfactan® group and Surfacten® group. 55% of the infants in Newfactan® group, and 52% of the infants in Surfacten® group were extubated by the 96 hr after surfactant instillation (Table 2). In ≥1,500 g group, the total doses of surfactant instilled were not significantly different between Newfactan® group and Surfacten® group (one dose 87 vs. 90%, two doses 13 vs. 10%). No infants in either group received three doses of surfactant. There was no significant difference in the response type to surfactant between Newfactan® group and Surfacten® group ('Favorable' 83 vs. 84%, 'Poor' 11 vs. 11%, 'Relapse' 6 vs. 5%). The extubation rate by the 96 hr after surfactant instillation was not significantly different between Newfactan® group and Surfacten® group (83 vs. 89%) (Table 2). The time courses of FiO2, MAP, a/APO2 and VI over the 96 hr after surfactant instillation were not significantly different between Newfactan® group and Surfacten® group in both birth weight groups (Fig. 2, 3). The incidences of air leak, patent ductus arteriosus, pulmonary hemorrhage, and high-grade intraventricular hemorrhage, and short-term mortality during the 96 hr after surfactant instillation were not significantly different between Newfactan® group and Surfacten® group in both birth weight groups (Table 3). BODY.RESULTS.LONG-TERM OUTCOME AND COMPLICATIONS: Total duration of intubation was not significantly different between Newfactan® group and Surfacten® group (14.3±18.6 days versus 12.2±18.8 days) in <1,500 g group, as well as in ≥1,500 g group (3.7±3.2 vs. 3.6±2.6 days). The incidences of BPD and periventricular leukomalacia and the ultimate mortality were not different between two surfactant groups in both birth weight groups (Table 4). BODY.DISCUSSION: Our data showed that there were no significant differences in the efficacy in the treatment of RDS and in the incidence of acute complications occurring around the surfactant instillation therapy between Newfactan® and Surfacten®. In the ultimate mortality and long-term complications including BPD and periventricular leukomalacia, there were also no differences between the two surfactants. There has been an urgent need for the clinical data that could support the clinical comparability of Newfactan®, a less expensive domestically-developed surfactant, to Surfacten® for Newfactan® to be used nation-wide as a primary pulmonary surfactant agent in the treatment of RDS in Korea. The results of the first clinical comparative study were already published in 2000 (8). However, because it had a relatively small sample size and lacks the population of extremely low birth infants whose birth weight was less than 800 g, further large-scale clinical study has been required. Our study has a clinical significance in that it is a multi-center study enrolled as many as 492 newborn infants, especially 112 extremely low birth weight infants whose birth weight was less than 1,000 g. Although the data were not shown, like in <1,500 g group and ≥1,500 g group, there were also no statistically significant difference in the short-term responses and acute complications and long-term outcome and complications between Newfactan® group and Surfacten® group in the subgroup of infants whose birth weight was less than 1,000 g. The two surfactants were alternatively allocated with stratified block randomization, which is <1,500 g and ≥1,500 g block. However, at a part of participating hospitals where the infants with a birth weight of less than 1,500 g were in the minority, the allocation was done without stratification. This seems to be the origin of the size discrepancy between the two surfactant groups in <1,500 g group. Due to the vague reliance on Surfacten® over Newfactan®, the attending physicians seem to have prescribed Surfacten® instead of Newfactan® in Newfactan's turn of the original allocation order at those hospitals when the birth weight of newly enrolled infant was less than 1,500 g, resulting in the disruption of the original allocation order. This problem of disordered randomization may be a major drawback of our study. Nevertheless, the results of our study are not thought to be nullified. We also compared the outcome by stratifying the subject infants by birth weight. After the stratification, there were no statistically significant differences in demographic and perinatal variables between the two surfactant groups in both <1,500 g group and ≥1,500 g group. This implicates that the two surfactant groups in each stratum are similar in baseline conditions and therefore comparable to each other. Therefore, the results of our study are considered to remain to be valid. The reason why we stratified the subject infants by birth weight is that the etiology and clinical course of RDS in each subgroup were considered to be different. The etiology of RDS in <1,500 g group may be primarily attributable to absolute deficiency of pulmonary surfactant pool and its course may be complicated due to a number of problems arising from the prematurity itself. On the other hand, the etiology of RDS in ≥1,500 g group may be more complex. Besides the absolute deficiency of surfactant pool, conditions that lead to secondary surfactant inactivation such as asphyxia, infection and aspiration of meconium or blood might be responsible (1, 13). However, the clinical course of RDS in ≥1,500 g group may be, if not all, uncomplicated. Once the RDS has resolved, the majority of infants in this group seem to go uneventful until the discharge. This rationale for the stratification seems to be partially validated by our data. The extubation rate, the incidence of patent ductus arteriosus, pulmonary hemorrhage, and high-grade intraventricular hemorrhage, and short-term mortality during the 96 hr after surfactant instillation, total duration of intubation, and the ultimate mortality were significantly different between <1,500 g group and ≥1,500 g group. However, contrary to our expectation, the total doses of surfactant instillation and the response to surfactant instillation therapy were not different between the two birth weight groups. This stratification in the outcome analyses are considered to have made our results more clinically relevant. In the time courses of a/APO2 and VI in ≥1,500 g group, there is a tendency that respiratory status is aggravated again later in the course after surfactant instillation. However, this finding might have originated from the fact that a/APO2 and VI were obtained only from the infants who took the blood gas analysis. Because the infants who took blood gas analysis until later in the course after surfactant instillation might have had relatively severe and complicated course of RDS, these later aggravations observed in the time courses of a/APO2 and VI might be the reflections of these infants. In the present multi-center post-marketing comparative study, Newfactan® showed no differences in the efficacy in the treatment of RDS and in the incidences of acute and long-term complications, compared to Surfacten® in both <1,500 g group and ≥1,500 g group.

TITLE: Comparison of the effectiveness and toxicity of neoadjuvant chemotherapy regimens, capecitabine/epirubicin/cyclophosphamide vs 5-fluorouracil/epirubicin/cyclophosphamide, followed by adjuvant, capecitabine/docetaxel vs docetaxel, in patients with operable breast cancer ABSTRACT: The aim of this study was to compare the effectiveness and toxicity of neoadjuvant chemotherapy regimens, xeloda/epirubicin/cyclophosphamide (XEC) vs 5-fluorouracil/epirubicin/cyclophosphamide (FEC), followed by adjuvant chemotherapy regimens, capecitabine/taxotere (XT) vs taxotere (T), in axillary lymph node (LN)-positive early-stage breast cancer. In this randomized, Phase III trial, 137 patients with operable primary breast cancer (T2-0, N0-1) who were tested axillary LN positive through aspiration biopsy of axillary LNs were randomized (1:1) to four 3-weekly cycles of XEC or FEC. Patients underwent surgery within 4–6 weeks after the fourth cycle, followed by four adjuvant cycles of 3-weekly XT or T. The primary end point was tumor pathological complete response. Toxicity profiles were secondary objectives. In total, 131 patients had clinical and radiological evaluation of response and underwent surgery. Treatment with XEC led to an increased rate of pathological complete response in primary tumor (18% vs 6%, respectively, P=0.027) and objective remission rate (87% vs 73%, P=0.048) compared to FEC. Clinical complete response occurred in 20% and 7% for XEC and FEC, respectively. Compared to FEC, XEC was associated with more hand-foot syndrome (57% vs 11%, P<0.001) and 3/4 grade nausea/vomiting/diarrhea (30% vs 14%, P=0.034) but less phlebitis (3% vs 14%, P=0.035). XT and T adjuvant chemotherapy regimens were well tolerated: treatment-related 3/4 grade adverse events occurred in 28% and 17% of patients receiving XT and T, respectively. BODY.INTRODUCTION: Breast cancer is a type of most commonly highly metastatic malignant tumor among females.1 Neoadjuvant chemotherapy was first used as induced chemotherapy or primary chemotherapy to treat locally advanced and inoperable breast cancer. Patients with breast cancer with pathological complete response (pCR) after neoadjuvant chemotherapy were able to achieve better overall survival rate.2,3 Therefore, pursuing the pCR becomes the primary goal in neoadjuvant chemotherapy. Neoadjuvant chemotherapy can be used to evaluate the therapeutic effect of tumors, and the evaluation of neoadjuvant chemotherapy has advantages in providing prognosis and adjusting treatment strategy compared to other treatments. Therefore, with the development of research in neoadjuvant chemotherapy, its application has expanded from locally advanced to early and medium stage in breast cancer.4 Neoadjuvant chemotherapy is the best in vivo chemosensitivity test because an effective follow-up plan can be made according to the reaction of tumor during chemotherapy treatment, and it is an important method of achieving individualized treatment in operable breast cancer.5,6 The advantage of neoadjuvant chemotherapy in operable breast cancer before operation can not only decrease the size of large tumors and clinical staging but also narrow the area of operative surgical resection and kill small metastasized micrometastases prior to surgery. An objective evaluation of resistance to chemotherapy in cancer and prognosis by clinical and pathological response is another advantage.7 The pCR rate can be mainly used to predict the long-term outcomes of neoadjuvant chemotherapy and also as a surrogate end point in clinical trials.8 The best preoperative treatment should achieve a high pCR rate by neoadjuvant chemotherapy. The effect of the chemotherapy regimen, capecitabine/epirubicin/cyclophosphamide (XEC), as a neoadjuvant chemotherapy regimen has been reported to be better than that of 5-fluorouracil (5-FU)/epirubicin/cyclophosphamide (FEC), with a good tolerance; thus, it is advisable to use XEC instead of FEC during neoadjuvant or adjuvant chemotherapy treatment.9 Lee et al10 claimed that the efficacy of capecitabine (Xeloda®, F. Hoffmann La-Roche, Basel, Switzerland)/docetaxel (Taxotere®, Sanofi-Aventis, Paris, France) (XT) neoadjuvant chemotherapy was better than doxorubicin/cyclophosphamide (AC), because the pCR rate of the former was significantly improved and treatment-related G3/4 adverse events were relatively lower. Other data11,22 proved that capecitabine which treated patients with metastatic breast cancer was a valid single reagent and well tolerated. A meta-analysis also reported that capecitabine should be used in neoadjuvant chemotherapy, and treatment with XT was the first-line chemotherapy regimen with an effective rate of 42%.13 Another meta-analysis also reported that treatment with XT can effectively improve the efficacy of adjuvant chemotherapy and is highly likely to become a new adjuvant chemotherapy regimen.14 The primary objective of this study was to compare the tumor pCR rate achieved with four 3-weekly cycles of XEC vs FEC when used as neoadjuvant chemotherapy for patients with axillary lymph node (LN)-positive stage II/III and operable breast cancer. BODY.PATIENTS AND METHODS.STUDY DESIGN: This was a randomized, open-label, single-center clinical trial comparing XEC with FEC as a neoadjuvant chemotherapy for patients with axillary LN-positive stage II/III and operable breast cancer. All the patients provided written informed consent before enrollment. Furthermore, all the procedures performed in this study involving human participants followed the ethical standards of the Medical ethics committee of the Affiliated Cancer Hospital of Guangxi Medical University who approved the study and followed the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The trial profile is shown in Figure 1. Patients were randomized to one of two treatment arms: capecitabine (Xeloda®; Shanghai Roche Pharmaceuticals Ltd., Shanghai, People's Republic of China) 1,000 mg/m2 orally twice daily on days 1–14, plus epirubicin (Pfizer, Inc., New York, NY, USA) 100 mg/m2 infusion on day 1, plus cyclophosphamide (Endoxan®; Baxter Oncology GmbH, Halle, Germany) 500 mg/m2 infusion on day 1 every 3 weeks for four cycles, or 5-FU (Shanghai Xudong Haipu Pharmaceutical Co. Ltd., Shanghai, People's Republic of China) 500 mg/m2 infusion on day 1, plus epirubicin 100 mg/m2 infusion on day 1, plus cyclophosphamide 500 mg/m2 infusion on day 1 every 3 weeks for four cycles before surgery. The first assessment of curative effect was performed within 24–48 hours before the third cycle of chemotherapy when the second cycle was over, and the second assessment was performed within 24–48 hours after the fourth cycle. If the evaluation was considered invalid, then the patients were categorized as invalid group and the others underwent surgery after the fourth cycle of neoadjuvant chemotherapy and then crossed over to receive the other treatment regimens as adjuvant therapy. In the XEC arm, four-cycle adjuvant chemotherapy of XT was given: capecitabine 1,000 mg/m2 orally twice daily on days 1–14, plus docetaxel (Jiangsu Hengrui Medicine Co. Ltd., Lianyungang, People's Republic of China) 75 mg/m2 infusion on day 1 every 3 weeks. In the FEC arm, four-cycle adjuvant chemotherapy of T was given: docetaxel 75 mg/m2 infusion on day 1 every 3 weeks. All patients positive for human epidermal growth factor receptor 2 (HER2) were not using trastuzumab while receiving adjuvant chemotherapy because it might increase the cardiac toxicity of chemotherapy drugs. Upon completion of the adjuvant chemotherapy, all patients received radiotherapy and were concurrently treated with tamoxifen or letrozole when hormone receptor (HR) was positive. BODY.PATIENTS AND METHODS.ELIGIBILITY CRITERIA: To be included in the study, patients should have biopsy-proven, newly diagnosed stage II/III, and operable breast cancer with axillary LN involved. Operable breast cancer was defined as a tumor with a diameter of >1 cm diagnosed by ultrasonography or magnetic resonance imaging (MRI). The axillary LN positivity was determined by fine-needle aspiration cytology of axillary LN. Further, the eligibility criteria included patients aged 18–70 years, with an Eastern Cooperative Oncology Group (ECOG) performance status of ≤1 and adequate hematologic (absolute neutrophil count ≥1,500/mm3, platelet count ≥10,000/mm3, hemoglobin ≥10 g/dL), renal (serum creatinine ≤1.5 mg/dL), cardiac (confirmed by normal or nonspecific ECG or multigated acquisition scan taken within 1 month of enrollment), and hepatic (total bilirubin ≤1.5 mg/dL, aspartate aminotransferase, alanine transaminase, alkaline phosphatase ≤2.5× upper normal limit) functions. Patients were excluded if they had undergone prior surgery, hormonal treatment, chemotherapy, or radiotherapy, or had a history of cancer except for in situ uterine cervical cancer or non-melanotic skin cancer, any distant metastasis, or any serious concomitant systemic disorder. BODY.PATIENTS AND METHODS.PATIENT EVALUATIONS: The three-dimensional size of the primary breast tumor was measured by physical examination, ultrasound, and molybdenum target or MRI. These measuring methods started within 1–2 days before the first cycle of neoadjuvant chemotherapy, before the third cycle, and after the fourth cycle. Clinical response was assessed using the response evaluation criteria in solid tumors15 and was categorized into invalid, partial response, clinical complete response (cCR), and pCR. In this study, pCR was defined such that invasive carcinoma was not found in both primary site and axillary node; however, pCR was considered if the ductal carcinoma remained in situ in the primary site.16 Toxicities were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events Version 3.0. BODY.PATIENTS AND METHODS.STATISTICAL METHODS: The primary objective of this study was to compare pCR rates between the XEC and FEC groups. Chi-square test was used in the comparison of clinical and pathological response rates and toxicities in the two treatment groups, while Fisher's exact test was used when the cell expectation was less than six. Breslow–Day test for homogeneity was performed to determine whether significant treatment by subgroup interactions existed with respect to pCR. All statistical analyses were performed using SPSS Version 20.0 (IBM Corporation, Armonk, NY, USA) with significance determined at P<0.05. BODY.RESULTS.PATIENT CHARACTERISTICS: A total of 137 patients were enrolled in this study between January 2011 and December 2013. Of these, 131 patients had clinical and radiological evaluation of response and completed surgery and were randomly assigned to the XEC (n=61) and FEC (n=70) groups. The reasons why the six subjects were withdrawn from the study were as follows: three patients refused further chemotherapy or surgery after the third cycle of FEC, one patient refused surgery after the fourth cycle of FEC, and two patients refused any further therapy after the first cycle of XEC. Patients' baseline characteristics were balanced between the two treatment arms (Table 1). Patients in each treatment arm were well matched for age, ECOG performance status, clinical T-stage, pathological pattern, clinical N-stage, estrogen receptor, progesterone receptor, and HER2 status. Invasive ductal carcinoma (n=47, 77%) was the most common pathological pattern. All patients had an ECOG performance status of 0 or 1. HR and HER2 were positive in 64% (XEC vs FEC: 64% vs 64%) and 29% (XEC vs FEC: 28% vs 30%) of patients, respectively. BODY.RESULTS.EFFICACY: Of the 131 patients evaluable for response, one patient in the XEC arm and two patients in the FEC arm did not complete treatment due to tumor progression or stable disease and received alternative chemotherapy prior to surgery. Treatment with XEC led to an increased rate of pCR (18% vs 6%, P=0.027), cCR (20% vs 7%, P=0.033), and objective remission rate (ORR; 87% vs 73%, P=0.048) compared with FEC (Table 2). No patient progressed in the XEC arm, whereas eight patients progressed in the FEC arm (13%, P=0.004). Tumor ORRs were not significantly different among the various molecular types of breast cancer between the two arms (P>0.05) as shown in Table 3. The ORR was greater in XEC than in FEC in triple-negative breast cancer (TNBC) without statistical significance (92% vs 73%, P=0.571). An exploratory analysis of pCR rate according to major subgroups (age, T-stage, N-stage, HR status, HER2 status, molecular type, and clinical response) was performed (Table 4). Although the subset analysis might not have enough power to detect the differences, XEC appeared more effective than FEC, particularly in patients with TNBC (83% vs 22%; P=0.009; interaction P=0.028). BODY.RESULTS.SAFETY: Safety was assessed in all 131 patients. The tolerance in XEC and FEC regimens was good (Table 5). In terms of 3/4 grade treatment-related clinical adverse events, there were more cases of hand-foot syndrome (HFS; 57% vs 11%, P<0.001) and 3/4 grade nausea/vomiting/diarrhea (30% vs 14%, P=0.034) but less cases of phlebitis (3% vs 14%, P=0.035) in XEC compared with FEC. The incidence of 3/4 grade leukopenia (18% vs 16%, P=0.723), arthralgia/myalgia (5% vs 8%, P=0.502), alopecia (85% vs 89%, P=0.572), and 3/4 grade mucositis (8% vs 10%, P=0.721) was lower in XEC than in FEC, but the difference between the two groups was not statistically significant. XT and T adjuvant chemotherapy regimens were well tolerated (Table 6). In terms of 3/4 grade treatment-related clinical adverse events, there were more cases of HFS (16% vs 0%, P<0.001) in XT compared to T, and the treatment-related 3/4 grade adverse events occurred in 28% and 17% of patients receiving XT and T, respectively. XT and T were associated with a similar rate of 3/4 grade leukopenia (20% vs 14%, P=0.411), 3/4 grade nausea/vomiting/diarrhea (7% vs 1%, P=0.126), 3/4 grade mucositis (3% vs 1%, P=0.922), and alopecia (100% vs 99%, P=1.000). BODY.DISCUSSION: The incidence of breast cancer has a rising tendency in developed cities in the People's Republic of China and becomes the first morbidity of malignant tumor among females.17 The treatment is conventional surgery assisted with chemotherapy and radiotherapy, while distant metastasis and local relapse are the main reasons for mortality in breast carcinoma. Neoadjuvant chemotherapy before surgery is one of the main treatments for locally advanced breast cancer. It not only narrows the primary lesion and regional LNs, degrades clinical staging, increases the chances in patients who could not be operated before, and improves the resection rate and the breast conserving rate but also controls subclinical lesions effectively, reduces tumor loading, and improves the prognosis. Furthermore, it could reduce the chances of distant metastasis by destroying distantly potential micrometastasis and provide evidence for sequential treatment through judging the sensitivity of tumors to chemotherapy.18,19 Currently, neoadjuvant chemotherapy has become a standard treatment for invasive breast cancer in stage II/III, but there is no unified standard in the specific treatment including cyclophosphamide/methotrexate/5-FU, 5-FU/adriamycin/cyclophosphamide, FEC, and docetaxel/doxorubicin/cyclophosphamide.20,21 Neoadjuvant chemotherapy has provided an opportunity for studying the biological effects of systemic treatment in breast cancer and can be used to choose effective index for predicting the prognosis in clinical settings. Early systemic chemotherapy can induce apoptosis of tumor cells and reduce the metastasis rate in surgery.22 The pathological response in the primary tumor after neoadjuvant chemotherapy is closely related to prognosis. Patients with pCR have an 86% 5-year survival rate and achieve an obvious survival benefit. Therefore, we can evaluate the effect of chemotherapy through pathological response condition of the primary tumor and predict the prognosis.18,20,23,24 Patients who responded well to neoadjuvant chemotherapy especially those with pCR, have a significantly improved disease-free survival rate.25 Capecitabine, as an antimetabolite, is a new-generation oral derivative of FU. After ingestion, it is rapidly nearly completely absorbed as an active compound in the gastrointestinal tract and is converted into 5-FU via a three-step enzymatic pathway.26,27 After two intermediate steps involving carboxylesterase in the liver and cytidine deaminase in the liver and/or tumor tissue, the final metabolite is converted into 5-FU by thymidine phosphorylase. Thymidine phosphorylase as the last enzyme in the activation of capecitabine has a higher concentration in malignant cells compared to healthy tissue, especially in breast cancer and gastric carcinoma causing DNA dyssynthesis in cancer tissue to achieve an antitumor effect, thus having selective and targeted anti-tumor effect.28 Therefore, cancer tissue can convert more capecitabine into 5-FU, with less concentration in healthy tissue, so that 5-FU does not injure the healthy tissue and has less toxic and side effects. In conclusion, capecitabine is an oral chemotherapy drug with less toxicity and has a unique advantage in the treatment of breast cancer. Patients with stage II/III breast cancer were treated prior to surgery by neoadjuvant chemotherapy, including treatment regimens XEC and FEC where both were compared for efficacy and adverse events in this study. It was concluded from the results that compared to the FEC arm, XEC significantly increased both the tumor pCR rate (18% vs 6%, P=0.027) and the clinical response rate (87% vs 73%, P=0.048). A large-scale, multiple-center clinical research reported that patients with primary or metastatic breast cancer who could not be treated with anthracycline- and/or taxol-containing regimens can be treated with capecitabine. The effective rate of single-drug first-line treatment was found to be 15%–37%, and the median progression-free survival was from 3 to 5 months.29 In the study by Kamal et al,30 257 patients with metastatic breast cancer were treated with capecitabine or taxol. The results showed that the overall survival rate and tumor specificity in patients treated with capecitabine or taxol were similar, and there was no significant difference in the survival benefit. In addition, the results from a multiple-center, randomized Phase III clinical trial showed that the effect of capecitabine was as good as the combination of vinorelbine plus gemcitabine, when compared between these drugs in patients who were treated ineffectively with anthracycline- and/or taxol-containing regimens.31 Therefore, capecitabine played an important role in both single drug and combination therapy. Capecitabine monotherapy or capecitabine-containing regimens can be used not only as first-line treatment in advanced breast cancer but also as second- or third-line treatment in locally advanced breast cancer due to its good tolerance to side effects.32–35 After success with combination therapy, sequential treatment until single-drug maintenance could prolong the survival time in advanced breast cancer patients. One research36 showed that TNBC was more sensitive to capecitabine and could achieve better pCR and cCR as neoadjuvant chemotherapy compared to non-TNBC. Other studies37,38 suggested that HR-positive tumors show resistance to chemotherapeutic drugs and were difficult to achieve pCR. In this study, the fact that 92% of TNBC achieved ORR in XEC while only 73% in FEC implied that treatment with XEC would have better curative effect to TNBC than FEC. Since there was no statistically significant difference, the result may change if the number of subjects was increased. In addition, XEC achieved more tumor pCR (ten patients, 83%) compared with FEC (two patients, 22%) in TNBC (P=0.009; interaction P=0.028). At the same time, XEC had a trend toward increased pCR in patients with HR negative and HER2 negative/unknown. Similarly, due to small sample, the result may change after increasing the number of subjects, although there was no statistically significant difference. In addition to superior efficacy, the tolerability of XEC was also compared to that of FEC. From the comparison of adverse events, the incidence rate of HFS in the XEC arm was higher (57% vs 11%, P<0.001) compared to the FEC arm. Several studies showed that the incidence rate of HFS in capecitabine-containing chemotherapy regimens was higher because of the characteristics of the drug, which was consistent with the results in this study.39,40 Severe HFS may lead to reduction in drug dosage; therefore, it is important to prevent and reduce the incidence rate and degree of this syndrome, and it is known that vitamin B6 can relieve the symptom of HFS caused by 5-FU. In this study, it is reported that oral capecitabine with a large dose of vitamin B6 (300 mg/d) can reduce the incidence rate and symptom of HFS. In addition, a retrospective study suggested that cyclooxygenase-specific inhibitor might have the same effect as the combination of capecitabine and vitamin B6, as well.41 There were more patients with 3/4 grade nausea/vomiting/diarrhea in the XEC arm than in the FEC arm (30% vs 14%; P=0.034), because capecitabine was absorbed in the gastrointestinal tract and needs to be taken orally for 2 weeks. The side effects such as phlebitis, arthralgia/myalgia, alopecia, leukopenia, and 3/4 grade mucositis were lower in XEC than in FEC. Among them, there was a statistically significant difference in phlebitis. XT and T adjuvant chemotherapy regimens were well tolerated: treatment-related 3/4 grade adverse events occurred in 28% and 17% of patients receiving XT and T, without statistical significance, respectively. This indicated that the XEC followed by XT regimen can be safely implemented because of less side effects and toxicity. BODY.CONCLUSION: This study showed that the XEC regimen was effective and had similar curative effect compared to the FEC regimen. Due to its good tolerability and low incidence and severity of preventable adverse events, XEC regimen should be promoted and implemented in clinical practice.

TITLE: Effectiveness of an Oral Health Education Program for Obstetrician/Gynecologist Residents at Tufts Medical Center ABSTRACT.AIM AND OBJECTIVES:: To assess Tufts Medical Center obstetrician/gynecologist (OB/GYN) residents' knowledge, beliefs, and previous training in oral health and to assess the effect of an oral health educational seminar on their knowledge and beliefs. ABSTRACT.MATERIALS AND METHODS:: A preseminar questionnaire was distributed to the residents. The same questionnaire was distributed immediately after the seminar and 3 months later. SPSS Version 21 was used for the data analysis. ABSTRACT.RESULTS:: Convenience sample of 25 residents were included in the study. The mean (standard deviation) age of participants was 29.08 (2.47) years. Only 1 (4%) participant reported receiving >8 h previous training in oral health and 7 (28%) reported receiving <1 h of training. The nonparametric Friedman test showed a statistically significant difference between administrations in terms of total score on knowledge-based questions (P < 0.001) and some of the belief-based questions. The post hoc Wilcoxon signed-rank test with Bonferroni correction showed statistically significant improvement in the knowledge-based questions between pre- and post-seminar questionnaire (P = 0.002) and between preseminar and 3-month follow-up (P = 0.003). ABSTRACT.CONCLUSIONS:: OB/GYN residents at Tufts Medical Center received limited training in oral health. Their knowledge improved significantly following the oral health educational seminar. Similar training modules can be brought to other OB/GYN residencies and OB/GYNs in an effort to enhance the symbiotic relationship between medical and dental professionals. BODY.I: Pregnancy is a unique period of women's life, in which unique hormonal and eating habit changes take place. Therefore, pregnant women have a higher incidence of periodontal disease, tooth mobility, tooth erosion, and dental decay.[123] Although the association between periodontal diseases during pregnancy and adverse pregnancy outcomes, including preterm birth, intrauterine growth restriction, preeclampsia, and delivery of low birth weight infants, has been reported in several studies,[456789] additional randomized clinical trials are required to confirm the association. Prenatal oral health education for pregnant mothers has shown a great impact on improving a mother's oral health and enhancing a positive attitude to their infant's oral health later on.[10] However, in a study including four states, most mothers reported that they did not receive dental care during their pregnancy, and among those who reported having dental problems, half of them did not get the dental care they needed.[11] For many women, obstetrician/gynecologists (OB/GYN) are the most frequently accessed healthcare professional. Unfortunately, a national consensus statement with the American College of Obstetricians and Gynecologists (ACOG) Committee Opinion and the American Dental Association (ADA) reported that many times, neither the pregnant women nor OB/GYN professionals understand that oral health care is an essential component of a healthy pregnancy.[12] In another study done by Curtis et al., in 2013, most of the deans and dental program directors reported limited clinical exposure to prenatal oral health during residency.[13] Limited data are available on the OB/GYNs' knowledge, attitude, practice, and training regarding oral health care during pregnancy and the association between oral health and pregnancy outcomes. In a study conducted to evaluate OB/GYNs and dentists' knowledge regarding prenatal dental care, 34% of the OB/GYNs did not know the potential association between periodontal disease and the delivery of preterm low birth weight babies.[14] Morgan et al. in 2009[15] and Wilder et al. in 2007[16] reported that most OB/GYNs recognized the importance of maintaining proper oral hygiene during pregnancy and believed that the treatment of periodontal disease during pregnancy can have positive impact on pregnancy, but limited incorporation of dental care during their prenatal care of pregnant women was reported. With the hope to start a collaborative effort among pediatric dentists and OB/GYNs for the management of oral health of their pregnant patients and to improve the OB/GYNs' oral health knowledge, the aim of our study was to assess Tufts Medical Center OB/GYN residents' knowledge, beliefs, and previous training in oral health. In addition, the study aimed to evaluate the effect of a PowerPoint oral health educational seminar on their knowledge and beliefs immediately after the educational seminar and at a 3-month follow-up. BODY.M: This study was approved by the Institutional Review Board (IRB) at Tufts Medical Center and Tufts University Health Sciences Campus (IRB # 10709). Before the scheduled oral health educational seminar, a 24-item baseline preseminar questionnaire was distributed among OB/GYN residents at Tufts Medical Center after informed consent form explaining the aim of the study was signed by each participating OB/GYN resident. The preseminar questionnaire aimed to assess the residents' knowledge, beliefs, and previous training in oral health. The questionnaire included 16 knowledge-based and eight belief-based multiple-choice questions. Knowledge-based questions were in a true/false format and aimed to evaluate the OB/GYN resident's knowledge in oral health during pregnancy. Each correct answer received a +1 score and each wrong answer received a zero score. A total was calculated for each participant and for each question. The total number and percentage of OB/GYN residents who correctly answered each knowledge-based question were calculated at before and after the educational seminar and at the 3-month follow-up. A five-point scale ranging from strongly disagree (1) to strongly agree (5) was used for the belief-based questions. Data regarding the age, gender, race, years in OB/GYN residency and the amount of previous training received in oral health were also collected. The questionnaire was developed with the aid of previous studies and from the ACOG and ADA guidelines on oral health during pregnancy.[17] Then, a PowerPoint oral health educational seminar was provided by a trained pediatric dentist resident and followed by 15-min discussion. The oral health educational seminar discussed pregnant women and infant oral health, with emphasis on oral health care, prevention, and diet during pregnancy, while promoting the concept of establishing oral hygiene regimens for the baby once the baby's first tooth erupts. Immediately following the seminar, the same postseminar questionnaire was administered to assess the effect of the oral health educational seminar on the resident's oral health knowledge and beliefs. Three months later, the same questionnaire was administered to the same residents. All participants completed the pre/post-seminar and the 3-month follow-up questionnaire. For participating in the study, each resident received a $10 gift card. The study period was from July 2013 to February 2015. SPSS version 21 (IBM SPSS Statistics 21.0) was used for the data analysis. The Friedman test was used to compare pretest, posttest, and 3-month follow-up results regarding answers to the belief-based questions as well as total score on the knowledge-based questions. The Wilcoxon signed-rank test with Bonferroni correction was used for post hoc comparisons. Statistics regarding the residents' gender, age, residency year, and previous oral health training were also calculated. BODY.R.D: A convenience sample of 25 residents participated in the study. Of the 25 participants, 23 (92%) were female and 2 (8%) were male. The mean (standard deviation) age of the participants was 29.08 (2.48) years. The plurality of participants was 1st-year residents 10 (40%). When the participants were asked about their previous training in oral health during medical school years and residency, only 1 (4%) participant reported receiving >8 h training in oral health. Six (24%) reported receiving no training and another 7 (28%) reported receiving <1-h training [Table 1]. Table 1 Demographics Characteristics of the Participants ( N =25) BODY.R.R: The participants scored the lowest on the question regarding the association between preeclampsia and periodontal disease during pregnancy; the question was answered correctly by only 6 (24%) of the participants. Only 11 (44%) of the participants knew that young children can acquire caries-causing bacteria from their mother's saliva and that intrauterine uterine growth restriction can be associated with periodontal disease during pregnancy. Twenty-four (96%) of the participants correctly answered the questions regarding dental decay being one of the most common diseases of childhood and that it is important to establish oral hygiene regimens for the baby once the baby's first tooth erupts correctly [Table 2]. Table 2 Total number and frequency of correct answers for knowledge-based questions ( n =25) The median (interquartile range [IQR]) of the preseminar, postseminar, and 3-month follow-up for correctly answered knowledge-based questions was 9 (4), 11 (2), and 11 (3), respectively. The nonparametric Friedman test showed a statistically significant difference in the total score of knowledge-based questions between the pre- and post-educational seminar and 3-month follow-up (P < 0.001). Furthermore, Wilcoxon signed-rank test with Bonferroni correction showed statistically significant improvement in the knowledge-based questions total score between pre- and post-oral health educational seminar questionnaire (P = 0.002) and between preoral health educational seminar and the 3-month follow-up (P = 0.001). The difference in the total scores between postoral health educational seminar and 3-month follow-up was not statistically significant (P = 0.270). At the preseminar, participants strongly agreed that it is important to receive routine dental care during pregnancy (median 5.0, IQR 1.0). At the 3-month follow-up, participants strongly agreed (median 5.0) when they were asked about their beliefs in the importance of routine dental care during pregnancy; periodontal disease's adverse effect on pregnancy; and including dental screening in prenatal care. The Friedman test showed a statistically significant difference in the belief-based questions regarding conducting an examination of the oral cavity during pregnancy being outside the routine practice of an OB/GYN, including dental screening as part of parental care provided for pregnant mothers, being up to date on the topic of oral health and pregnancy, and receiving adequate training concerning screening and assessment of oral health issues during medical school and residency [Table 3]. Table 3 Participant's responses to belief-based questions ( n =25) Post hoc Wilcoxon signed-rank test with Bonferroni correction showed a statistically significant difference in the question regarding participants' belief in conducting an examination of the oral cavity during pregnancy being outside the routine practice of an OB/GYN between the pre- and post-educational seminar (P = 0.008) and the preeducational seminar and the 3-month follow-up (P = 0.011). The question about including dental screening as part of parental care provided for pregnant mothers was statistically significant between the pre- and post-educational seminar (P = 0.034) and the preeducational seminar and 3-month follow-up (P = 0.0067). Statistically significant differences between pre- and post-educational seminar (P < 0.001) and preeducational seminar and 3-month follow-up (P < 0.001) were observed when participants were asked about their beliefs in being up to date on the topic of oral health and pregnancy and receiving adequate training concerning screening and assessment of oral health issues during medical school and residency. BODY.D: The aim of our study was to assess Tufts Medical Center OB/GYN residents' knowledge, beliefs, and previous training in oral health, as well as to assess the effect of a PowerPoint oral health educational seminar on their knowledge and beliefs immediately after the educational seminar and at a 3-month follow-up. At the baseline preseminar questionnaire, OB/GYN residents showed adequate knowledge about oral health during pregnancy. Most of the OB/GYN residents (84%) at Tufts Medical Center knew that periodontal disease during pregnancy is associated with preterm birth. Similar results were reported in the previous studies done by Suri et al., 2015[18] and Zanata et al., 2008.[14] Suri et al. in 2015 reported that 85.4% of OBs identified the association between periodontal disease during pregnancy and preterm delivery,[18] compared to 65.8% of the OB/GYNs in the study reported by Zanata et al., in 2008.[14] On the other hand, our study showed that 76% of the OB/GYN residents at Tufts Medical Center failed to answer the question regarding the association between periodontal disease during pregnancy and preeclampsia as compared to 62% of the OB/GYNs in a study done by Suri et al. in 2015 to evaluate OBs' knowledge, attitude, and practice in oral health and pregnancy.[18] Furthermore, a study conducted by Roche et al., in 2011 reported that 80 % of the participated OBG identified periodontal disease as a risk factor for preterm birth and low birth weight.[19] Although previous studies reported that OB/GYNs had adequate knowledge about oral health during pregnancy, most of the OB/GYNs did not apply their knowledge in their practice.[161719] Suri et al., 2015 reported that only 40% of the OB/GYNs included in the study advised routine dental visits during pregnancy and only 47% advised their patients about maintaining oral hygiene during pregnancy.[18] Wilder et al. in 2007 reported that up to 49% of the practicing OB/GYNs rarely or never recommended a dental examination for their pregnant patients.[16] In our study, while the knowledge and beliefs of the participating residents were evaluated before the educational seminar and immediately after the seminar and at a 3-month follow-up, the practical implications of the acquired knowledge were not evaluated. Most of the OB/GYN participating residents were 1st-year residents and most of them reported limited previous training in oral health. Similar results were reported by a previous national survey done by Ferullo et al., 2011 who assessed the extent of oral health education among United States medical schools. The majority of the United States medical schools reported that they offer very little oral health education to their students.[20] Another study by Morgan et al. in 2015 assessed how OB/GYNs in the United States addressed oral health during pregnancy and reported that most of the OB/GYNs responding to the mail questionnaire reported nonexistent (52%) or inadequate (33%) training in oral health during medical school and residency.[15] This emphasizes the importance of providing more oral health education to the OB/GYN residents during their education years. To our knowledge, this is the first study to evaluate the effect of an oral health educational seminar on OB/GYN residents' knowledge and beliefs after the educational seminar and at a 3-month follow-up. Although a significant improvement in the OB/GYN residents' knowledge was reported at the postseminar and at the 3-month follow-up, the long-term retention of the information and the practical implications of the acquired knowledge are unknown. Therefore, further studies, including those with a larger sample size and with long-term follow up, are recommended. Limitations of the study include the method of sampling: A convenience sample of Tufts Medical Center OB/GYNs was taken. The questionnaire was self-report, and belief-based questions may be subject to response bias. The questionnaire was not validated, and due to limited numbers of OB/GYN residents at Tufts Medical Center, the questionnaire was not pilot tested. BODY.C: Within the limitations of the study, OB/GYN residents received limited training in oral health. Their knowledge improved and their beliefs changed significantly following the oral health educational seminar immediately and at 3-month follow-up. Thus, similar training modules can be brought to other OB/GYN residencies and OB/GYNs in an effort to enhance the symbiotic relationship between medical and dental professionals. BODY.C.F: This work was supported in part by the U.S. Department of Health and Human Services Health Resources and Services Administration Grant ##D84HP19955. BODY.C.C: There are no conflicts of interest.

TITLE: The Evaluation of Otilonium Bromide Treatment in Asian Patients With Irritable Bowel Syndrome ABSTRACT.BACKGROUND/AIMS: Antispasmodics including otilonium bromide (OB) are recommended to treat irritable bowel syndrome (IBS). However, reports about OB experience in Asia is sparse. The purpose of present study was to provide the efficacy of OB in treating Asian IBS patients. ABSTRACT.METHODS: Overall, 117 IBS patients meeting Rome II criteria were enrolled in an 8-week, double-blind, active-controlled and single center trial. Randomized participants received either OB 40 mg or mebeverine 100 mg 3 doses daily. The primary endpoints were to evaluate the net changes of abdominal pain/discomfort frequency score (APDFS) and safety profile, while the secondary endpoints were to assess the changes in abdominal pain/discomfort intensity, flatulence, abdominal bloating, satisfied stool frequency etc. ABSTRACT.RESULTS: Finally, 49 OB and 52 mebeverine subjects were eligible for efficacy analysis. Compared to baselines in per protocol populations, the reduced APDFSs in OB and mebeverine were 0.55 ± 1.20 (P = 0.011) and 0.37 ± 1.11 (P = 0.042), respectively, to show similarly reduced scores. The most reported side effects included dry mouth, nausea and dizziness. Besides, the improved APDFSs at 4th week visit, final alleviations in abdominal pain intensity, flatulence, abdominal bloating and satisfied stool frequency with global assessments filled by both patients and investigators were significantly achieved by both treatments, and OB was not inferior to mebeverine in treating these parameters. ABSTRACT.CONCLUSIONS: In Orientals, OB is as effective as mebeverine for alleviating IBS symptoms in terms of abdominal pain, flatulence, abdominal bloating etc. However, obvious side effects are also observed. A large-scaled trial and post-marketing surveillance are recommended to confirm its efficacy and safety. BODY.INTRODUCTION: Irritable bowel syndrome (IBS) refers to a functional gastrointestinal (GI) disorder characterized by the abdominal pain/discomfort that is associated with disturbed bowel movement (BM) in terms of stool frequency and consistency.1,2 Currently, the true IBS pathophysiology remains enigmatic since many mechanisms have been addressed but not well agreed. For example, the most mentioned mechanisms include GI dysmotility, autonomic nervous dysfunction, visceral hypersensitivity, gut immune dysfunction, neuropeptide receptor dysfunction, brain-gut dysfunctional linkage, genetic polymorphism, psychological disorders etc.3-7 Clinically, IBS per se has a profound impact on the living and quality of life leading to the excessive use of medical resources.2,8-10 The up-to-date IBS treatment strategy recommends a positive diagnosis, consideration of the patients' agenda and emotional state, continuous care and evaluations on graded therapeutic responses.1,3,6,10,11 Unfortunately, many available treatments are not globally agreed and accepted between countries, medical payers and patients, while IBS subjects are often not satisfactory to those treatments.5,11,12 Newly developed drugs targeted on receptors are emerging, however, current IBS guidelines still recommend antispasmodics in diminishing pain/discomfort severity although these agents have been launched for decades.2,3,5,6,10,13 Meta-analysis indicates again that smooth muscle relaxants are effective in reducing abdominal pain and global symptom compared to the placebo.14 Among the antispasmodics, otilonium bromide (OB) is claimed to reduce the IBS pain severity effectively.3,10,12 Pharmacologically, OB is one of the quaternary ammonium derivatives with GI smooth muscle spasmolytic activity via inhibition of calcium ion influx through L-type voltage operated calcium channels.15,16 European studies already confirmed OB in relieving IBS pain/discomfort.17,18 Interestingly, reports on its experiences in Asia are not existed. Therefore, we conducted a single center, double-blind, randomized, active drug-controlled trial in looking its efficacy. The primary endpoints were to evaluate the improvement of abdominal pain/discomfort frequency score (APDFS) and drug safety under an 8-week OB treatment. The secondary endpoints were to assess the OB efficacies by (1) the net change of the 4th week evaluation compared to the baseline APDFS, (2) the net change of the 8th week evaluation compared to the baseline abdominal pain/discomfort intensity severity, (3) the changes of other IBS parameters including flatulence, abdominal bloating and satisfied stool frequency and (4) the global assessments after treatment filled by both the investigators and patients. BODY.MATERIALS AND METHODS.ENROLLED SUBJECTS: This study was conducted between November 2003 and January 2006. Briefly, eligible subjects aging 20-80 year who presented with bowel symptoms fulfilling Rome II criteria in the Outpatient Department of Taipei Veterans General Hospital were consecutively invited and considered to participate in this trial.19 Alarm symptoms and signs such as fever, bloody stool, persistent diarrhea, marked body weight loss and anemia were excluded. Colon imaging study using either barium enema or colonoscopy within the previous year should be normal in the subjects (age ≥ 40 years). Other criteria of exclusion included pregnant or breastfeeding women, history of malignancy within 5 years, gut surgery except appendectomy, malabsorption diseases, hyper- or hypothyroidism, abnormal renal or liver function tests, inflammatory bowel diseases, connective tissue diseases, severely progressive diseases, diabetes, obvious psychiatric disorders, substance abuse, unable to discontinue drugs known to influence gut motility, using iron supplements or colchicine, concurrently participating in another study etc. This study was approved by the Institutional Review Board of Taipei Veterans General Hospital and Department of Health, ROC, while informed consent was obtained from all participants prior to the recruitment. BODY.MATERIALS AND METHODS.STUDY DESIGN: This study was designed as a double-blind, randomized, parallel and comparative trial. We expected to assign about 40 assessable subjects to each of 2 study arms. Under consideration of probable withdrawal, at least 100 IBS subjects (50 in each arm) were planned to enroll. During the screening visit (14 days prior to the first treatment day), demographic data, vital signs, general physical examinations, concomitant medications, medical history, concurrent diseases, laboratory and pregnancy tests and current IBS symptoms were obtained. After this visit, all the prohibited drugs were held at least 1 week prior to the randomization. On day 1 of visit 2, treatment assignment was based on pre-generated permuted block randomization scheme. The block size of four was used to enroll subjects with a 1:1 ratio of the OB arm to controlled mebeverine arm. Clinical evaluation was performed again to confirm the eligibility and to record the IBS symptomatic baselines. After randomization, the eligible subjects were provided with a patient diary record and were treated using either OB or mebeverine for a total of 8 weeks in a double-blind method. They were instructed to come back for the 3rd visit at the end of 4th week (within ± 5 days treatment window) after randomization. Similar clinical evaluation was performed to assess their treating efficacy and safety. Patient's diary record was collected and a new card was given again. Unused study medications were collected to document drug accountability. Each subject was prescribed with the similarly coded medications for another 4-week treatment period. Final visit was performed at the end of 8th week after randomization. The efficacy and adverse events were recorded again. The global assessments filled by the investigators and patients were acquired. Consumption of concomitant medications during the entire study period was documented. Laboratory and pregnancy tests were repeated to confirm the study safety. BODY.MATERIALS AND METHODS.STUDY DRUGS AND RESCUE AGENTS: A double-blind and double-dummy method was used in this study to achieve the double masking. For instance, OB active tablet (40 mg, batch no. ML157011; YL115128/YL115129) and its matching placebo tablet were prepared to be identical in all aspects. While mebeverine HCl active tablet (100 mg, bath no. 310991) and its matched placebo tablet were similarly manufactured to be unidentifiable. Subjects in the OB arm were instructed to consume one tablet of OB plus 1 tablet of mebeverine placebo simultaneously 3 times daily 30 minutes before the meals. Subjects assigned to the mebeverine arm were instructed to consume one mebeverine tablet plus one OB placebo tablet 3 times daily before the meals. All the study products and placebo were supplied under the responsibility of TTY Biopharm Co, Ltd (Taipei, Taiwan). The frequency to supply study drugs to the study site was adapted to the expiry date of products. Rescue agent such as loperamide one tablet daily was allowed to the subject who suffered from intractable diarrhea (> 3/day for 3 days) during the trial. On the other hand, bisacodyl 2 tablets daily was allowed for subject who suffered from annoying constipation (no BM > 3 days). BODY.MATERIALS AND METHODS.SYMPTOMATIC ASSESSMENTS AND SAFETY RECORDING: The primary efficacy variable was to measure the change in APDFS after 8 weeks of treatment. Based on the diary card, the APDFS was categorized into four grades: score 0 = none; score 1 = ≤ 3 episodes per week; score 2 = 4-7 episodes per week; and score 3 = > 7 episodes per week. The APDFS scored at visit 2 served as baseline and was re-evaluated at the ends of 4th and 8th week after treatment, respectively. The abdominal pain/discomfort intensity was subjectively scored into four categories: score 0 = absent; score 1 = mild; score 2 = moderate; and score 3 = severe. Besides, a 10 cm visual analog scale (VAS) to record grades of flatulence, abdominal bloating and satisfied stool frequency by the subjects was instructed by oral and written information before the assessment. The 0 cm end means "no symptom" or "normal," whereas the 10 cm end indicates the "worst bowel symptom" or "most intolerable." Abdominal pain/discomfort intensity and VAS scores at visit 2 served as the baselines and the differences of pre- and post-treatment VAS scores at final visit were used for the secondary endpoints. At the final visit, subjects and investigators completed a global assessment to the overall responses after trial, respectively. The global assessment was a 5-point scale, using the following definition: 0 = worsened, 1 = no change, 2 = slightly improved, 3 = improved and 4 = significantly improved. Throughout the study, the investigators closely monitored and recorded the probable occurrences of any adverse event (AE) after visit 2. All the AEs that occurred in subjects who took at least 1 dose of study medication were recorded in detail. The relationship of AE to study medication was also defined. BODY.MATERIALS AND METHODS.STATISTICAL METHODS: All subjects who had consumed at least one dose of study medication and had at least one post-treatment evaluation on the primary endpoints were included in the intent-to-treat (ITT) population analysis. Furthermore, the per-protocol (PP) population consisted of all ITT subjects who did not take any violated medications and had completed at least 28 days of treatment. The primary endpoint was APDFSdifference or APDFS at baseline APDFS at 8th week. The primary hypothesis was: H0: μt-μs ≤ -0.35 Ha: μt-μs > -0.35. While H0 means null-hypothesis and Ha means alternative-hypothesis; μt means the APDFSdifference of the net OB response and μs denotes the APDFSdifference of the net mebeverine response. Treatment arm was declared as non-inferior if the difference in lower limit of 97.5% one-sided confidence interval (CI) calculated by either t test or Wilcoxon rank sum test between treatments was greater than -0.35. Results were expressed as mean ± SD or median with inter-quartile range (IQR) when appropriate. Categorical difference was based on chi-square test or Fisher's exact test when appropriate. The net changes of various parameters used for secondary endpoints were analyzed using either t test or Wilcoxon rank sum test to compare between treatments. Intra-assay difference was calculated using Wilcoxon signed rank test. Global assessment by the investigators and subjects was analyzed using Mantel-Haenszel test to compare the differences between treatments. All the statistical tests were 2-tailed and the P-value less than 0.05 was considered significant. BODY.RESULTS: Totally, 132 IBS subjects fulfilling Rome II criteria gave informed consent and were screened, and only 117 subjects were eligible to be randomized and treated (Figure). All these randomized subjects had consumed at least 1 dose of study medication. However, 16 subjects had violated ITT criteria and only 101 subjects were enrolled in the ITT population including 49 in OB arm and 52 in mebeverine arm. Twenty subjects in the ITT population were excluded from the PP population based on our definition. Finally, 81 subjects (38 OB:43 mebeverine) were eligible in the PP population. Table 1 depicts the demographic characteristics and baseline IBS symptoms of both arms. There were no differences in terms of age, gender, body mass index, IBS duration, proportions of IBS subtypes, abdominal pain/discomfort severity etc. Overall, the treatment compliances of OB and mebeverine arms were 88.9% ± 13.0% and 90.4% ± 11.6%, respectively (NS), while the mean daily doses of using study drugs were 2.60 ± 0.47 and 2.66 ± 0.44 units, respectively (NS). On the other hand, the numbers of patient who consumed rescue agent at least once during the trial were 49 (100%) and 51 (98.1%), respectively (NS). The total numbers of consumed loperamide among both arms were 2.89 ± 0.32 and 3.05 ± 0.53, respectively (NS), whereas the numbers of consumed bisacodyl were 3.13 ± 0.97 and 2.93 ± 0.26, respectively (NS). Briefly, about 40 % of subjects had APDFS improvement under either OB or mebeverine treatment based on ITT and PP populations (Table 2). Likewise, the mean reduced APDFS scores in the OB arm were 0.39 ± 1.32 (P = 0.056) and 0.55 ± 1.20 (P = 0.011) based on ITT and PP populations, respectively. However, the net APDFS reduced scores in the mebeverine arm were 0.40 ± 1.16 (P = 0.023) and 0.37 ± 1.11 (P = 0.042) based on ITT and PP populations, respectively. None of the improved data showed statistically significant differences between 2 treatments. The lower bound of 95% CI of treatment difference was over the hypothesized set margin (-0.35) in both ITT and PP populations. Accordingly, we concluded that OB was at least comparable to mebeverine in reducing APDFS at final visit. Regarding the drug safety, 118 AEs had been reported including 65 in OB and 53 in mebeverine. Among them, dry mouth was the most reported event in both treatments followed by nausea and dizziness particularly in OB treatment arm (Table 3). Most AEs were mild to moderate in nature (89.2% in OB; 88.7% in mebeverine), whereas 3 subjects (5.1%) in OB and 5 subjects (8.6%) in mebeverine were withdrawn owing to the intolerable AEs. Besides, 2 OB subjects (1 renal stone; 1 coronary heart disease) and 2 mebeverine subjects (1 amnesia; 1 herniated inter-vertebral disc) were hospitalized during the trial. All these serious AEs judged by the investigators were unlikely related to the study. On regarding the changes in terms of hematology, biochemistry, vital signs and physical examinations of 2 arms, there were no differences compared to the baselines. With regard to the 2nd endpoints, Table 4 illustrates the net APDFS changes at 4th week evaluation. The mean APDFS reduced scores for OB and mebeverine were 0.12 ± 1.32 (P = 0.545) and 0.31 ± 1.08 (P = 0.072), respectively, based on ITT populations without differences between 2 arms. The mean changed scores of PP populations were 0.24 ± 1.36 (P = 0.276) and 0.33 ± 1.04 (P = 0.70), respectively, without differences between 2 arms. The net changes of the patient numbers at final evaluation on the abdominal pain/discomfort intensity are illustrated in Table 5. Overall, the mean reduced intensity scores in OB arm were 0.41 ± 0.57 (P < 0.001) and 0.45 ± 0.60 (P < 0.001), and the mean reduced intensity scores in mebeverine arm were 0.33 ± 0.62 (P = 0.001) and 0.30 ± 0.60 (P = 0.004), respectively, based on ITT and PP populations. None of the above data showed statistical difference between 2 arms. Other changes of IBS symptoms in terms of flatulence, abdominal bloating and satisfied stool frequency are depicted in Table 6. Briefly, these evaluated bowel symptoms had marked improvement after both treatments, on either ITT or PP analysis, while the changes in abdominal bloating and satisfied stool frequency were similar in both groups (data were not shown). Table 7 denotes the global symptom assessments filled at final visit. Regarding ITT population, the proportions of at least slight improvement of OB and mebeverine evaluated by the investigators were 87.8% and 76.9%, respectively (NS), whereas those of PP population were 86.8% and 74.4%, respectively (NS). By the patients, the proportions of subjects with at least slight improvement of OB and mebeverine were 89.8% and 78.8% (P < 0.05), respectively, in the ITT populations and 86.8% and 79.1% (P < 0.05), respectively, in the PP populations. BODY.DISCUSSION: Mebeverine has long been marketed in Asia as an antispasmodic to treat IBS for decades. Using it as an active-controlled agent, our study based on the primary endpoint mainly indicated that OB was as effective as mebeverine in improving the APDFS of represented IBS main symptoms during an 8-week period treatment. Our previous study pointed out that mebeverine was effective in reducing BM frequency and stool consistency for the diarrhea-predominant IBS patients but no obvious effect for abdominal pain.20 Besides, a meta-analysis also indicates that mebeverine has no global effect to treat IBS abdomen pain compared to the placebo although it is tolerable without obvious side effects.21 It is believed that placebo does exhibit an amazing efficacy to treat IBS patients with range of 20%-50%.3,22 Accordingly, an agent shown superior to placebo in treating IBS should be obtained from a large-scaled study or meta-analysis, eg, the tegaserod trials.23 Owing to the comparable efficacy to placebo, it is likely that many mebeverine studies including our previous trial do not confirm its perfect efficacy to treat abdominal pain, one of the IBS main symptoms. Since abdominal pain/discomfort has been a very subjective complaint to IBS patients, it would be difficult to quantify it using any reliable parameters.2 In addition, as the abdominal pain scales in many trials are often recorded by the recalled manner, it is unknown whether any pain character recording does reflect the true sensation on the assigned visits after several days or weeks. In order to diminish this drawback, we employed the APDFS based on recorded pain frequency in the distributed diary card and tried to describe the pain character more objectively for the primary endpoint. Based on APDFS, our primary objective did confirm a fair but not perfect efficacy of OB comparable to mebeverin in reducing the IBS abdominal pain frequency. In addition, another parameter of abdominal pain intensity score also confirmed the at least equal OB efficacy to mebeverine. The rationale of using antispasmodics to treat IBS is likely based on the observations of disturbed GI motility among these patients.2,4,12,24 Actually, bowel dysmotility does result in functional abdominal pain among the IBS patients.3,25 Unfortunately, the central sensation of visceral pain is not uniquely related to the disturbed GI motility. It means that many biopsychosocial factors like mind, environmental factors, visceral hypersensitivity and brain-gut interaction may modify the final pain perception for the patients with functional GI disorders including IBS.1,26 Because of the excessive confounding factors in modifying the central pain sensation with heterogeneity in published studies, many antispasmodic trials cannot conclude a perfect efficacy to treat the abdominal pain, while the comparable placebo does achieve a similar efficacy. Sometimes, only the meta-analysis can summarize its possible efficacy.3,14 Among researches on the OB efficacy on IBS patients, an early small-scaled study conducted in Italy indicated that OB did reduce the abdominal pain and bloating but no differences were observed from placebo.27 Consequently, a large-scaled placebo controlled trial among the Italian IBS patients indicated that the reduced episode of abdominal pain was higher in OB treated patients (55.3%) than in placebo group (39.9%), while the fair efficacies were observed for the abdominal distension and general well-being among those on OB treatment.17 Nevertheless, the authors did not particularly address the probable side effects in that trial. A review based on 4 OB trials then concluded that OB may relieve IBS symptoms with a relative risk of 0.55 (95% CI, 0.310-0.097) over placebo.28 Currently, OB has been recommended as an effective and safe agent to treat IBS abdominal pain and distension, particularly in reducing diarrhea, whereas the most addressed side effect is urticaria.29 In addition to the reduction in IBS abdominal pain, our OB study did confirm the similar efficacies including improved abdomen bloating, flatulence, satisfied BM frequency and global assessments. Apart from blocking the calcium ion influx through L-type voltage operated calcium channels in GI smooth muscle cells, OB pharmacologically inhibited central/peripheral tachykinin-2 receptor which not only showed antispasmodic activity but also reduced the afferent transmission of sensory signals from the periphery to central nervous system.15,30 It is of interest whether this tachykinin receptor blocking ability in the afferent transmission reduce the IBS pain severity or not. In fact, a clinical trial did confirm that OB enhanced the perceptual threshold of IBS subjects during the anorectal balloon distension.31 Since visceral hypersensitivity has been one of the main components of the IBS pathogeneses,2,3 OB is therefore possible to treat the IBS main symptom apart from its antispasmodic effect. In addition, OB also shows a high affinity in binding to various muscarinic receptor subtypes in terms of M1, M2, M3, M4 and M5, respectively.32,33 Owing to its potent muscarinic blockade, OB not only exhibit an antispasmodic but also antisecretory ability through M3 sub-receptor which is abundantly found in human colonic crypt cells to mediate secretion coupled with calcium channels.33 It is why OB can improve the stool consistency.18,29 Furthermore, both M1 and M3 sub-receptors have been the main targets of nervous impulse to mediate salivary gland secretion.34 Because OB also targets on these muscarinic sub-receptors, the most observed AE of dry mouth among our trial was likely the unexpected OB effect on the salivary glands. Antimuscarinic drugs used to treat overactive bladder are occasionally associated with central nervous system side effects, eg, cognitive dysfunction, dizziness, fatigue etc.35 Nausea and dizziness reported under OB treatment were suggested to result from the central antimuscarinic effect. However, other trials did not report these side effects. The limitation of our OB study has been the single center and small-scaled trial. Usually, the small-scaled study may have inadequate sample size and study power to achieve an expected result, whereas single center trial sometimes leads to study bias.36 Clinically, functional GI disordered subjects are often not very satisfactory to the recommended treatments because of the associated somatic complaints, functional disturbances existed in other organ systems, psychiatric disorders and social impacts.2,3,13,37 Our enrolled subjects almost had consumed rescue agents during the trial. It is likely that they were very to concerned about the abnormal BM and had no confidence in relieving the abnormal BM activity even been informed of an active treatment. Since this is the first OB trial conducted in Asia, it remains uncertain whether OB could achieve an ideal efficacy to treat IBS patients comparable to that of European study.17 Concerning its major antimuscarininc side effects, a large-scaled trial and post-marketing surveillance are recommended based on our preliminary results. In conclusions, OB effectively improved the IBS symptoms in terms of abdominal pain, flatulence, abdominal bloating, satisfied stool frequency and global assessment in Asian patients. Unfortunately, obvious side effects like dry mouth, nausea and dizziness were also observed. A large-scaled trial and post-marketing surveillance are recommended to confirm its true efficacy and safety.

TITLE: To Evaluate the Efficacy of Topical Propolis in the Management of Symptomatic Oral Lichen Planus: A Randomized Controlled Trial ABSTRACT.INTRODUCTION:: Lichen planus (LP) is a chronic inflammatory, autoimmune, mucocutaneous disease of unknown etiology. The first line of treatment for oral LP (OLP) has been corticosteroids, but because of their adverse effects, alternative therapeutic approaches are being carried out, of which the recent natural alternative is propolis. ABSTRACT.AIM:: This study aims to evaluate the efficacy of topical propolis in the management of OLP. ABSTRACT.MATERIALS AND METHODS:: The research group consisted of 27 patients diagnosed with symptomatic OLP, among which 15 patients were in the control group and the rest 12 were in the study group. The patients in the control group received triamcinolone acetonide 0.1% (topical application) while the patients in the study group received propolis gel. Both the groups were evaluated for pain and erythema at baseline (1st visit), first follow-up (7th day), and second follow-up (14th day) using numerical rating scale and modified oral mucositis index. ABSTRACT.RESULTS:: The patients in both the study and control groups showed a statistically significant reduction (P = 0.000 for the study group and P = 0.000 for the control group) in pain and erythema scores from baseline to second follow-up visit. However, on comparison of the reduction in pain and erythema scores between the two groups, the difference was found to be statistically insignificant (P = 0.255). ABSTRACT.STATISTICAL ANALYSIS USED:: Chi-square and Cramer's V test were used. ABSTRACT.CONCLUSION:: The topical propolis was found to be of comparative effectiveness with respect to triamcinolone acetonide 0.1% in the management of OLP. BODY.INTRODUCTION: The mouth is a mirror of health or disease, a guardian, or early warning system. The oral cavity is considered as a window to the body because oral manifestations accompany many systemic diseases. In many instances, oral involvement presages the appearance of other symptoms or lesions at other locations.[1] Oral lichen planus (OLP) is a chronic inflammatory, autoimmune, mucocutaneous disease of unknown etiology. The strange name of the condition was provided by the British physician Erasmus Wilson, who first described it in 1869. He named it so as the lesions on the skin looked similar to the tree mosses growing on the rocks. In Greek, "lichen" means tree moss, and in Latin, "planus" means flat.[2] As the exact causative factor for OLP is a matter of conflict, the failure to achieve appropriate or exact treatment for it may be the reason for its incomplete regression. The first line of treatment for OLP has been corticosteroids,[3] but because of their adverse effects, alternative therapeutic approaches are being carried out. Recently, use of natural drugs, such as propolis, has gained considerable interest. It is a sticky, resinous substance which is collected by the honey bees from the sap, leaves, and buds of plants, and then mixed with secreted beeswax.[4] It has been used in folk medicine for thousands of years and is also known as Russian penicillin.[5] Propolis being extremely high in bioflavonoid content has antioxidant, antibacterial, antifungal, antiviral, immunomodulatory, and anti-inflammatory properties.[4] These properties have prompted investigators to check its efficacy on various oral diseases, namely lichen planus (LP), oral candidiasis, recurrent aphthous stomatitis, radiation mucositis, denture stomatitis, and herpes labialis.[6] A study conducted in the past has obtained favorable results in the management of OLP using topical propolis. With this background in mind, this study was designed to evaluate the efficacy of topical propolis in the management of OLP and to further strengthen the previously obtained results. BODY.MATERIALS AND METHODS.SOURCE OF DATA: The study participants comprised of dental outpatients visiting the Department of Oral Medicine and Radiology, JSS Dental College and Hospital, Jagadguru Sri Shivarathreeshwara University, Mysore. BODY.MATERIALS AND METHODS.METHOD OF COLLECTION OF DATA: The study sample was collected through purposive sampling. Twenty-seven individuals of either gender, satisfying the following eligibility criteria, and those willing to participate in the study were selected for the study. BODY.MATERIALS AND METHODS.INCLUSION CRITERIA: Patients with clinically diagnosed atrophic/erosive OLP(based on modified WHO clinical criteria, 2003)[7]Individuals willing to be a part of the study, who sign the informed consent form, and who find it convenient to appear for follow-ups as required by the study.Patients who had not used systemic or topical glucocorticosteroids for at least past 2 weeksPatients who agree not to use any other medication such as analgesics and anesthetics in either topical form or systemic form during the study. BODY.MATERIALS AND METHODS.EXCLUSION CRITERIA: Patients not willing to be a part of the studyPatients with lichenoid lesions thought to arise as a hypersensitivity reaction to drugs and dental materialsPatients on long-term glucocorticosteroid therapyPregnant and lactating female patientsPatients allergic to bee products. The patients were informed about the study parameters, and signed informed consent was taken. The patients in the control group received triamcinolone acetonide 0.1% while the patients in the study group received 5% propolis. Patients in both the groups were instructed to apply the paste on the lesion three times a day for 15 days and were asked to refrain from eating, drinking, and rinsing for at least 30 min after topical application. Symptom score for OLP was considered at baseline using numerical rating scale (NRS) ranging from 0 (no oral discomfort) to 10 (worst imaginable oral discomfort) [Figure 1].[8] Figure 1Numerical rating scale The clinical signs of OLP were measured at baseline using a semi-quantitative scale, modified oral mucositis index (MOMI), validated for measurement of clinical signs of OLP. An oral examination was conducted and atrophic and erosive changes were quantified based on severity and the number of sites involved. An intensity score for erythema ranging from 0 to 3 was used: 0 = normal1 = mild erythema2 = moderate erythema3 = severe erythema. The score for ulcerations was based on area of ulceration: 0 = no ulcerations1 = between 0 and 0.25 cm2 2 = between 0.25 and 1 cm2 3= ≥1 cm2. The following clinical parameters were assessed during follow-up: Pain: NRSErythema and ulceration: MOMISide effects. The patients were followed up after 7 and 15 days. Readings for all clinical parameters for each patient from baseline to subsequent visits were recorded. The patients were inquired for side effects if any. The data were entered into the computer using Microsoft excel and were analyzed using SPSS software for windows (IBM Corp. Released 2013. IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp). The data were tabulated and subjected to the following statistical analysis, i.e., the Cramer's V and Chi-square test. P < 0.05 was considered as statistically significant. BODY.MATERIALS AND METHODS.PREPARATION OF PROPOLIS: Preparation of propolis was done in the Department of Pharmaceutics, JSS College of Pharmacy, Mysore. BODY.MATERIALS AND METHODS.PREPARATION OF PROPOLIS.CHEMICALS USED: Ethanol, starch powder, carbopol 934, triethanolamine (TEA), methylparaben, propylparaben, and peppermint oil. BODY.MATERIALS AND METHODS.PREPARATION OF PROPOLIS.EQUIPMENT USED: Digital weighing balance, Magnetic stirrer, and Propeller mixer. The propolis was cleaned using sterile hand gloves and was cut into small pieces, and 400 g of cleaned propolis was considered for the present study. A sterilized 1000 ml beaker was filled with about 500 ml of absolute alcohol and approximately 400 g of propolis was added. It was then covered with aluminum foil which was kept in a warm dark place for 7 days to achieve complete extraction. After 7 days, the contents in the conical flask were filtered using filter paper, and the solution was collected in a clean beaker. It was then subjected to evaporation using the magnetic stirrer to remove excess of solvent. The temperature was maintained at 40°C. The resultant thick dark brown-colored liquid of 100 ml was collected in a clean beaker, and 500 gm of starch (99% pure) was added to remove the stickiness as well as to transform the extract to a powder form. Then, 1% carbopol solution (2 g in 200 ml of water) was prepared separately in a sterilized beaker to which 0.40 g of methylparaben, 0.30 g of propylparaben, and 0.75 ml of peppermint oil were added and stirred continuously to get a homogeneous mixture. The starch-mixed propolis powder extract was then added to the beaker and homogenized using propeller mixer at 250 rpm for 15 min. After that, 1 ml of TEA (neutralizing agent/thickening agent) was added to thicken the solution into a gel of desired consistency. Finally, the preparation was packed in the preweighed sterilized aluminium tubes and sealed. It was packed in such a way that 1 aluminium tube contains 25 gm of the formulation and 1 g of the formulation consists 0.2 g of the extract. Therefore, each aluminium tube consists of 5 gm of propolis extract. BODY.RESULTS: Of the 27 patients enrolled in the study, 15 were in the control group and 12 were in the study group. The patients in the control group received triamcinolone acetonide 0.1%, and the patients in the study group received propolis. They were instructed to apply the paste on the lesion three times a day for 15 days and were asked to report on the 7th and the 15th day. Demographics and clinical characteristics are shown in Table 1 and Graph 1. There were no significant differences between the two groups with regard to age, sex, clinical characteristic, pain, and erythema scores at baseline. Table 1 Demographic data of the study participants and patient characteristics Graph 1Change in pain scores from baseline to first follow-up, first follow-up to second follow-up visit, and baseline to second follow-up visit in the study and control groups The patients in both the groups, i.e., the study group and the control group reported a complete reduction in the intensity of pain at the second follow-up visit [Table 2]. An overall statistically significant improvement in the pain scores was found from baseline to second follow-up visit in both the groups (P = 0.000), but no significant differences were observed between the two groups (P = 0.255). Table 2 Comparison of distribution of individuals according to the improvement in pain scores from baseline to second follow-up visit in the study and control groups In the control group, all the patients reported complete resolution of erythema at the second follow-up visit [Table 3 and Graph 2]. An overall statistically significant improvement was found in the erythema scores in the control group patients from baseline to second follow-up visit (P = 0.000). Table 3 Comparison of distribution of individuals according to the improvement in erythema scores from baseline to second follow-up visit in the study and control groups Graph 2Change in erythema scores from baseline to first follow-up, first follow-up to second follow-up visit, and baseline to second follow-up visit in the study and control groups Among 12 patients in the study group, 11 patients reported complete resolution of erythema and 1 patient had a score of 1 (mild erythema). There was an overall improvement in the erythema scores which was statistically significant [Table 2]. However, no significant differences were observed between the two groups (P = 0.255). The patients in the study and control group showed a statistically significant reduction (P = 0.000 for the study group and P = 0.000 for the control group) in the pain and erythema scores from baseline to second follow-up visit. However, on comparison of the reduction in pain and erythema scores between the two groups, the difference was found to be statistically insignificant (P = 0.255) [Table 4a and b]. In other words, an intragroup analysis showed significant differences in each group, but the intergroup analysis did not show any significant differences. Table 4a Comparison of baseline, first, and second follow-up values for various parameters of study and control groups Table 4b Comparison of baseline, first, and second follow-up values for various parameters of study and control groups BODY.DISCUSSION: LP is a chronic inflammatory, autoimmune, mucocutaneous disease of unknown etiology. As the exact causative factor for OLP is a matter of conflict, the failure to achieve appropriate or exact treatment for it may be the reason for its incomplete regression. The first line of treatment for OLP has been corticosteroids, but because of their adverse effects, alternative therapeutic approaches are being carried out.[3] Recently, use of natural drugs, such as propolis, has gained considerable interest. It is a sticky, resinous substance which is collected by the honey bees from the sap, leaves, and buds of plants, and then mixed with secreted beeswax. It has been used extensively in ayurvedic medicine for centuries, as it has a diversity of therapeutic properties including antioxidant, anti-inflammatory, antibacterial, antiviral, antifungal, antitumor, and immunomodulatory effect.[4] These properties have prompted investigators to check its efficacy on various oral diseases, namely LP, oral candidiasis, recurrent aphthous stomatitis, radiation mucositis, denture stomatitis, and herpes labialis.[9] The role of the immune system as a primary factor in the pathogenesis of LP has become established in recent years. It is attributed to the basal layer degeneration and band-like infiltration of T lymphocytes and macrophages. T helper 1 and T helper 2 are the well-known independent subdivisions of T cells. Recently, a third "T helper" subdivision has been recognized, which plays a principal role in defense against extracellular pathogens. This subdivision of T cells controls immune and inflammatory responses through secretion of cytokines such as Interleukin 17. This family of T cells provides a new route for cooperation between innate and acquired immunity. The major role of IL-17 is to increase the expression of threatening factors for colony chemokines, metalloproteinase, and IL-6. Therefore, IL-17 is a strong stimulator for recalling, activating, and immigration of neutrophils, production of INF-alpha, IL-B from macrophages, and recalling eosinophils.[10] In a study by Zenouz et al., it was proven that propolis administration significantly decreased IL-17 serum levels, VAS means, and the maximum lesion sizes in patients with symptomatic OLP.[11] A study conducted by Zyada et al. also obtained favorable results in the management of OLP using topical propolis.[12] Hence, we hypothesized that propolis could minimize the underlying inflammatory mechanism by lowering the levels of IL-17, thereby preventing the destruction of the basement membrane by the lymphocytes. With this background in mind, this study was designed to evaluate the efficacy of topical propolis in the management of OLP and to further strengthen the previously obtained results. The demographic pattern and clinical profile of OLP patients were also recorded in our study. According to our study, the patients were in the age group of 28–60 years and their mean age was 45.3 years which was almost similar to the study conducted by Tak, Gumru, Mostafa, and Chitturi. The study conducted by Chitturi et al. comprised of 58 patients from the age group of 11–70 years and their mean age was 45.72 years.[13] According to the study conducted by Mostafa and Ahmed, Tak and Chalkoo, and Gümrü, the mean age was found to be 48 years, 43 years, and 49.8 years, respectively.[141516] In the present study, the mean age was 45.3 years which was more than the mean age reported by Keshari et al. and Munde et al. and lower than the mean age reported by Ingafou et al., Gandolfo et al., and Xue et al. This variation might be due to the difference in ethnicity and geographic locations.[1718192021] The mean age group reported by Keshari et al., Munde et al., Ingafou et al., Gandolfo et al., and Xue et al. was 39.9 years, 36.9 years, 50.4 years, 52 years, and 56.7 years, respectively. According to our study, no sex predilection (male: female – 1.08:1) was noted which was similar to the studies conducted by Chitturi et al., Ingafou et al., Lacy et al., and Anjum et al. where the male to female ratio was found to be 1:1.[2122] Many studies claim that OLP is more predominant in females as they are more prone to stress and hormonal imbalance. However, in our study, we observed an equal distribution between males and females and this might be due to the small sample size as compared to other studies. In the studies conducted by Munde et al., Tak and Chalkoo, and Chitturi et al., the most common symptomatic form of LP was the erosive form. However, in the present study, the atrophic form was found to be more prevalent which is similar to a study conducted by Keshari et al. This variation could be due to the smaller sample size.[131518] The most common site where OLP was seen in our study was in the buccal mucosa, followed by gingiva. This is in accordance with the studies conducted by Tak and Chalkoo, Munde et al., and Mostafa and Ahmed.[141519] The study conducted by Chainani-Wu et al. has reported that more than one mucosal surface can be involved which is in accordance with our study wherein a combination of gingiva and buccal mucosa was seen.[23] In the present study, the patients in both the groups, i.e., the study group and the control group reported a complete reduction in the intensity of pain at the second follow-up visit. An overall statistically significant improvement in the pain scores was found from baseline to second follow-up visit in both the groups, but no significant differences were observed between the two groups. In other words, an intragroup analysis showed significant differences in each group, but the intergroup analysis did not show any significant differences. In the control group, all the patients reported complete resolution of erythema at the second follow-up visit. An overall statistically significant improvement was found in the erythema scores in the control group patients from baseline to second follow-up visit [Figure 2a–c]. Figure 2(a) Patient with atrophic lichen planus at baseline visit included in the study group. (b) Reduction in severity of erythema following treatment with propolis at first follow-up visit. (c) Further reduction in erythema at the second follow-up visit. (d) Patient with atrophic lichen planus at baseline visit included in the control group. (e) Reduction in severity of erythema following treatment with triamcinolone acetonide 0.1% at first follow-up visit. (f) Further reduction in erythema at the second follow-up visit Among 12 patients in the study group, 11 patients reported complete resolution of erythema and 1 patient had a score of 1 (mild erythema). There was an overall improvement in the erythema scores which was statistically significant. However, no significant differences were observed between the two groups [Figure 2d–f]. The result of our study is comparable to the study conducted by Zydaa et al. to evaluate the efficacy of propolis in the management of LP. They also proved that propolis showed to be a promising pharmacological agent for inhibiting epithelial cell proliferation and has anti-inflammatory effect.[12] According to our study, propolis is comparative in its efficacy to corticosteroids. It must be enunciated that topical propolis does not accord any adverse effects, unlike topical corticosteroids. Chiefly, the fatalistic effects demonstrated in topical steroid use such as oral candidiasis, mucosal atrophy, telangiectasia, hypersensitivity reactions, hypopigmentation, and delayed wound healing were eliminated in topical propolis use.[24] To the best of our knowledge, this is the first clinical trial to be conducted using propolis obtained from South Asia as a topical preparation in the management of OLP. Considering its safety, wide availability, and low cost, propolis could be a novel alternative therapeutic modality in the management of inflammatory conditions such as OLP. BODY.DISCUSSION.LIMITATIONS AND FUTURE RECOMMENDATION: A major limitation of this study was the small sample size. A larger sample size would have allowed for stronger statistical analysis. Another limitation of the study was the dependence on patient's compliance which could not be monitored. Furthermore, since the patients were followed up only till 14 days, the recurrence rate of LP could not be elicited. Future studies can be conducted using a larger sample size to further authenticate the effectiveness of propolis. A longer follow-up period will help in demonstrating a difference in the recurrence rate of LP among the study and the control groups. BODY.CONCLUSION: The current study comprised of 27 patients diagnosed with OLP, among which 15 patients were in the control group and the rest 12 were in the study group. The patients in the control group received triamcinolone acetonide 0.1% while the patients in the study group received 5% propolis. Both the groups were evaluated for pain and erythema at baseline (1st visit), first follow-up (7th day), and second follow-up (14th day). The following conclusions were drawn: the topical propolis was found to be as effective as triamcinolone acetonide 0.1% in the management of OLP. It has both antioxidant and anti-inflammatory effects, which may significantly contribute to its clinical effects. No adverse reactions were noted with the use of topical propolis, and it was also found to be effective at the prescribed dose, i.e., 5% propolis. Considering the chronicity of the disease and the need for the long-term treatment modalities, propolis can be proposed as a better treatment modality for OLP. Considering the safety, wide availability, and low cost, propolis should be considered as a novel alternative therapeutic modality in the management of OLP. Hence, we conclude that our results provided practical hints for the better management of OLP. However, more research with larger sample size is necessary for a full evaluation of the efficacy of propolis. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Cholecalciferol Additively Reduces Serum Parathyroid Hormone Levels in Severe Secondary Hyperparathyroidism Treated with Calcitriol and Cinacalcet among Hemodialysis Patients ABSTRACT: We evaluated the improvement of intact parathyroid hormone (iPTH) levels and bone parameters by supplementing nutritional vitamin D (cholecalciferol) to combined calcimimetic (cinacalcet) and active vitamin D analog (calcitriol) among severe secondary hyperparathyroidism (SHPT) hemodialysis (HD) patients. A randomized, controlled open-label study was undertaken in 60 HD patients with serum iPTH > 1000 pg/mL or persistently high iPTH ≥ 600 pg/mL even after >3 months of calcitriol (3 μg/week). The study group received oral cholecalciferol (5000 IU/ day) and the control group received a placebo. All patients received fixed dose cinacalcet (30 mg/day, orally) and calcitriol. Calcitriol was reduced if iPTH ≤ 300 pg/mL and cinacalcet was withdrawn if serum iPTH was persistently low (iPTH ≤ 300 pg/mL) for 4 weeks after the reduction of calcitriol. A significantly lower iPTH level was noted from the 20th week in the study group compared to the placebo group, and the target iPTH ≤ 300 pg/mL was achieved at the 24th week in the study group. Most patients achieved serum 25-(OH)D3 ≥ 30 ng/mL in the study group. Nearly 40% of study patients gained >10% improvement in femoral neck (FN) bone mineral density (BMD). We conclude that cholecalciferol additively reduced serum iPTH levels, improved 25-(OH)D3 levels and improved FN BMD when used together with cinacalcet/calcitriol in severe SHPT HD patients. BODY.1. INTRODUCTION: Secondary hyperparathyroidism (SHPT) is a major complication among dialysis patients which can have renal osteodystrophy and cardiovascular consequences [1,2]. Dietary control, phosphate binders and active vitamin D analogs are used in earlier SHPT whereas calcimimetic agent, cinacalcet, is indicated in later stages of SHPT cases who have markedly elevated parathyroid hormone (PTH) levels or failed to respond earlier treatment. The doses of active vitamin D analogs are reduced or cinacalcet is added when the patients have progressively high calcium, phosphate and Ca × P products to prevent cardiovascular and soft-tissue calcification [3,4]. Many studies demonstrated that cinacalcet improves PTH control and achieves recommended serum calcium and phosphorus values when used in combination with active vitamin D analogs and phosphate binders [5,6,7,8,9]. In our previous study, we revealed additional PTH lowering and anti-inflammatory effects of nutritional vitamin D (cholecalciferol) supplementation to active vitamin D analogs in SHPT patients [10]. However, data regarding the addition of cholecalciferol supplementation to combined cinacalcet and active vitamin D analogs in severe SHPT patients is still lacking. Circulating PTH levels were found to be inversely correlated with serum 25(OH) D3, a circulating vitamin D metabolite which indicated the vitamin D status [11,12]. 25(OH)D3 was an important substrate for the local generation of 1,25(OH)2D with the help of local 1α-hydroxylase (1α-OHase) activity [13,14]. Most end-stage renal disease (ESRD) patients had low serum 25(OH)D3 levels [15,16]. Segersten et al. [17] revealed 1α-OHase expression in parathyroid glands which presumably suppressed the PTH gland hyperplasia in an autocrine/paracrine manner. Previous research revealed coincident increased expression of 1α-OHase (approximately increased in 10 folds) and reduced 24-hydroxylase in most SHPT glands [18] and highlighted the requirement of more 25(OH)D3 in these patients. CS Ritter et al. [19] proved that the local effect of 25(OH)D3 on PTH suppression possibly occurs through direct activation of the vitamin D receptor (VDR) in parathyroid glands. Furthermore, 25(OH)D3 played less role in systemic hypercalcemia and related complications. These findings explain the possible additive role of nutritional vitamin D (cholecalciferol) supplementation in SHPT patients. Among two types of parathyroid cells (chief cells (CC) and oxyphil cells (OC)), OC markedly increased in chronic kidney disease (CKD) [20]. Lomonte C et al. revealed calcitriol therapy significantly increases the OC content in parathyroid glands [21]. Cinacalcet acts through CaR in the CC of parathyroid glands and exerts antiproliferative and proapoptotic action [22,23]. Studies revealed that cinacalcet significantly increases the OC/CC ratio (approx. increase 3.42 times) [24] and increases the oxyphil area [25]. These OC excessively express 1α OHase enzyme and increase local calcitriol production [26], which further carries out autocrine/paracrine regulation of PTH synthesis and release. Thus, cinacalcet use further increases the requirement of substrate chocalciferol for 1α OHase to produce local calcitriol production in OC. Calcimimetics also up-regulate decreased parathyroid CaR and VDR in both in vitro and in vivo studies [27,28] which further mediates parathyroid proliferation. Therefore, we speculate that combining cinacalcet to calcitriol therapy increases the parathyroid OC/CC ratio, increases local 1αOHase activity, and increases VDR expression which additively needs more 25(OH)D3 for local calcitriol synthesis. We hypothesize that the cholecalciferol supplementation in SHPT patients together with calcitriol and cinacalcet therapy increase local calcitriol production, which further suppresses intact parathyroid hormone (iPTH) secretion. We further describe the changes in bone turnover markers and bone densities with or without cholecalciferol supplementation in SHPT hemodialysis patients. BODY.2. MATERIALS AND METHODS.2.1. STUDY DESIGN: The trial was designed as a randomized, controlled open-label study. A total of 80 patients were eligible and agreed to participate. Group matching with gender, age (within 5 years), and duration of hemodialysis (within 1 year) was conducted for every pair group and they were then randomly assigned to either treatment with cinacalcet, calcitriol and cholecalciferol (CCC, study group) or control group, treated with cinacalcet, calcitriol and placebo (CCP, control group). G*power was used to calculate the required sample size [29] and effects were detected in a two-sided test with a power of (1 − β) = 80% at a significance level of 0.05. Other calculation settings were as follows: (1) the randomization process was based on 1:1 proportion of this study; (2) the effect size was set as 0.8. The required sample size for calculating was at least 25 subjects in the CCC group and 25 subjects in the CCP group. BODY.2. MATERIALS AND METHODS.2.2. PATIENT ELIGIBILITY AND RANDOMIZATION: Patients aged above 18 years treated with maintenance hemodialysis three times per week for at least 3 months before screening were eligible. Patients with severe SHPT (serum iPTH > 1000 pg/mL or persistantly high serum iPTH ≥ 600 pg/mL even after more than 3 months of calcitriol treatment) were enrolled. All patients needed to stop receiving an active vitamin D at least 30 days before entering the study. Patients using calcimimetic agents and/or native vitamin D analogs were excluded. The patients were excluded if they were pregnant, breastfeeding or of childbearing potential and not practicing birth control. Those with malignancies, severe malnutrition, and inflammatory or infectious disorders diagnosed for more than 3 months before the study were also excluded. Other exclusion criteria included surgical interventions and vascular diseases, including acute coronary syndrome, unstable angina, cerebrovascular accident, transient ischemic attack, deep vein thrombosis, pulmonary embolism, or congestive heart failure within 3 months of the study period. The patients were also excluded if they had a history of allergy to medications (Figure 1). Sixty hemodialysis (HD) patients fulfilled the criteria and were randomized into the CCC study group (N = 30) and CCP control group (N = 30). Patients were well matched by treatment allocation (Figure 1). Protocol and informed consent were approved by the authorities of the Institutional Review Board of Cardinal Tien Hospital and Taipei Medical University (CTH106A-2B01 and CTH-104-3-5-022). All the patients provided written consent before study enrollment. BODY.2. MATERIALS AND METHODS.2.3. TREATMENT INTERVENTION: All patients were given oral cinacalcet (30 mg/day) from the start of the study. Patients in the study group were given an oral form of cholecalciferol 5000 IU per day (Healthy Origens, Pittsburgh, PA 15241, USA); and cholecalciferol placebo (olive oil) was given in the control group. During the treatment, the calcitriol dose could be decreased when iPTH was ≤ 300 pg/mL and Ca × P was >55 mg2/dl2. The doses of Ca-based and other phosphate binders could be adjusted throughout the study. Ca-based phosphate binders could be increased when the serum Ca was <8.4 mg/dL or the patient had symptoms of hypocalcemia. Cinacalcet was given in a fixed low dose (30 mg/day) during the whole study period, and was withdrawn if the serum iPTH was persistently low (iPTH ≤ 300 pg/mL) for 4 weeks after the reduction of calcitriol or serum calcium <8.4 mg/dl or the patient had symptoms of hypocalcemia. All patients used low dialysate Ca 2.5 mEq/L and adjusted according to serum Ca levels throughout the study. A baseline visit was performed just before the start of the study, and further study visits were performed at 4, 8, 12, 16, 20 and 24 weeks after the medication. Serum biochemical parameters and bone turnover markers were assessed at baseline and during follow-up. The femoral neck (FN) and lumbar spine (LS) bone mineral density (BMD) were determined by dual X-ray absorptiometry (DXA) before and at the end of the study period. Hospital records were obtained and examined by two practicing nephrologists. BODY.2. MATERIALS AND METHODS.2.4. SERUM BIOCHEMICAL AND BONE METABOLISM PARAMETERS: Blood samples were collected and serum was separated within 1 h of collection and immediately frozen until analysis. Serum levels of iPTH, 25(OH)D3, phosphorus, calcium and other bone metabolism parameters were determined. Serum iPTH levels were measured in an immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). Serum 25(OH)D3 was determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Immundiagnostik AG, Bensheim, Germany). Serum bone specific alkaline phosphatase (BAP), a marker of bone formation was determined by ELISA (Quidel, Inc., San Diego, CA, USA); whereas tartrate-resistant acid phosphatase (TRACP)-5b, a marker of bone resorption was measured by ELISA (Quidel®, Tecomedical Group, Sissach, Switzerland). The doses of calcitriol, cinacalcet and phosphate binders were recorded at each visit. Adverse events were collected from patients' reports and in response to non-directed questioning at each study visit. BODY.2. MATERIALS AND METHODS.2.5. OBJECTIVES AND OUTCOMES AND MEASURES: Our study used the Kidney Disease Outcomes Quality Initiative (K/DOQI) [30,31] targeted bone metabolism levels; serum Ca 8.4–9.5 mg/dL, P 3.5–5.5 mg/dL, iPTH 150–300 pg/mL. Interpretation of DXA scans using lumbar spine BMD was not precise in our patients since it might have been interfered with by aorta calcification and degenerative joint diseases. Thus, we used 10% improvement in femoral BMD instead of lumbar spine BMD as our outcome. Primary Outcome: The primary outcome measure was serum iPTH ≤ 300 pg/mL. Secondary Outcome: The secondary outcome measures were serum 25-(OH)D3 ≥ 30 ng/mL and 10% improvement in femoral neck BMD from baseline. BODY.2. MATERIALS AND METHODS.2.6. DATA COLLECTION AND STATISTICAL ANALYSIS: The results were expressed as mean ± standard deviation or median (interquartile range). Parametric or non-parametric tests were used for analysis; for paired data, the Student t or Wilcoxon tests, respectively, and for between-group comparisons, the Student t, one-way ANOVA or Mann–Whitney U tests were used. Unilateral correlation analysis was performed using Pearson (r) or Spearman correlation (rs), as appropriate. All the tests were two-sided, and p < 0.05 was considered statistically significant. Statistica (Version 11, Stat Soft, Inc., 2300 East 14th Street, Tulsa, OK 74104, USA) was used for calculations. BODY.3. RESULTS.3.1. PATIENT RECRUITMENT AND ANALYSIS SETS: Table 1 shows the demographic characteristics of the study patients. All the patients were under HD with severe SHPT (serum iIPTH > 1000 pg/mL) or persistently high SHPT (serum iIPTH ≥ 600 pg/mL) even with 3 months of calcitriol treatment. Dialysis vintage, body mass index (BMI), underlying etiologies of ESRD and prior calcitriol usage were not significantly differing between two groups. All patients had high serum iPTH levels [CCC, 1034 ± 270 pg/mL vs. CCP, 1016 ± 252 pg/mL] and low 25(OH)D3 levels [CCC, 18.2 ± 8.4 ng/mL vs. CCP, 19.2 ± 7.4 ng/mL]. Serum albumin, ALP, cCa and P levels were not significantly different. BODY.3. RESULTS.3.2. CHANGES IN SERUM INTACT PARATHYROID HORMONE (IPTH) LEVELS DURING THE STUDY PERIOD: At 24 weeks, an average target iPTH level (≤300 pg/mL) was achieved in the CCC group but not in the CCP group (Table 2). The serum iPTH levels were significantly lower in the CCC group compared to the CCP group from the 20th week of the study, 336.4 ± 124.1 pg/mL in the CCC group vs. 404.4 ± 107.0 pg/mL in the CCP group (p = 0.034) at 20th week (Table 3 & Figure 2). The median changes in iPTH levels from baseline were significantly different as early as the 16th week of the study, −528.5 ± 148.1 pg/mL in the CCC group vs. −451.6 ± 116.0 pg/mL in the CCP group (p = 0.036) (Table 3). In subgroup analysis of the patients with severe vitamin D deficiency (25(OH)D3 < 12.5 ng/mL), serum iPTH levels were significantly different between the two groups at the 12th week, 486.5 ± 203.9 pg/mL in the CCC group vs. 841.5 ± 209.1 pg/mL in the CCP group (p = 0.024) (Table 4). The target iPTH level (≤300 pg/mL) was achieved from the 20th week in the CCC group compared with the CCP group in this subgroup of patients (Table 4). The mean dose of calcitriol use during the course of treatment was reduced progressively in the CCC group to control serum iPTH levels (Table 5). Dialysate calcium and doses of phosphate binder use did not differ significantly between the groups (data not shown). We further found that 11.1%, 29.6%, 55.6% and 81.5% of the study CCC group and 7.1%, 10.7%, 14.3% and 25% of the control CCP group achieved the target iPTH level at the 12th, 16th, 18th, 20th and 24th weeks, respectively (Table 6). Significantly more patients in the study group achieved the target level from the 20th week of the study (15/27 vs. 4/28, p = 0.046 at 20th wk; 22/27 vs. 7/28, p = 0.033 at 24th wk (chi-square test)) (Table 6). Most of our patients tolerated 30 mg of cinacalcet and none of the patients had hypocalcemia (cCa < 8.4 mg/dL) during the study period. BODY.3. RESULTS.3.3. CHANGES IN SERUM 25(OH)D3 LEVELS DURING THE STUDY PERIOD: In the CCC group, 25(OH)D3 levels increased significantly from 18.2 ± 8.4 to 37.4 ± 9.6 (p < 0.01) at the end of the study. However, in the CCP group, 25(OH)D3 levels did not change significantly (from 19.2 ± 7.4 to 23.4 ± 7.5 (p = 0.46)) (Table 2). A significant number of patients in the CCC group achieved target 25(OH)D3 level (≥30 ng/dL) as early as the 12th week compared to the CCP group (21/27 (78%) vs. 2/28 (7%), p = 0.001 at 12th week); and nearly 89% of the CCC group vs only 10.7% in the CCP group achieved the target level at the end of the study (24/27 vs. 3/28, p = 0.001 (chi-square test)) (Table 6). BODY.3. RESULTS.3.4. CHANGES IN SERUM BONE TURNOVER MARKERS AND BONE MINERAL DENSITIES: Serum BAP and TRACP-5b levels were not significantly different between the groups (Table 2). No obvious changes in serum Alk-P, cCa and P were noted. Although no significant differences in femoral neck and lumbar spine BMD were noted between the two groups (Table 7), the number of patients who had 10% increase in FN BMD was insignificantly higher in the CCC group ((13/27) 40%) compared with the CCP group ((5/28) 6.7%) (Table 6). BODY.4. DISCUSSION: Our results demonstrated that more than 80% of patients with severe SHPT achieved the primary target serum iPTH ≤300 pg/ml level with cholecalciferol supplementation. Nearly 90% of patients achieved the target serum 25-(OH)D3 (≥30 ng/mL) with high-dose cholecalciferol supplementation (5000 IU/day). About 40% of patients receiving cholecalciferol achieved a 10% increase in FN BMD by the end of the study. To our knowledge, this is the first study to reveal the beneficial effects of cholecalciferol supplementation in further lowering iPTH and bone density improvement when combined with cinacalcet and calcitriol among severe SHPT patients. As previously described, cinacalcet significantly increases the oxyphil cells (OC) and chief cells (CC) OC/CC ratio (approx. increase 3.42 times) [24], further increases local calcitriol production [26], and regulates local PTH secretion in a paracrine/autocrine manner. Thus, the need for local 25(OH)D3 substrate increases with cinacalcet treatment. We proved in our study that addition of nutritional vitamin D (cholecalciferol) improves the 25(OH)D3 requirement by cinacalcet treatment, and further inhibits PTH secretion among those patients with severe SHPT when used in combination with cinacalcet/active vitamin D analogs. About 55% of the patients achieved the iPTH target (iPTH ≤ 300 pg/mL) at the 20th week and almost 81% of patients achieved that target after the 24th week of cholecalciferol supplementation. This effect was also seen among those patients with severely deficient vitamin D levels (serum 25(OH)D3 < 12.5 ng/mL). Furthermore, a higher percentage of patients achieved the primary endpoint in the CCC group, irrespective of the prior use of active vitamin D, calcitriol. Although the CCP group also significantly improved iPTH levels, the target iPTH level was not achieved in most patients by the end of the study. Previous studies also proved that cinacalcet therapy improved the targets of bone metabolism (PTH, Ca, P, and the Ca-P product (Ca-P)) when used alone [32] or in combination of low-dose active vitamin D analogs [7]. However, all these studies used titrated doses of cinacalcet (30–180 mg per day) according to biochemical measures. We used a single daily dose of cinacalcet (30 mg per day) throughout our study, since the drug is not covered by health insurance in Taiwan and patients took the drug by self-payment. We also found that lower doses of calcitriol were needed among our study group with time (Table 5). Most of the CCC group patients achieved KDOQI targets [33] (serum PTH 150–300 pg/mL, Ca 8.4–9.5 mg/dL, P 3.5–5.5 mg/dL, CaxP product < 55 mg2/dL2) and none had hypocalcemia. Since hypocalcemia is the major event prohibiting the use of cinacalcet, and hypercalcemia is a major complication of active vitamin D analogs, the supplementation of nutritional vitamin D avoids both hypo- and hypercalcemic events in such combination therapy. We found that up to 70% of CCC patients achieved the target 25(OH)D3 level (≥30 ng/mL) from the 12th week of supplementation and nearly 90% had the target level by the end of the study. However, no such improvement was seen in the CCP group. Clinical studies reported that the combined use of nutritional vitamin D might reduce the active vitamin D doses [3,34] and improve adverse effects. Accordingly, we found our CCC group patients used progressively lower doses of calcitriol compared to the CCP group. Studies regarding the effective dosage of vitamin D supplementation are still controversial [35,36]. Our previous study used low-dose cholecalciferol (5000 IU/week) and about 60% of patients achieved the target 25(OH)D3 level (≥30 ng/dL) at 16 weeks [10]. Growing evidence shows that active vitamin D analogs may aggravate 25(OH)D3 deficiency because of the feedback inhibition of hepatic 1α-OHase and 25α-OHase [37,38] and induction of the 24-OHase enzyme [39]. Thus, we used quite a high dose of cholecalciferol (5000 IU/day) in combination with calcitriol in our study. We closely monitored 25(OH)D3 levels to avoid toxicity, and all of our patients tolerate high-dose cholecalciferol supplementation (5000 IU/day) well. The next major observation of our study was that about 40% in the CCC group gained >10% improvement in femoral BMD compared with the CCP group (~7%). Other clinical studies also revealed that calcimimetic agents reduced the elevated bone formation rate/tissue area with improvement in high turnover bone disorders [40,41]. In a recent Indian study among CKD patients, researchers found a significant reduction in iPTH and bone turnover markers, bone-specific alkaline phosphatase (BAP) and serum C-terminal cross-linked collagen type I telopeptides (CTX-1) with cholecalciferol supplementation [42]. Previous studies [43,44] also proved that cinacalcet treatment improves the BMD of the FN without affecting the LS BMD. How and whether the combination of these agents interacts to improve bone parameters needs to be explored in future studies. Our study has several limitations. Firstly, we cannot apply our findings or draw definitive conclusions in the general HD population due to the relatively small sample size; however, the results were indeed promising and clinically important. We used fixed low doses of cinacalcet in both study and control arms, which needed to be followed longer for efficiency. Furthermore, we excluded patients with severe malnutrition and inflammatory or infectious disorders who might have benefited the most from the intervention due to vitamin D deficiency. However, our cohort had significantly low vitamin D levels in both arms of the study. Next, we did not determine the morbidity or mortality benefits of combination therapy. Although we performed some questionnaires on various bone fractures, the results were inconsistent and non-significant due to shorter follow-up duration. The optimal serum 25(OH)D3 target level in dialysis patients remains unknown; however, we supposed some additive PTH lowering effects with 25(OH)D3 ≥ 30 ng/dL. Although no toxicity levels and signs were noted in our patients, the beneficial role and possible side effects of high-dose cholecalciferol in dialysis patients still requires a multifaceted long-term approach and needs further study in larger clinical trials. BODY.5. CONCLUSIONS: Our findings provide in vivo evidence that 25(OH)D3 repletion with high-dose cholecalciferol acts additively with low-dose cinacalcet and active vitamin D analogs to gain K/DOQI targets of bone metabolism markers and improve femoral neck bone mineral density in dialysis patients. We recommend that the addition of cholecalciferol is more favorable than the titration of cinacalcet and calcitriol doses among severe SHPT patients.

TITLE: Effect of carbonated water manufactured by a soda carbonator on etched or sealed enamel ABSTRACT.OBJECTIVE: The purpose of this study was to determine the effects of carbonated water on etched or sealed enamel according to the carbonation level and the presence of calcium ions. ABSTRACT.METHODS: Carbonated water with different carbonation levels was manufactured by a soda carbonator. Seventy-five premolar teeth were randomly divided into a control group and 4 experimental groups in accordance with the carbonation level and the presence of calcium ions in the test solutions. After specimen preparation of the Unexposed, Etched, and Sealed enamel subgroups, all the specimens were submerged in each test solution for 15 minutes three times a day during 7 days. Microhardness tests on the Unexposed and Etched enamel subgroups were performed with 10 specimens from each group. Scanning electron microscopy (SEM) tests on the Unexposed, Etched, and Sealed enamel subgroups were performed with 5 specimens from each group. Microhardness changes in different groups were statistically compared using paired t-tests, the Wilcoxon signed rank test, and the Kruskal-Wallis test. ABSTRACT.RESULTS: The microhardness changes were significantly different between the groups (p = 0.000). The microhardness changes in all experimental groups except Group 3 (low-level carbonated water with calcium ions) were significantly greater than those in the Control group. SEM showed that etched areas of the specimen were affected by carbonated water and the magnitude of destruction varied between groups. Adhesive material was partially removed in groups exposed to carbonated water. ABSTRACT.CONCLUSIONS: Carbonated water has negative effects on etched or sealed enamel, resulting in decreased microhardness and removal of the adhesive material. BODY.INTRODUCTION: Carbonated water, colloquially called sparkling water, is an aqueous solution of carbon dioxide.1 Some of these have additives, such as sweeteners or fruit flavors, but plain carbonated water is composed mostly of water and carbon dioxide, with no other additives. For this reason, carbonated water is considered as a healthier beverage than conventional soft drinks and its consumption is increasing. According to a statistical survey on the production of food and food additives conducted by the Ministry of Food and Drug Safety in Korea,2 carbonated water sales in 2014 saw a 100.63% increase from those in 2013, as compared to an 8.32% increase in conventional soft drink sales within the same period. Despite the increase in consumption, the general public is not well-informed of the negative effects of carbonated water on dental health. Erosion of enamel is defined as the physical result of dental hard tissue being chemically etched away from the tooth surface by acid.3 Previous studies have shown that dietary habits accompanied by excessive consumption of acidic foods and beverages increase enamel erosion.3456 A healthy enamel surface is an important factor for a favorable prognosis during orthodontic treatment; since erosion of the enamel can occur due to increased consumption of acidic drinks, it can negatively affect the outcome of orthodontic treatment, especially on teeth that have been etched for orthodontic appliance bonding. Not only conventional soft drinks, but also plain carbonated water is an acidic liquid. In previous studies, commercial carbonated waters showed a wide range of pH (pH 4.18-5.87)78 and some showed a pH level that is below the critical level, pH 5.5, which is required for enamel demineralization. Since appliances that can carbonate water easily, such as soda carbonators, have become more common recently, carbonated water can easily be prepared and consumed at home. Consumers can manufacture carbonated water with varying degrees of carbonation, and a high degree of carbonation results in a high level of acidity, which can be harmful to teeth. Some oral health officials are aware of this problem and have suggested that the intake of carbonated water containing calcium ions is better than that of carbonated water without calcium ions. Numerous studies9101112 on the negative effects of conventional soft drinks on etched and sealed enamel have been conducted, and have shown that the drinks could cause erosion of etched enamel and dissolve adhesive material; however, studies that focus on the effects of plain carbonated water in this respect remain scarce. The purpose of this study was to determine the in vitro influences of carbonated water on etched or sealed enamel in accordance with carbonation level and the presence of calcium ions. BODY.MATERIALS AND METHODS.SPECIMEN PREPARATION AND TREATMENT: This in vitro study was approved by the institutional review board at the Dental Hospital of Wonkwang University (No. WKDIRB201610-01). Seventy-five human premolars, extracted from young adults (age range, 20–29 years) for orthodontic purposes, were used in this study. Teeth with caries, erosion and/or other damage, or restorations were excluded. These teeth were obtained from various private practices and from clinics at the Wonkwang University Dental Hospital (Iksan, Korea). All teeth were cleansed and stored in normal saline solution. The roots of the teeth were cut off with a water-cooled, low-speed diamond saw for ease of use. The teeth were cleansed with nonfluoridated pumice and a rubber cup, thoroughly washed, and then air-dried. The left side of the buccal surface of each tooth was incisogingivally covered with melted acid-resistant wax (Sticky Wax; Kerr Co., Orange, CA, USA), and after cooling, the area was isolated from the test solution or artificial saliva during the experiment (Figure 1A and 1D). This established the Unexposed enamel subgroups prior to immersion of the teeth in the test solutions, and allowed comparison of the exposed surfaces. The right side of the buccal surface, i.e., the unwaxed part of each tooth, was etched with 37% phosphoric acid (DB-Etching 37; Denbio, Gwangju, Korea) for 20 seconds, followed by cleansing for 20 seconds, and drying for 10 seconds (Figure 1B). When the white, chalky surface appeared, Transbond XT Light Cure Adhesive Primer (3M Unitek, Monrovia, CA, USA) was applied on the cervical half of the etched surface, and this was then cured with an light emitting diode (LED) curing light (Ortholux; 3M Unitek) for 20 seconds (Figure 1C). Thus, the occlusal part of the etched surface was designated as the Etched enamel subgroup of each tooth, and the cervical part of the etched surface was designated as the Sealed enamel subgroup of each tooth (Figure 1D). A soda carbonator with a 3-level LED fizz indicator (Source Sparkling Water Maker, SodaStream, NJ, USA), which is a popular commercial apparatus for manufacturing carbonated water, was used in this study. The LED fizz indicator shows the 3 stages of carbonation levels, by changes in LED lights when the carbonated level reaches a certain range. This was used to prepare carbonated water with different carbonation levels according to the manufacturer's instructions. The tooth specimens were then randomly divided into 5 groups, in accordance with the carbonation level and the presence of calcium ions in the test solutions (i.e., a control group and 4 experimental groups): Control group: Ultrapure deionized water Group 1: Low-level carbonated water Group 2: High-level carbonated water Group 3: Low-level carbonated water with calcium ions Group 4: High-level carbonated water with calcium ions The product name, manufacturing method, and pH value of each test solution are listed in Table 1. Over a period of 7 days, all the specimens were submerged in each test solution for 15 minutes, 3 times a day, with 2-hour intervals. When the teeth were not submerged in test solutions, they were stored in artificial saliva at a constant temperature of 37°C. Before being submerged in the test solution, all the specimens were washed with ultrapure deionized water. The artificial saliva was prepared with 0.4 g NaCl, 1.21 g KCl, 0.78 g NaH2PO4·2H2O, 0.005 g Na2S·9H2O, 1 g CO(NH2)2, and 1,000 mL of distilled and deionized water in the laboratory, as described previously.4 Then, sodium hydroxide was added to this solution until the pH value was measured electrometrically as 6.75.4 The test solutions and artificial saliva were remade and refreshed at the beginning of every experimental session. After the immersion procedure, the specimens were washed with ultrapure deionized water and the wax was removed mechanically as a whole block without any effect on the enamel surface. BODY.MATERIALS AND METHODS.MEASUREMENT OF MICROHARDNESS: After the immersion procedure, 10 specimens from each group were cross-sectioned mesiodistally to divide the tooth into occlusal and cervical specimens. The cross-section was made at the level of the occlusal third (Figure 1E). Each occlusal specimen was embedded in acrylic resin, such that the cut surface was exposed for the microhardness test (Figure 1F). The surface of each specimen was ground to achieve a favorable flat enamel surface, using 600, 1,000, 1,200 grit silicon carbide abrasive paper sequentially, followed by polishing with 0.05-mm alumina slurry. All the specimens were kept in ultrapure deionized water prior to the microhardness test. Microhardness tests on the Unexposed enamel subgroup and the Etched enamel subgroup were performed using a Vickers indentor attached to a microhardness tester (MXT-70 Microhardness tester; Matsuzawa, Tokyo, Japan). Microhardness tests were conducted under a 100-g load for 20 seconds dwell time. Three indentations per test were made on the outer enamel surfaces which were 10, 55, or 100 μm apart, making rows toward the dentin. Three tests were performed on each subgroup (Figure 1F). Three rows of indentations were made with a distance of at least 300 μm. The length and width of each indentation were measured and the Vickers hardness number was calculated from the obtained values by the indentation apparatus of a measuring microscope. The microhardness was determined using the average value of 3 indentations per test and 3 rows were assessed for each subgroup. BODY.MATERIALS AND METHODS.OBSERVATION OF SPECIMEN SURFACE: After the immersion procedure, 5 specimens from each group were cross-sectioned incisogingivally to divide the tooth into buccal and palatal specimens (Figure 1G). Each buccal specimen was kept in a desiccator containing CaCl2 for 3 days, affixed to scanning electron microscopy (SEM) stubs, coated with platinum twice with a 108 Auto Sputter Coater (Cressington, Watford, UK), and was then examined with a JSM-6360 SEM (JEOL, Tokyo, Japan) at 15 kV, at different magnifications. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The microhardness of each Unexposed enamel subgroup was also compared (Table 2). The Shapiro-Wilks test found a lack of normality of the distribution of the microhardness of each Unexposed enamel subgroup. Thus, the nonparametric Kruskal-Wallis test was used for analysis, and showed no significant difference (p > 0.05). Descriptive statistics were calculated for the microhardness of each subgroup. Statistical analyses were performed using the Predictive Analytics Software (version 18.0; SPSS Inc., Chicago, USA). To assess microhardness changes of each group after the exposure to test solutions, microhardness of the Unexposed enamel subgroup and the Etched enamel subgroup were compared (Table 2). Difference values between subgroups in the Control group and Groups 1, 3, and 4 were normally distributed. Paired t-tests were used for the Control group, and Groups 1, 3, and 4, while the Wilcoxon signed rank test was used for Group 2. The nonparametric Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used as post-hoc comparisons to compare difference values between subgroups (Tables 3 and 4). BODY.RESULTS.MICROHARDNESS CHANGE: Descriptive data and statistical comparisons of microhardness between the Unexposed enamel subgroup and Etched enamel subgroup in each group are given in Table 2, and microhardness changes in specimens after exposure to test solutions are given in Tables 3 and 4. The results showed statistically significant differences between groups (p = 0.000). All experimental groups, except Group 3, showed greater statistically significant change than the Control group. High-level carbonated water (Group 2, 90.18 ± 33.16) resulted in greater statistically significant changes than low-level carbonated water, without or with the presence of calcium ions (Group 1, 49.57 ± 26.13; Group 3, 32.60 ± 19.83). High-level carbonated water with calcium ions (Group 4, 71.33 ± 38.28) resulted in a greater statistically significant change than low-level carbonated water with calcium ions (Group 3, 32.60 ± 19.83). Low-level carbonated water with calcium ions (Group 3, 32.60 ± 19.83) showed lesser statistically significant change than low-level carbonated water without calcium ion (Group 1, 49.57 ± 26.13). There was no significant difference in microhardness changes between high-level carbonated water with calcium ions (Group 4, 71.33 ± 38.28) and low/high-level carbonated water without calcium ions (Group 1, 49.57 ± 26.13; Group 2, 90.18 ± 33.16). BODY.RESULTS.SPECIMEN SURFACE CHANGE: The enamel surface of the Unexposed enamel subgroup in each group was observed. All groups showed a smooth enamel surface (Figure 2). Demineralized areas were observed in each group that had been etched and exposed to test solutions. The etched enamel surfaces in Groups 1–4 lost more tissue than the Control group (Figure 3). Dissolution of the peripheral prism was observed in the Control group (Figure 3A). Apparent dissolution of the prism core and a slightly flattened honeycomb-like appearance were observed in Group 1 (Figure 3B), and a flattened and agglomerated honeycomb-like structure was observed in Group 2 (Figure 3C). Dissolution of the prism core and a well-defined honeycomb-like appearance were observed in Group 3 (Figure 3D), while a fuzzy and worn honeycomb-like structure was observed in Group 4 (Figure 3E). Different amounts of adhesive primer removal were observed on the sealed enamel surfaces (Figure 4). Intact adhesive primer was observed in the Control group (Figure 4A). Adhesive primer with a roughened surface remained in Group 1 (Figure 4B) and some adhesive primer was stripped off (Figure 4C) in Group 2. The border of the adhesive primer was partially removed in Group 3 (Figure 4D) and the border of the adhesive primer was irregular and some part of it had disappeared in Group 4 (Figure 4E). BODY.DISCUSSION: The objective of this study was to determine the in-vitro effect of carbonated water on erosion of the etched enamel and degradation of the adhesive material in accordance with the carbonation level and the presence of calcium ions. This study revealed that carbonated water with different carbonation levels affects etched and sealed enamel to varying degrees. Higher carbonation levels show a higher tendency for enamel erosion, and addition of calcium alleviates this tendency (Table 3). In this study, the waxed part of the teeth was used as the Unexposed enamel subgroups for comparing conditions after etching and exposure to test solutions. Prior to the experiment, we conducted a pilot study, which showed that coating and mechanical removal of the wax had no effect on microhardness and the surface structure of the enamel. We designed this study to reproduce the oral environment in vivo, including the immersion time, protocol, and storage medium in intervals, based on previous studies.1112 Since meal time was assumed to be 15 minutes, the teeth used in the experiment were submerged in test solutions for 15 minutes at a time and in artificial saliva between experimental sessions to simulate the oral environment. This procedure was repeated 3 times a day to simulate every meal. This study had some limitations because of the in-vitro study design. We considered that the post-eruptive age of teeth could affect enamel surface structures and microhardness.13 Thus, the premolars extracted from young adults were used in this study. Nonetheless, exposure level to acidic beverages or food before extraction and its impact on the teeth could not be estimated. Furthermore, we used artificial saliva at a constant temperature of 37°C to simulate the oral environment. To exclude the possibility of remineralization effects by calcium ions in saliva and to focus on the role of calcium ions in the test solutions, we used artificial saliva that did not include calcium ions.5 Previous studies have shown that saliva has protective effects against demineralization of teeth, not only due to its buffering capacity, but also by enhancing ion-induced remineralization.614 Thus, in the oral environment in vivo, with normal saliva secretion and function, protective mechanisms against demineralization might have a stronger effect than the in-vitro environment. Additionally, the effects of biofilms on the reactivity of the enamel in the oral environment could decrease demineralization by the carbonated water. The effects of carbonated water on the etched and sealed enamel must be further studied in vivo to be verified in an oral environment. To assess dental erosion, the microhardness test and SEM test were used. Microhardness tests directly assess the condition of a tooth surface and quantify dental erosion.15 SEM tests, on the other hand, allow visual observation of the enamel surface change. These 2 test methods are complementary. Microhardness of teeth showed significant differences and different features were observed in SEM images. The result from this study showed that the teeth exposed to carbonated water had more enamel erosion than those of the Control group. Other studies41112 have analyzed the effect of acidic drinks, such as soft drinks on the teeth, but they have not mainly dealt with the effect of carbonated water. The microhardness values of teeth exposed to acidic drink were significantly lower than those of the control group.16 Erosion of teeth exposed to carbonated water (Groups 1–4) as observed by SEM was greater than that observed in a previous study.11 In a previous study, commercial sparkling mineral water was used, whereas carbonated water made by a soda carbonator was used in this study. In our pilot study, we found that carbonation levels vary by products similar to previous findings817 and a soda carbonator can manufacture water with higher levels of carbonation. Accordingly, the carbonated waters used in this study were predicted to result in a higher tendency for enamel erosion than commercial sparkling waters. The critical pH of demineralization, defined as the highest pH at which saliva crosses the saturation line, has been established as pH 5.5.18 When exposed to an acidic solution with a lower pH than this critical pH, enamel dissolution is initiated. We used manufactured carbonated water of two different carbonation levels, and their pH levels were lower than the critical pH, even at the lowest carbonation level. Furthermore, in this study, the teeth were intermittently exposed to carbonated waters and artificial saliva during 7 days, and demineralization occurred even during this short period. Since the orthodontic treatment period is estimated to be about 1.5 years or more, carbonated water can cause greater harmful effects. A greater amount of erosion was seen in the high-level carbonated water group than in the low-level carbonated water group. This tendency was not influenced by the presence of calcium ions. This demonstrated that higher levels of carbonation have a more destructive effect on enamel surface. In this study, higher levels of carbonation resulted in water with a more acidic pH value. However, this result might have been overestimated because the study design did not include a positive control. Nevertheless, it could provisionally be concluded, based on similar results for potential of erosion after consumption of acidic drinks that have previously been reported.111619 In particular, the amount of erosion differed significantly between Group 1 and Group 3, whereas there was no significant difference between Group 2 and Group 4. These results reflect that calcium ions reduced erosion of the enamel, such that the microhardness was decreased in Group 3 compared to the Control group, and in Group 4 as compared to Group 1. The presence of calcium ions influenced the dissolution equilibria of the dental enamel. Nonetheless, addition of calcium ions seemed to have a limited effect in high-level carbonated water. This result corresponded with that of a previous study.20 In terms of assessing the effect of the calcium ions in carbonated water, the calcium ion concentration was adjusted to 100 mg/L that resulted in an approximately 50% reduction in dissolution of hydroxyapatite in the previous study20; this reflects the highest calcium ion concentration among commercial mineral waters. Additionally, if we used human saliva, the calcium ions influence of on reduction of enamel erosion could be decreased due to remineralization. Therefore, further studies with human saliva are needed to confirm the role of calcium ions in carbonated water. In a previous study,21 calcium ions were released into the carbonated water after bovine teeth were immersed in carbonated water. The concentration of calcium ions from the bovine teeth increased over time and continued, and moreover detection of the calcium ion was performed after several hours of immersion in carbonated water. In this study, the immersion time was shorter than that in the previous study,21 and the test solutions were remade and refreshed at the beginning of every experimental session to minimize the effect of the calcium ions released from the teeth. In addition, calcium ions were added as calcium chloride, based on a previous study,20 because sodium chloride has been reported to have a negligible influence on the potential of erosion. A SEM test showed significant features associated with these erosive tendencies. Phosphoric acid etchant changed the enamel morphology of the Etched enamel subgroup in the Control group, and this showed a typical etching pattern, type 2, as a predominant pattern.22 When the etched enamel surface was exposed to carbonated water, this well-defined pattern was collapsed by the destructive effect of the carbonated water. The level of influence varied according to the carbonation levels and the presence of calcium ions. These observations accorded with the results of the microhardness test. In Sealed enamel subgroups, the adhesive primer was also affected by carbonated water. The adhesive primer was roughened and partially removed in Groups 1–4, and some parts of the etched surface were revealed. In this context, the revealed etched surface had a more important significance in practice, because of demineralization. In the Sealed enamel subgroup, calcium ions had little effect on the protective effect of the adhesive primer. During orthodontic treatment with fixed appliances, frequent intake of carbonated water could increase the risk of erosion of enamel around the brackets. Erosion of enamel around the brackets, which had been etched for bonding,2324 could cause dental caries and decrease the retention of the appliances. Even though the etched enamel had been sealed with adhesive material, carbonated water could strip off the adhesive and reveal the etched enamel. Therefore, clinicians should instruct orthodontic patients that carbonated water has negative effects on teeth, especially those with fixed appliances. BODY.CONCLUSION: Carbonated water has negative, destructive effects on teeth, and result in decreasing microhardness and removal of the adhesive material on etched or sealed enamel. Erosion occurred when the etched enamel of teeth was exposed to carbonated water, particularly in groups exposed to high-level carbonated water. Alleviation of this destructive effect is observed in groups exposed to carbonated water with calcium ion. Partial removal of the adhesive material on sealed enamel could be observed after exposure to carbonated water.

TITLE: Comparison of Superficial Mycosis Treatment using Butenafine and Bifonazole nitrate Clinical Efficacy ABSTRACT: Superficial fungal infections are commonly encountered by the physician. And the continuously changing epidemiology of invasive fungal infections results in the need for an expanded armamentarium of antifungal therapies. This study was designed to evaluate the safety and efficacy of Butenafine (BTF) versus Bifonazole (BFZ) in the treatment of superficial mycosis in a randomized, double-blind, parallel-group trial. Of 96 patients, 48 applied (BTF) cream and 48 applied (BFZ) cream for 2 weeks to tinea versicolor, corporis and cruris treat, while tinea of feet & hands was treated for 4 weeks duration. Efficacy was assessed after the end of treatment and 2 weeks later. At the end of therapy, we find somewhat more patients using (BTF) than using (BFZ) had a mycologic cure ((BTF), 87.5%; (BFZ) 83.3%) and effective clinical response ((BTF), 91.7%; (BFZ), 83.3%). (BTF) provides rapid and persistent antifungal activity and symptom relief in patients with superficial mycosis during treatment. And patients continued to improve for at least 2 weeks after treatment. The Rates of mycologic cure and effective treatment with (BTF) were higher than with (BFZ) at cessation of treatment and 2 weeks later. However, no significant difference found between the two drugs (p> 0.05). BODY.1. INTRODUCTION: Superficial fungal infections, including rare infections and commons skin disease confine to be limit to groups of patients or geographical areas. Superficial mycosis has been considered to be as major world public health problem and one of the most frequent diseases in human beings and animals (Burns et al., 2004; Kibbler et al., 1996; Midgley et al., 1997; Merlin et al., 1990). Hence; the superficial mycosis clinical treatment has been introduced by many of antifungal agents (Zarowny et al., 1995; Fredriksson, 1983; Kokoschka et al., 1986; McVie et al., 1986; Groll & Tragiannidis, 2010; Nolting et al., 1992; Reyes et al., 1998; Del Palacio et al., 1999). Nevertheless the increased use of such agents in recent years has caused the development of resistance to available drugs. For this reason, a constant effort toward the synthesis of new antifungal agents has been made in the last few years. In particular, the class of azoles (imidazole and Triazole derivatives) has supplied many effective antifungal drugs currently in clinical use, and newer azoles with expanded spectrum of activity are at the moment in continuous development (Han et al., 2011). Bifonazole (BFZ), an imidazole derivative, has a broad spectrum of action used currently for topical therapy of mycoses and dermatomycoses (Gupta & Sauder, 1994; European Pharmacopoeia Commission, 2001). Butenafine (BTF) is a synthetic benzylamine antifungal agent with fungicidal activity against susceptible organisms that have been approved in many countries worldwide (Georgopapadakou, 1998). The therapeutic efficacy of these two agents was compared in this study. BODY.2. MATERIALS AND METHODS: Our hospital received 96 patients with superficial mycosis disease complaints. Informed consent was obtained. We begin with complete medical history for each patient and record all data, including sex, age, and symptoms and signs of the disease. A physical examination included total body mass, weight and dermatological inspection. A clinical examination with standard laboratory tests was also carried out at inclusion and repeated after the end of treatment and 2 weeks later. In addition, vascular disease, particularly diabetes and other medical condition's history are record. Hence, any patients received antibiotic or antifungal treatment for previous 90 days are excluded. For this study 96-subjects with superficial skin mycosis by carry out KOH and fungal cultures positive randomized, parallel group; double-blind. The study group (subjects, including female 54 Male 42, age 14~72; duration of disease 3~10 months) divided into two treatment groups. Of 96 patients, twice daily for 2 weeks each 48 applied (BTF) hydrochloride 1% and 48 applied (BFZ) 1% cream to the affected area to treat versicolor, corporis and cruris tinea and tinea of feet and hands need 4 weeks treatment. Efficacy was assessed after the end of treatment and 2 weeks late. In this paper, the skin scraping by curetting of the affected webs was used for mycological study. For each patient, the Potas-sium hydroxide preparation was performed and Sabouraud dext-rose agar (SDA) was used for all specimens. Firstly, SDA with 0.005% chloram-phenicol to inhibit bacterial contamination and 0.05% cycloheximide to inhibit contaminating fungi were used for selective isolation of dermatophytes. Secondly, SDA without cycloheximide used to grow non-dermatophyte fungi that are commonly considered saprophytes or contaminants. With the aid of a straight needle, small fragments of skin were placed on the surface of the agar in Petri's dishes containing 40 mL of culture medium and pressed onto the surface to make a good contact. The samples were adjusted into four different areas, or at least in two spots in the case of a scanty sample. Petri's dishes were surrounded by paraffin seal to prevent dehydration and incubated at 30 °C for 4 weeks before being considered negative for fungus. Media were tested every 3–4 days for growth of fungus. Plates overgrown with contaminants growing from Petri dish edges were eliminated. Media were examined macroscopically and when applicable, the texture and surface color of the colony, as well as any pigment diffusing into the medium, were carefully noted and recorded. An adhesive tape strip was applied onto the surface of the colony, and then mounted in a drop of lactophenol cotton blue stain. BODY.2. MATERIALS AND METHODS.2.1 DRUG ADMINISTRATION: The first group was treated with 1% (BTF) hydrochloride cream. The second group was treated with 1% (BFZ) cream. Both of them were treated by applying the cream twice daily for 2 weeks to treat tinea cruris, tinea corporis and Tinea versicolor. And 4 weeks for tinea of feet and hands Where: 1% (BTF) hydrochloride cream Brand Names: brand name: mai ke shu (Seaside Pharmaceutical Company) 1% (BFZ) cream Brand Names: brand name: mei ke (Yongda drug manufactory) BODY.2. MATERIALS AND METHODS.2.2 EFFICACY CRITERIA: A) Clinical Assessment Beginning symptoms and signs for each Patient's before, after treatment had been record and two-week later accord to 4 levels from zero to three depending on the degree of itching, redness, papules & pustules before as follows. Zero=no, one=mild, two=moderate and three for server one. where Symptom score reduces the index: SSRI Total Score before Treatment: TSBT Total Score after Treatment: TSAT Cure: rash fade away; the symptoms disappear and microscopic examination of fungus and culture were negative, efficacy index 100%. Obvious effect: rash markedly improved, symptoms relieved significantly; microscopic examination of fungus and culture were negative, the index effect 70%. Improved: rash and symptoms were improved; microscopic examination of fungus and culture were negative or positive effect index 30% ~ 70%. Not effective: skin rash did not disappear and, fungi examination and / or culture were positive, effective index less 30%. B) Laboratory Assessment Mycological Cure: Clear: Both microscopically and fungal cultures were negative. Not cleared: fungi examination and / or culture were positive. C) Safety Criteria Clinical checkup had been done before treatment, and patient's blood, creatinine, urea, urine ALT, etc. then two weeks later all analysis done at cessation of treatment. All withdrawal treatment by laboratory abnormal parameters and vital signs also recorded. D) Results Analysis SPSS software was performed to analyse an intention-to-treat basis and to determine Quantitative variables such as means and standard relatively by class based on dichotomous variables as absolute or both deviations and categorical. The two tests (t-test and Mann–Whitney test) had been used to determine the significance of differences between mean values of two continuous variables, and to determine non-parametric distribution respectively. ANOVA as one-way analysis of variance was employed for comparison of several groups means and to determine the means significant differences between the two groups. In addition, multiple regression analysis was used to predictor the best diagnosis as a dependent variable. P < 0.05 was considered significant. BODY.3. RESULT.3.1 CLINICAL EFFECTIVENESS: There was high efficiency noticed for both drugs after end of treatment and 2 weeks later as shown in Table 1 and Table 2. Table 1 The effectiveness of clinical at of treatment Group cases Cured obvious effectiveness Improved efficiency BTF group 48 12 32 4 91.7% BFZ group 48 7 34 7 83.3% Significant difference was p> 0.05 for the two groups Table 2 Clinical effectiveness 2 weeks after stooping treatment Group cases Cured obvious effectiveness Improved efficiency BTF group 48 36 9 3 93.7% BFZ group 48 33 11 4 91.7% Also no significant difference found by comparing the two groups there was p> 0.05 BODY.3. RESULT.3.2 MYCOLOGICAL CURE: High clearance rate is also noticed for both drugs after end of treatment and 2 weeks later as shown in Table 3 and Table 4. Table 3 Mycological cure at end of treatment Group cases cleared clearance rate BTF group 48 42 87.5% BFZ group 48 40 83.3% There wasn’t any significant difference (p> 0.05) Table 4 Mycological cure weeks after stooping treatment Group cases cleared clearance rate BTF group 48 46 95.8% BFZ group 48 44 91.7% Similarly, there wasn’t any significant difference (p> 0.05) There was not any serious adverse effects. For (BTF) cream there was no noticeable adverse reaction; also for (BFZ) cream apart from local skin erythema and mild itching which require no treatment. Finally, no abnormality was found by laboratory examination for the two group's treatment. BODY.4. DISCUSSION: The progress in managing of fungal infections, the incidence increased and response rates remain insufficient and this may led to the development a new antifungal agent. Recent antifungal chemotherapy drugs show new advances in offer clinician's fewer toxic alternatives and more effective at conventional therapy. BTF hydrochloride has similar action a synthetic benzylamine derivative mode to that of the allylamine class of antifungal drugs (Gerald & Mc Evoy, 2008). The evaluation of randomized, double-blind, controlled trial for the safety and efficacy of 2 weeks, twice-daily treatment with (BTF) versus (BFZ) for each patient with versicolor, cruris, and corporis tinea; and 4 weeks to treat tinea of feet & hands. The final comparing the treatment of BTF group mycologic cure higher than a BFZ show group (87.5, vs. 83.3%, p> 0.05). Similarly, the mycologic cure rates at the 2 weeks follow-up were 95.8% vs. 91.7%, respectively (p >0.05). Clinical efficiencies were 91.7% for (BTF) group, compared to 83.3% for BFZ group in the (p>0.05). Then after 2 weeks clinical efficiency was 93.7% for (BTF) group, compared to 91.7% for BFZ group (p> 0.05). These results show that both (BFZ) and the significant advance for newer (BTF) treatment of fungal infections. Their expanded activity gives the clinician efficacious, safe, inexpensive and available more choices other than conventional treatment. Particularly, the optimal treatment of superficial mycosis still remains in the resistant cases with neither drug class offering an overwhelming advantage. Slightly higher rates of mycologic cure and effective treatment, (BTF) group offers a little advantages over (BFZ) group. The BTF main benefits are appeared as safety, ease of use, and offset by its current additional costs. No serious side effects were noted with (BTF) except for mild itching and skin rash, but it was tolerable and no patient discontinued therapy due to an adverse event. In the adverse events for most commonly uncontrolled trials, associated with the use of (BTF) group 1% cream included erythema, contact dermatitis, itching, and irritation, for each occurring in less than 2% of patients (Syed et al., 1998). BODY.5. CONCLUSION: In this paper, during patient treatment (at least 2 weeks) BTF provides rapid improving antifungal activity and symptom relief with superficial mycosis The Rates of mycologic cure and effective treatment with (BTF) were higher than with (BFZ) at cessation of treatment. However, statistically there was not any difference significant between the two drugs (p> 0.05).

TITLE: Effect of eicosapentaenoic acids-rich fish oil supplementation on motor nerve function after eccentric contractions ABSTRACT.BACKGROUND: This study investigated the effect of supplementation with fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the M-wave latency of biceps brachii and muscle damage after a single session of maximal elbow flexor eccentric contractions (ECC). ABSTRACT.METHODS: Twenty-one men were completed the randomized, double-blind, placebo-controlled, and parallel-design study. The subjects were randomly assigned to the fish oil group (n = 10) or control group (n = 11). The fish oil group consumed eight 300-mg EPA-rich fish oil softgel capsules (containing, in total, 600 mg EPA and 260 mg DHA) per day for 8 weeks before the exercise, and continued this for a further 5 days. The control group consumed an equivalent number of placebo capsules. The subjects performed six sets of ten eccentric contractions of the elbow flexors using a dumbbell set at 40% of their one repetition maximum. M-wave latency was assessed as the time taken from electrical stimulation applied to Erb's point to the onset of M-wave of the biceps brachii. This was measured before and immediately after exercise, and then after 1, 2, 3, and 5 days. Changes in maximal voluntary isometric contraction (MVC) torque, range of motion (ROM), upper arm circumference, and delayed onset muscle soreness (DOMS) were assessed at the same time points. ABSTRACT.RESULTS: Compared with the control group, M-wave latency was significantly shorter in the fish oil group immediately after exercise (p = 0.040), MVC torque was significantly higher at 1 day after exercise (p = 0.049), ROM was significantly greater at post and 2 days after exercise (post; p = 0.006, day 2; p = 0.014), and there was significantly less delayed onset muscle soreness at 1 and 2 days after exercise (day 1; p = 0.049, day 2; p = 0.023). ABSTRACT.CONCLUSION: Eight weeks of EPA and DHA supplementation may play a protective role against motor nerve function and may attenuate muscle damage after eccentric contractions. ABSTRACT.TRIAL REGISTRATION: This trial was registered on July 14th 2015 (https://upload.umin.ac.jp/cgi-open-bin/ctr/index.cgi). BODY.BACKGROUND: It is widely recognized that unaccustomed muscle movement, including eccentric contraction (ECC), causes muscular damage [1, 2]. It has been shown that ECCs result in a loss of muscle strength, delayed onset muscle soreness (DOMS), a limited range of motion (ROM), muscle swelling, increases in serum creatine kinase and myoglobin levels, prolonged transverse relaxation time (T2) on magnetic resonance imaging (MRI), and echo intensity on ultrasound imaging [1, 3, 4]. Interestingly, because these effects differ in the time taken to reach their peak [1, 2], the relationship with each muscle damage marker is not clear. For example, it has been shown that strength loss and limited ROM peak immediately after performing ECCs, whereas DOMS reaches its peak at 1–3 days after the ECCs and T2 and echo intensity, which indicate the distribution of free water and/or interstitial edema, are at their maximum after 3–6 days [1, 2, 4, 5]. Nerve conduction velocity, M-wave latency, and amplitude are often measured to assess motor nerve function [6, 7]. M-wave latency is measured as the time between electrical stimulation and the onset of an M-wave, but this can be influenced by a number of factors, including various processes such as nerve conduction, neuromuscular transmission, and muscle fiber conduction, and sarcolemmal excitability [8]. Previous studies have used M-wave latency to examine nerve disorders such as neuropathy and neural muscular atrophy [6, 9]; these indicated that an increase in M-wave latency reflected the impairment of motor nerves. It has been shown that ECCs caused histological damage in rats, not only in myofibrils, the extracellular matrix, and the triads of the cytoplasmic membrane system [10, 11], but also in nerve fibers and in the thinning of myelin sheaths [12]. Kouzaki et al. [13] reported that M-wave latency delayed by 12% at 24 h and 24% at 48 h after 60 ECCs of the elbow flexors performed by women, suggesting musculocutaneous nerve impairment. However, it is not known whether nutritional strategies have a role for preventing temporal muscular dysfunction after ECCs. When fish oil is consumed in the diet, the concentration of the long-chain omega-3 polyunsaturated fatty acid increases proportionately in the cellular membranes of muscles [14, 15] as in other organs [16]. In mice, omega-3, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has several important roles in reducing the risk of cardiovascular diseases [17, 18], reducing inflammatory markers [19, 20], and improving vision and cognitive functions including Alzheimer's disease [21]. Recently, we have shown that EPA and DHA supplementation ameliorated reductions in muscle strength, the development of DOMS, and limited ROM following ECCs [22]. We have hypothesized that, as one of the mechanisms underlying this phenomenon, EPA and DHA play an important role related to nerve structure and/or function. However, as yet no study has investigated the effect of EPA and DHA supplementation on ECC-induced nerve damage. The aim of the present study was to investigate the effect of 8 weeks of EPA and DHA supplementation on M-wave latency of the biceps brachii after elbow flexion ECCs. We hypothesized that EPA and DHA supplementation for 8 weeks would inhibit the strength loss, limited ROM, DOMS, and muscle swelling after the ECCs and would increase M-wave latency. BODY.METHODS.SUBJECTS: A total of 21 healthy men (age, 21.0 ± 0.8 years; height, 170.9 ± 5.7 cm; weight, 64.3 ± 6.1 kg; body mass index, 25.0 ± 1.7) were recruited for this study. The sample size was determined by a power analysis (G*power, version 3.0.10, Heinrich-Heine University, Dusseldorf, Germany) by setting the effect size as 1, α level of 0.05 and power (1-β) of 0.80 for the comparison between groups, which showed that at least ten participants were necessary. None had participated in any regular resistance training, restriction of exercise, or other clinical trial, had food allergies to fish, or were taking any supplement or medication. The subjects were requested to avoid interventions such as massage, stretching, and strenuous exercise, and the excessive consumption of food and alcohol, during the experimental period. Prior to participation, they were provided with detailed explanations of the study protocol, and all signed an informed consent form. The study was conducted in accordance with the principles of the Declaration of Helsinki; it was approved by the Ethics Committee for Human Experiments at Juntendo University (ID: 27–66) and has been registered at the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR, identifier: UMIN000018285). BODY.METHODS.STUDY DESIGN: The study was conducted following the double-blind, placebo-controlled, parallel-group trial design. The subjects were randomly assigned to two groups using a table of random numbers in such a manner as to minimize the inter-group differences in age, body fat, body mass index. According to the previous studies [22, 23], the control group consumed daily placebo capsules for 8 weeks prior to an exercise experiment and for 5 days after the exercise whereas the EPA group consumed EPA supplement capsules as described in the following section. The capsules were taken for a total of 62 days (including the exercise day). The sequence allocation concealment and blinding to subjects and researchers were maintained throughout this period. Compliance to intake was assessed by daily record and a pill count at the end of the study. To help the reliability of the pill count, subjects were given an excess number of pills and asked to return any remaining pills at the end of the study. On the day of exercise testing, M-wave latency, maximal voluntary isometric contraction (MVC) torque, elbow joint ROM, upper arm circumference, and muscle soreness assessed by a visual analog scale, were assessed in the non-dominant arm before exercise. Immediately after these baseline measurements, the subjects performed ECCs with the same arm. All measurements were repeated immediately after the exercise, and then at 1, 2, 3, and 5 days later. Before the subjects started taking the supplement or placebo capsules, we surveyed their nutritional status using the food frequency questionnaire based on food groups (FFQg version 3.5, Kenpakusha, Tokyo, Japan). This was repeated after the 8 weeks supplementation. The primary outcome measures were MVC torque, ROM, M-wave latency, and muscle soreness. BODY.METHODS.SUPPLEMENTS: From the previous studies [20, 22, 24] and considering the safety factor [25], the EPA group consumed eight 300-mg EPA-rich fish oil softgel capsules (Nippon Suisan Kaisha Ltd., Tokyo, Japan) per day, a total of 2400 mg per day containing 600 mg EPA and 260 mg DHA. The CON group consumed eight 300 mg corn oil softgel capsules (Nippon Suisan Kaisha Ltd., Tokyo, Japan) per day (not containing EPA and DHA in a total of 2400 mg). The subjects consumed the supplements 30 min after meals with water. BODY.METHODS.ECCENTRIC CONTRACTIONS: For the eccentric exercise, the subject sat on a preacher curl bench with his shoulder joint angle at 45° flexion. The dumbbell used was set to weigh 40% of his one repetition maximum arm curl weight. The exercise consisted of six sets of ten maximal voluntary ECCs of the elbow flexors with a rest of 120 s between each set as described in our previous study [22]. The subject was handed the dumbbell in the elbow flexed position (90°) and instructed to lower it to a fully extended position (0°) at an approximately constant speed (30°/s), in time (3 s) with a metronome. The investigator then removed the dumbbell and the subject returned his arm without the dumbbell to the start position for the next eccentric contraction. BODY.METHODS.MAXIMAL VOLUNTARY ISOMETRIC CONTRACTION TORQUE: For the measurement of MVC torque, the subject performed two 5-s MVCs at a 90° elbow joint angle with a 15-s rest between the contractions. The peak torque of the two contractions was used as the MVC torque. The torque signal was amplified using a strain amplifier (DPM-611B; Kyowa, Tokyo, Japan). The analog torque signal was converted to digital signals with a 16-bit analog-to-digital converter (Power-Lab 16SP; ADInstruments, Bella Vista, Australia). The sampling frequency was set at 2 kHz. The measurement was based on a previous study [26]. BODY.METHODS.RANGE OF MOTION OF THE ELBOW JOINT: To examine elbow joint ROM, two elbow joint angles (extended and flexed) were measured using a goniometer (Takase Medical, Tokyo, Japan). The extended joint angle was recorded while the subject attempted to fully extend the joint with the elbow held by his side and the hand in supination [22] The flexed joint angle was determined while the subject attempted to fully flex the joint from an equally fully extended position with the hand supinated. The ROM was calculated by subtracting the flexed joint angle from the extended joint angle. BODY.METHODS.UPPER ARM CIRCUMFERENCE: The upper arm circumference was measured at 3, 5, 7, 9, and 11 cm above the elbow joint using a Gulick tape measure while the subject stood with his arm relaxed by his side. The mean value of five measurements was used for the analysis. Measurement marks were maintained throughout the experimental period using a semi-permanent ink marker, and a well-trained investigator made the measurements [22]. The mean value of measurements was used for analysis. BODY.METHODS.ULTRASONOGRAPHY: B-mode ultrasound pictures of the upper arm were taken of the biceps brachii using ultrasound (Aixplorer, SuperSonic, France), with the probe placed 9 cm from the elbow joint at the position marked for the circumference measurement. The gains and contrast were kept consistent over the experimental period. The transverse images were transferred to a computer as bitmap (.bmp) files and analyzed by a computer. The cross-sectional area of the elbow flexors was determined by measuring the distance between the subcutaneous fat layer and the edge of the humerus [27] on the transverse images of the biceps brachii. The average echo intensity for the region of interest (20 × 20 mm) was calculated by the computer image analysis software that provided a gray scale histogram (0, black; 100, white) for the region as described in previous study [27]. BODY.METHODS.MUSCLE SORENESS: Muscle soreness in the elbow flexors was assessed using a 10-cm visual analog scale in which 0 indicated "no pain" and 10 was the "the worst pain imaginable" [4]. The subject relaxed with his arm in a natural position; the investigator then palpated the upper arm using a thumb and the subject indicated his pain level on the visual scale. All tests were conducted by the same investigator, who had been trained to apply the same pressure over time and between subjects. BODY.METHODS.M-WAVE LATENCY: The musculocutaneous nerve was stimulated (for a pulse duration of 10 ms) by a bipolar surface electrode with an inter-electrode distance of 23 mm and a diameter of 0.8 mm (NM-420S, Nihon Kohden, Tokyo, Japan). The stimulation electrode was placed over Erb's point located on the supraclavicular fossa and sternocleidomastoid muscle and connected to an electrical stimulator (DS7AH, Digitimer Ltd., Welwyn Garden City, UK). The M-wave latency and amplitude were assessed according to the instructions in the "Manual of Nerve Conduction Studies" [28] and as described in our previous study [13]. The subject sat on a chair with both arms at his side and placed his forearm on his lap, resulting in the elbow joint being at approximately 10° flexion. This position was kept consistent between measures, and a plastic goniometer was used to reproduce the elbow joint angle. A monopolar surface electrode was placed at the mid-belly of the biceps brachii long head, a reference electrode was placed proximal to the antecubital fossa in the region of the junction of the muscle fibers and the biceps tendon, and a ground electrode was placed on the acromion [13]. The electrode locations were marked with a semi-permanent marker to ensure the same placement over time. The stimulation current was gradually increased to obtain the maximal M-wave (final stimulation current: 14–18 mA), and it was confirmed that a further increase in the current did not increase the M-wave amplitude. Data acquisition and analysis were performed using Lab Chart 7 (ADInstruments, Bella Vista, Australia). The relative increase in the M-wave latency from the pre-ECC value was then calculated. This measurement was based on a previous study [13]. BODY.METHODS.STATISTICAL ANALYSES: All analyses were performed using SPSS Statistics software version 22.0 (IBM Corp., Armonk, NY). Values are expressed as means ± standard deviation (SD). MVC torque, ROM, echo intensity, circumference, CSA, and nerve conduction latency values at post, day1, day2, day3, and day5 were calculated by relative changes from baseline (100%). MVC, ROM, upper arm circumference, echo intensity, cross-sectional area, muscle soreness, and M-wave latency over time were compared between the CON and EPA groups by two-way repeated-measure analysis of variance (ANOVA). When a significant main effect or interaction was found, Bonferroni's correction was performed for the post-hoc testing. A p-value of <0.05 was considered statistically significant. BODY.RESULTS.PHYSICAL CHARACTERISTICS AND NUTRITIONAL STATUS: No subject dropped out during the study period. No significant differences were observed between the EPA group and the CON group for age, weight, and body mass index (EPA; n = 10; age, 20.7 ± 0.7 years; height, 171.5 ± 6.6 cm; weight, 63.0 ± 6.3 kg; body mass index, 25.0 ± 2.0, CON; n = 11; age, 21.3 ± 0.9 years; height, 170.7 ± 5.0 cm; weight, 65.5 ± 5.9 kg; body mass index, 25.0 ± 1.5). The food frequency questionnaire results showed no significant difference in nutritional status between the EPA group (energy, 2578.1 ± 359.4 kcal; protein, 88.7 ± 18.7 g; fat, 91.9 ± 18.5 g; carbohydrate, 330.4 ± 60.0 g; omega-3 fatty acid, 2.4 ± 0.7 g) and the CON group (energy, 2273.4 ± 607.7 kcal; protein, 80.7 ± 20.7 g; fat, 92.9 ± 25.0 g; carbohydrate, 268.6 ± 90.8 g; omega-3 fatty acid, 2.3 ± 0.6 g) before the intake of supplements (Table 1). These parameters did not change during the experimental period. Blood analyses showed that EPA was significantly higher in the EPA group than in the CON group after 8 weeks intake of supplements, whereas DHA was no significantly difference between two groups (data not shown).Table 1Physical characteristics of subjectsAge (year)Height (cm)Weight (kg)BMICON (n = 11)21.3 ± 0.9170.7 ± 5.065.5 ± 5.925.0 ± 1.5EPA (n = 10)20.7 ± 0.7171.5 ± 6.663.0 ± 6.325.0 ± 2.0n.s. BODY.RESULTS.MAXIMAL VOLUNTARY ISOMETRIC CONTRACTION TORQUE: Compared with the pre-exercise value, MVC torque in the CON group had significantly decreased immediately after the exercise and 1 day later (post; p = 0.001, day 1; p = 0.003; Fig. 1a). MVC torque in the EPA group decreased immediately after the exercise compared with the pre-exercise level (p = 0.010). MVC was significantly higher in the EPA group than in the CON group at 1 day after the exercise (CON 76.8% ± 16.0%, EPA 90.5% ± 14.0%, p = 0.049). The results for the absolute MVC were similar to these.Fig. 1Changes (mean ± SD) of maximal voluntary isometric contraction (MVC) torque (a), range of motion (ROM) (b), muscle soreness (c), and echo intensity (d) measured before (pre) and immediately after (post) the eccentric contractions exercise and then 1, 2, 3, and 5 days later in the control and EPA groups. The values of MVC torque, ROM, and echo intensity at post, 1, 2, 3, and 5 days after eccentric contractions were calculated by relative changes from baseline (100%). * p < 0.05 for the difference between groups; † p < 0.05 for the difference from the pre-exercise value in the control group, # p < 0.05 for the difference from pre-exercise value in the EPA group BODY.RESULTS.RANGE OF ELBOW MOTION: As shown in Fig. 1b, a significant decrease in ROM was observed in the CON group immediately after the exercise (a reduction of 23.2%, p = 0.000), and ROM continued to be lower than baseline at 1 and 2 days (day 1; p = 0.003, day 2; p = 0.012). ROM in the EPA group decreased immediately after the exercise compared with the pre-exercise level (a reduction of 12.0%, p = 0.013). ROM was significantly greater in the EPA group than in the CON group immediately after exercise and 2 days later (post: CON 76.8% ± 10.4%, EPA 88.1% ± 7.2%, p = 0.006; day 2: CON 81.7% ± 17.4%, EPA 96.2% ± 6.3%, p = 0.014). BODY.RESULTS.MUSCLE SORENESS: Compared with the pre-exercise value, a significant level of muscle soreness was indicated by the CON group using the visual analog scale at 1 and 2 days after exercise (day 1; p = 0.006, day 2; p = 0.021; Fig 1c). In contrast, no increase in muscle soreness was indicated by the EPA group at any time points. Significantly greater muscle soreness was observed in the CON group compared with the EPA group at 1 day (CON, 5.7 ± 1.6 cm vs. EPA, 4.5 ± 1.0 cm; p = 0.049) and 2 days (CON, 5.4 ± 1.8 cm vs. EPA, 3.7 ± 1.2 cm; p = 0.023) after the exercise. BODY.RESULTS.ECHO INTENSITY: In the CON group, echo intensity increased at 1 day after exercise (133.8% ± 25.9%, p = 0.005; Fig. 1d), but there was no significant increase in echo intensity in the EPA group at any time point. The echo intensity was significantly higher for the CON group than the EPA group at 1 day after the exercise (CON, 133.8 ± 25.9% vs. EPA, 111.2 ± 18.2%; p = 0.045). BODY.RESULTS.UPPER ARM CIRCUMFERENCE AND CROSS-SECTIONAL AREA OF THE FLEXORS: No significant difference in the upper arm circumference from the pre-exercise values or between the two groups was observed at any time point (Fig. 2a). Similarly, there was no significant difference in the cross-sectional area of the elbow flexors at any time point (Fig. 2b).Fig. 2Changes (mean ± SD) of upper arm circumference (a) and cross-sectional area (CSA) of the elbow flexors (b) measured before (pre) and immediately after (post) the eccentric contractions exercise and then 1, 2, 3, and 5 days later in the control and EPA groups. The values of upper arm circumference and CSA at post, 1, 2, 3, and 5 days after eccentric contractions were calculated by relative changes from baseline (100%). n.s. not significant BODY.RESULTS.M-WAVE LATENCY: In the CON group, but not the EPA group, M-wave latency increased immediately after the exercise (p = 0.010; Fig. 3). In addition, M-wave latency was significantly longer in the CON group than in the EPA group immediately after the exercise (CON, 133.0 ± 32.1% vs. EPA, 113.3 ± 26.5%; p = 0.040).Fig. 3Changes (mean ± SD) of nerve conduction latency measured before (pre) and immediately after (post) the eccentric contractions exercise and then 1, 2, 3, and 5 days later in the control and EPA groups. The values of nerve conduction latency at post, 1, 2, 3, and 5 days after eccentric contractions were calculated by relative changes from baseline (100%). * p < 0.05 for the difference between groups, † p < 0.05 for the difference from the pre-exercise value in the control group BODY.DISCUSSION: This study investigated the effect of EPA and DHA supplementation on the M-wave latency of the biceps brachii and on muscle damage after single session of maximal elbow flexion ECC exercises. The results demonstrated that EPA and DHA supplementation inhibited the loss of muscle strength, limitation of ROM, development of DOMS, and increases in echo intensity and M-wave latency. These results support our original hypothesis. The reduction of isometric torque was significantly inhibited in the EPA group. Our recent study also showed that 600 mg EPA and 260 mg DHA supplementation for 8 weeks inhibited strength loss following elbow flexion ECC exercises [22]. To the best of our knowledge, that study was the first to demonstrate that EPA and DHA supplementation had a positive effect on MVC torque following ECC exercises, and the present study supports those results. Strength loss following ECCs has been considered to be the result of disruption of the myofibrils, muscle membranes, and neuromuscular junctions, and abnormal calcium (Ca2+) levels [3]. Temporary strength loss following ECCs has also been assumed to be due to excitation–contraction coupling failure [29]. Importantly, omega-3 polyunsaturated fatty acids are a major component of the cell membrane, with the total concentration in muscle cellular membrane significantly increasing after the ingestion of fish oil [30]. Thus, it is possible that the muscle membrane structure may be protected by EPA supplementation and that EPA and DHA thereby reduced the muscular dysfunction that resulted from the ECCs at day 1. Nerve injuries are classified as neurapraxia, axonotmesis, or neurotmesis [31–33]. In neurapraxia, nerve conduction is impaired by a segmental myelination of the nerve trunk without axonal degeneration. The causes of neurapraxia include mechanically induced lesions, ischemia, inflammation, toxic compounds, and metabolic disturbances [31, 34], and M-wave latency increases without an apparent change in M-wave amplitude [33]. In the present study, the M-wave amplitude did not significantly change after the ECCs (data not shown), and the prolonged M-wave latency was not observed at 2 days post-exercise. Thus, the effect of ECCs on the nerve may have been similar to neurapraxia. The present study also demonstrated that EPA and DHA supplementation inhibited the increased M-wave latency following the eccentric exercise. We believe that this is the first report to show that EPA and DHA have important roles in counteracting the muscle dysfunction resulting from ECCs. Braddom et al. [35] observed structural damage to musculoskeletal nerves and ischemic neuropathy with high-intensity weight lifting exercise. In addition, it has been shown in rats that repeated sessions of ECCs caused histological damage in nerve fibers and thinning of myelin sheaths [12]. It is therefore possible that the decreased M-wave latency was associated with a protective role for EPA and DHA with regard to nerve fiber damage and myelin sheaths thinning. As mentioned earlier, one cause of neurapraxia is mechanically induced inflammation [31, 34]; we assume that EPA and DHA reduce the inflammation resulting from nerve injury. Our study also demonstrated that EPA and DHA supplementation had a preventive effect on ROM and DOMS following eccentric exercise. These results are consistent with those of previous studies [22, 24]. Tartibian et al. [24] showed that daily supplementation with 324 mg EPA and 216 mg DHA attenuated the limitation of ROM after 40 min of bench stepping. Similarly, our recent study demonstrated that 600 mg EPA and 260 mg DHA supplementation for 8 weeks inhibited the limitation of ROM and the development of DOMS [22]. The limited ROM following ECC has been attributed to an inflammatory response within myofibrils leading to an increase in passive stiffness [36]. Although DOMS can be attributed to a combination of several factors, previous studies have suggested that its primary cause is a local inflammatory response [11, 37]. It is well established that EPA and DHA have anti-inflammatory effects that reduce levels of interleukin-6 [22]; we therefore suggest that their inhibition of limited ROM and the development of DOMS could be attributed to their anti-inflammatory effects. In this study, the echo intensity was significantly lower in the EPA group than in the CON group 1 day after the ECC exercise. Increased echo intensity is associated with edema of the muscle due to trauma, ischemia, or infraction [1]. Indeed, previous studies have confirmed that echo intensity in the biceps brachii and brachialis increases with elbow flexors after ECC exercise [1, 26, 38]. In addition, because echo intensity appears to be related to creatine kinase level after ECC [1], it has the potential to be a useful marker of muscle damage. Although the mechanism is unclear, the results in present study suggest that EPA and DHA supplementation may inhibit edema. However, no significant difference was observed in the upper arm circumference. This observation is similar to that of our previous study [22], and we assume that it related to a limitation of the study method. Although we used a Gulick tape measure, we could not exclude the effects of other muscles, fat, skin, etc. For this reason, in the present study we also calculated the cross-sectional area using ultrasonography. However, we observed no significant difference between groups, although the cross-sectional area showed a non-significant tendency to be smaller in the EPA group than in the CON group. The reason for this could be that the intensity of ECC and the reduction of muscle strength were lower than in the previous studies [2], and that the accuracy of ultrasonography is lower than that of MRI. A further study is needed that uses MRI to determine the response after severe ECC exercise. The present study had the following three limitations. First, we did not evaluate the inflammatory response such as C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-6 [22, 39]. Although the results demonstrated that EPA and DHA supplementation had a positive effect on DOMS and ROM following eccentric exercise, the actual inflammatory response was unknown. Second, we investigated only a single dose of EPA and DHA administration. It has been shown that the amount of EPA and DHA is limited 3000 mg in total per 1 day for the safety in human by natural medicines comprehensive database [25]. Regarding the effect of EPA and DHA supplementation on muscle damage, the minimal effect was with 540 mg/day (EPA and DHA) according to Tartibian et al [24]. Hence, we have decided to use the amount of supplementation (600 mg EPA and 260 mg DHA). However, further investigation is needed to study the different doses of EPA and DHA to elucidate the appropriate amount. Third, we used one bout of eccentric exercise model, which was a single-exercise, to untrained subjects. Therefore, our observations could not provide to athletes and multiple training sessions. Further study is required to investigate this point. BODY.CONCLUSIONS: In summary, we showed that 600 mg EPA and 260 mg DHA supplementation for 8 weeks inhibited the increased M-wave latency following a session of intense ECC exercise, as well as the strength loss, limitation of ROM, and development of DOMS. We speculate that the mechanism underlying these observations may be related to the improvement of nerve structure and function. These findings of the beneficial effects of EPA and DHA supplementation are of importance for applied sport scientists, nutritionists, and strength and conditioning professionals and could help them to design better nutritional interventions aimed at preventing temporary muscle strength loss, limited flexibility, and DOMS after exercise.

TITLE: Retroperitoneal laparoscopic pyelolithotomy in renal pelvic stone versus open surgery - a comparative study ABSTRACT.BACKGROUND: The introduction of endourological procedures such as percutaneous nephrolithotomy and ureterorenoscopy have led to a revolution in the the management of urinary stone disease. The indications for open stone surgery have been narrowed significantly, making it a second- or third-line treatment option. ABSTRACT.AIMS AND OBJECTIVES: To study the safety and efficacy of retroperitoneal laparoscopic pyelolithotomy in retroperitoneal renal stone. We compared the results of laparoscopic and open surgery in terms of easy accessibility, operative period, renal injuries, and early recovery. ABSTRACT.METHODS: This prospective study was conducted on renal pelvic stone cases from January 2009 to February 2016 in Suchkhand Hospital, Agra, India. The study included a total of 1700 cases with the diagnosis of solitary renal pelvic stones. In group A - 850 cases - retroperitoneal laparoscopic pyelolithotomy was performed, while group B - 850 cases – underwent open pyelolithotomy. ABSTRACT.RESULTS: The mean operative time was less in group B than group A (74.83 min vs. 94.43 min) which was significant (p<0.001). The blood loss was less in the laparoscopic group than in the open group (63 mL vs. 103mL). There were statistically significant differences in the post-operative pain scores, and postoperative complications compared to group B (p<0.001). The mean hospital stay was less in group A (p<0.03), which was significant. ABSTRACT.CONCLUSION: Laparoscopic surgery reduces analgesic requirements, hospital stay, and blood loss. The disadvantages include the reduced working space, the cost of equipment and the availability of a trained surgeon. BODY.INTRODUCTION: Retroperitoneal laparoscopy for renal surgery is a viable alternative to transperitoneal access. In spite of various significant advances in laparoscopy technologies, laparoscopic urologic surgery remains technically demanding regarding various surgical steps including the challenge of specimen retrieval and extraction [1]. The indications for open renal surgery to treat renal calculi are limited to special situations; it is needed in only 0.47% to 5.4% of the cases [2]. Laparoscopy is a preferable approach for large renal stones with an excellent stone-free rate, especially when it requires a single session [3]. The development of both approaches (transperitoneal/retroperitoneum) paralleled during the last two decades however, retroperitoneal laparoscopy witnessed a steep learning curve because of the constraint of working space [4]. The risk of spillage depends upon the size of the cyst, surgical technique, experience and the site from where specimen extracted [5]. It is yet to be established the role of retroperitoneal pyelolithotomy (RPL) though each modality has its specific role in the management of large renal stones [6]. The studies evaluated that the chances of bleeding and hospital stay are less with the retroperitoneal laparoscopic method for the management of complex renal stones [7]. Retroperitoneoscopic nephrectomy should also be offered as a standard treatment modality to patients needing pre-transplant or post-transplant native kidney nephrectomy for different indications [8]. Even after the advent of balloon dissection techniques, retroperitoneoscopic pyelolithotomy has not gained much popularity [9]. Our study compared the RPL versus open procedure for solitary large pelvic stones and evaluated the advantages and disadvantages of both procedures. BODY.MATERIAL AND METHODS: The present prospective clinical study was carried out in Department of Surgery, at Suchkhand Hospital, Agra, India from January 2009 to February 2016. Written informed consent was obtained from each patient, and it was approved by the ethical committee of our institution. In the present study, a total number of 1700 patients of either gender and in the age group of 12 to 70 years admitted to surgery was included. We compared the results of laparoscopic retroperitoneal and open pyelolithotomy. The study was divided into 2 groups. In Group A - 850 patients underwent Retroperitoneal Laparoscopic Pyelolithotomy (RLP) and in Group B - 850 patients underwent open pyelolithotomy. Patients' age ranged from 12 – 70 years, all unilateral and bilateral solitary pelvic stones (2–3 cm), intrarenal and extrarenal pelvis were included (Table I). Patients were excluded if age <12 and >70 years, pelvic stones less than 2 cm and more than 3 cm, multiple renal calculi and ureteric calculi either single or multiple. Patients with congenital or acquired anatomical abnormalities, previous history of renal surgery, bleeding disorders, pregnant cases, cardiac problem, disturbed renal function were excluded from the study. The pre-operative assessment of all the patients included the following investigations: plain X-ray and ultrasound of the KUB, renal function tests, urine routine and microscopy examination, intravenous urography. The patients with urinary infection received a course of antimicrobial therapy and they were taken up for the procedure after the urine culture was sterile. If required, plain CT scan (Computed Tomography), DTPA scan (Diaethylene Triamine Pentaetic Acid) were also performed. BODY.OPERATIVE DETAILS: All the patients received routine pre-operative and post-operative antibiotics (Ceftriaxone 1gm, Amikacin 500mg, and Metrogyl 100ml). The patient was later moved into a right or left lateral position, depending on the side of the patient on which the operation was performed. Three ports were made; the 1st port of 1.5 cm size was at the lateral border of the erecta spinae in the line of the umblicus. A long hemostatic artery was inserted into the retroperitoneal space to create the space followed by index finger to make sure correct plane by feeling the kidney. Then a working space was created retroperitoneally by a balloon (glove finger) filled with 150 ml of water and kept in place for a minimum of 3 minutes to achieve bloodless space. Two other working ports 5 mm in size, one in the renal angle just below the 12th rib at the lateral border of the sacrospinalis muscle, and the 3rd was anterior and 1 cm above the anterior superior iliac spine (Figure 1). The 5 mm one was converted into 8 mm port if required to insert the cold knife to give pelvic incision or directly pelvis can be opened with the scissor. Finally, a Hasson trocar was inserted in the middle of the port of 10 mm size and fixed to the musculature with a silk no 1 suture in order to avoid air leakage and subcutaneous emphysema. CO2 insufflation was performed until reaching 12-mm Hg tension. The ureter recognized and the renal pelvis identified, the incision was made with endoknife/cold knife or directly with scissor (Figure 2). The incision in the renal pelvis will be made as high as possible and will be linear or curvilinear depending upon the configuration of the stone and the exposure of the renal pelvis. If it was large in size, the 10 mm Mixter forceps was used (Figure 3). The stone was kept at retroperitoneal space. DJ stent was inserted through the 5 mm port with the help of the suction tip through the 5 mm port (Figure 4). We did not insert the DJ before proceeding for surgery. The pelvis closed with intracorporeal knot by absorbable 4.0 - Vicryl sutures. The cystoscope was inserted through the lower 5 mm port and under the guidance of the cystoscope the pelvic stone was removed by the 10 mm port incision site. Ureteric stent was kept for 4–6 weeks and confirmed on X- ray KUB on next day of Surgery. In group B - a standard transverse incision was given retroperitoneally and opened in layers. First ureter was identified and then pelvis recognized. Incision was made over the pelvis and stone removed. DJ stent placed inside and pelvis closed with the vicryl 3-0. An abdominal drain was placed in the retroperitoneum and exteriorized through one of the port incisions, and was subsequently removed when the drainage was lower than 30 mL/24 hours in both the groups. The ureteral catheter was removed on average the 2nd day after surgery. The patient will be discharged on the 7th day of surgery or according to his/her condition. The drain will be removed as soon as the drainage becomes minimal (<20 ml). All the patients will be followed up to six months, initially at 15 days and thereafter 1 month and then at 3 & 6 months. Criteria- X- Ray KUB and USG KUB can be done to rule out retained stone. Statistical Analysis – At the end of the study, the data will be collected and analyzed by using appropriate statistical methods. P value of less than or equal to .05 will be taken as the cut off point for statistical significance. BODY.RESULTS: The results were compared in both the groups in term of stone clearance, hospital stay, blood loss, analgesic requirement, and intra and post-operative complications. In the laparoscopic and open groups, the main complaint was flank pain; few cases presented with hematuria, and some presented recurrent urinary tract infections (UTI). The mean age and gender ratio was comparable in both groups. Also there was no significant difference observed in stone size in both groups. The mean stone size in group I was 1.9±0.9 and in group II 2.0±1.3 (Table I). The stone size less than of 2 cm, 2–2.5 cm, 2.5–3 cm in cases 340, 290, 220 in group A and in group B, in cases 355, 294, and 201 respectively. The operative time was substantially less in the group B (64.8±22.4) than in the group A (84.2±41.7) and the mean time was 74.83 min vs. 94.43 min respectively, and this difference was statistically significant (p<0.001). The time required for pelvis closure was lower in the group B (3.4±1.4) than group A (10.2±4.3). The time taken for the stent insertion again was less in group B (4.1±1.9) than group A (9.8±3.7). But our technique was different from other studies for insertion of the stent. We inserted the stent through the suction cannula by the 5 mm port without increasing the port or the size. The mean blood loss was less in the laparoscopic group A than in the open group B (63 mL vs. 103 mL), although this difference was not statistically significant. It was observed that the blood loss, post operative analgesia required and post operative hospital stay was significantly lower in the laparoscopic pyelolithotomy group except for the operative time. Also no blood transfusion was required in laparoscopic group and it was required in 12 cases in group B. The mean time for drain removal was 24.7±8.9 hours in laparoscopic pyelolithotomy as compared to 96.5±16.2 hours in open pyelolithotomy. The mean operative time was significantly higher in the laparoscopic pyelolithotomy group as compared to open pyelolithotomy group. The time required for wound closure was lower in the laparoscopic group (Table II). Minor intra-operative complications were seen in the laparoscopic group, including an inadvertent opening of the peritoneum in 9 patients, stone migration in 35 patients, and the inability to negotiate the DJ stent in three patients which were managed successfully. All of these procedures were completed laparoscopically and without much difficulty. In those cases where we were not able to place a stent the DJ stenting was done through the cystoscopy per urethra. The patient who experienced stone migration into the calyx was managed by localizing the stone using the cystoscope, and the procedure was completed laparoscopically. The patient whose stone could not be located despite an adequate dissection was switched to the open surgery group and was finally managed by open pyelolithotomy with double-J stenting. The patient for whom it was not possible to negotiate the stent from above was managed by stenting the ureter retrogradely using a cystoscope. In the open group, stone fragmentation and stone migration occurred in 40 cases. These cases were managed by thorough normal wash and using a rigid nephroscope for stone localization, respectively. There was no significant difference in the timing of the return of bowel function, the resumption of oral intake or drain removal between the open and the laparoscopic groups. There were a total of 141 post-operative complications, out of which 24 were in the laparoscopic group (A) and 117 in the open group (B). Three patients in the open group developed superficial wound infections, which were managed by a short course of empirical antibiotics against Staphylococcus aureus. One patient experienced a severe wound infection which necessitated skin-stitch removal, twice daily dressing and a broad spectrum antibiotic. The patient was subsequently scheduled for secondary suturing after discharge. Another patient who developed a wound infection experienced a prolonged urinary leak, which was managed by keeping both the drain and the indwelling catheter in place until the leak ceased. In the laparoscopic cohort, 7 complications occurred. One patient developed surgical emphysema, which was managed conservatively; 3 patients developed port site infections, which required daily dressings, and the remaining 2 experienced prolonged urinary leaks with port site infections, which were managed in the same way as in the open group. A patient who had a prolonged ileus was managed by Ryle's tube suction, and oral intake was started on the 4th post-operative day (Table III). Hospital stays and postoperative analgesia requirements: the length of the hospital stay was shorter in the laparoscopic group than in the open group (3.8 vs. 5.13 days; p<0.03). Post-operative pain was quantified using a visual analogue scale (VAS score) and the total quantities of analgesic and diclofenac sodium (intramuscular) used in the postoperative period. The post operative pain as observed by VAS score was significantly lower in the laparoscopic group A as analyzed till the third postoperative day as compared to group B (Table IV). Follow up, patient satisfaction and convalescence (the average period required to return to normal activity in weeks. Patients undergoing laparoscopic surgery rated their overall satisfaction higher. The mean period of convalescence in the open and the laparoscopic groups was 4.75 weeks and 2.64 weeks, respectively; this difference was statistically significant (p<0.001). The laparoscopic surgery was significantly more costly than the open procedure. However, considering the relatively short hospital stays, lower morbidity rates and shorter convalescences, the overall costs associated with the laparoscopic surgery are expected to be less than those associated with the open surgery. Laparoscopic pyelolithotomy is cosmetically superior to open pyelolithotomy. In laparoscopic pyelolithotomy, the average scar size was 3.5 cm (range 3–3.5 cm), while in open pyelolithotomy, the average scar size was 15 cm (range 9–17 cm). This difference was statistically significant (p<0.001). BODY.DISCUSSION: The most common entity worldwide is kidney calculi, a condition whose treatment is widely discussed by urologists. Laparoscopic Pyelolithotomy (LP) may be done where SWL and PCNL are not possible, and also feasible in cases that have renal abnormalities, or have a solitary large stone in renal-pelvic calculus [10]. The retroperitoneoscopic approach is spread widely in which space was created with the help of the balloon. It gives full vision to the urinary tract, easy to mange and prevents the leakage of urine into the peritoneum. The main problem in retroperitoneoscopic approach is lesser working area, due to which it is difficult to do the suturing for the urinary tract [11]. In our experience, to create the retroperitoneal space with help of the balloon is useful, bloodless. We didn't encounter any problem for suturing the pelvis but it is was difficult in intrarenal pelvis cases. Some studies observed that in retroperitoneoscopic surgery alongwith RP, it is sometime difficult to create the pneumoretroperitoneum by the needle and is slow as compared to the transperitoneal approach [12]. In our study, with the help of the curved long artery sheath was pierced to enter the retroperitoneum space and with the help of glove finger, (ballooning) the fat was got separated without any peritoneum breach. They stated that in laparoscopic retroperitoneal pyelolithotomy hospital stays is shorter with better cosmetic results. Even the chances of conversion from retroperitoneum laparoscopy into open surgery are less and few complications. Our results have also showed that LRP is a better option than open surgery. We observed that bleeding was less and one can even identify the ureteric stent if it gets misplaced from the ureter, while inserting it in the ureter. Other authors have reported that the LPL is harmless to the parenchyma so bleeding is higher in PNL which is a frequent complication. They stated that laparoscopic surgery should be considered over open surgery only when expertise is available (Table V) [13]. Our study also declared stone free rate and without any parenchymal or blood loss or major complications and quick recovery. Although in retroperitoneal approach the renal pelvis can be accessed directly which prevents extensive dissection, without any urine and blood leakage into the peritoneal space alongwith early recovery, they preferred the transperitoneal route to access the renal pelvis [14]. So to avoid peritoneal injuries or contamination, we preferred retroperitoneum approach for the renal stones. Though in RPL, space is less for surgery or approaching pelvis closure, these disadvantages may be overcome by the surgeon's experience. Another study stated that despite of absence of landmarks and paucity of space, no significant complications occurred in their study except peritoneal rent prolonged urinary leakage, subcutaneous emphysema and superficial port site infections [15], but in our study we did not encounter any difficulty with the port site position and neither any surgical emphysema or peritoneal tear. To avoid the surgical emphysema, we used the Hasson cannula which prevents gas leakage. Another study has reported that mean surgical time and hospital stay is longer in transperitoneal LP, hemoglobin loss and requirement of blood transfusion is significantly less than PCNL [16]. We fully agreed with this study and our results are comparable. Another study also preferred the retroperitoneal approach to achieve the renal pelvis as it allows direct access to the posterior aspect of the renal pelvis [17]. We agreed with another point that RPL is difficult in cases of past history of surgery. We observed that RPL allows direct access to the renal pelvis, avoids extensive dissection, postoperative recovery is faster and our results were also same as of above study. In our group A, the analgesic requirement was less as compared to group B. there were no radiation exposure in either group. The cosmetic result was again much better than the group B. BODY.CONCLUSION: Laparoscopic pyelolithotomy is a promising alternative for patients who are candidates for open surgery, with an acceptable stone-free rate. RPL is a non invasive procedure as we never used the C arm and is a feasible option that can be recommended for management of renal calculi.

TITLE: Uterine salvage management for atonic postpartum hemorrhage using “modified lynch suture” ABSTRACT.BACKGROUND: To assess the effectiveness of the new modified technique in order to control bleeding in women presenting with atonic, flabby uterus compared to the most commonly described technique of classic B-Lynch suture. ABSTRACT.METHOD: This study included 160 women of uncontrolled atonic postpartum hemorrhage delivered by cesarean section at Ain Shams University Maternity Hospital between January 2013 and October 2015. Participants were randomly assigned following simple randomization procedures (computerized random numbers) and divided into two groups. Group, I (80 patients) operated upon by the modified (new technique) stitch while group II (80 patients) operated upon by the classic technique. The ultimate goals were to stop blood loss after placement of the sutures and avoid life-saving hysterectomy thus preserving the life and fertility of the patient. ABSTRACT.RESULTS: The modified new technique was done in 80 patients with atonic postpartum hemorrhage and it was found to be superior to the classic technique with a success rate 95 % (4 cases needed hysterectomy as a lifesaving measure) compared to 85 % with the classic technique (in 12 cases, a life-saving hysterectomy was done). ABSTRACT.CONCLUSIONS: This technique can replace the classic B-lynch in flabby unresponsive atonic uteri as it has 8 shaped placement of the stitch which causes more firm compression on the uterus and simultaneous bilateral uterine artery ligation. This technique was proved valuable and successful in many patients who suffer from uncontrolled massive postpartum hemorrhage (PPH). BODY.BACKGROUND: Primary postpartum hemorrhage (PPH) refers to the bleeding from the genital tract that is more than 500 ml after vaginal delivery or more than 1000 ml following cesarean section during the first 24 h following delivery of the fetus [1]. However, intraoperative blood loss estimation is inaccurate in most cases; therefore, the American College of Obstetrics and Gynecology has defined it as a drop of hematocrit value of more than 10 % from the predelivery value [2]. PPH accounts for most cases of maternal morbidity and mortality in developing countries [3]. The causes of PPH include trauma, retained placenta, abnormal coagulation that may be congenital or acquired, and atonic uterus, which is one of the preventable causes of primary PPH and accounts for more than 80 % of cases of primary PPH [4]. In March 1997, Lynch [5] published his brace suture for controlling PPH when other primary measures failed. The procedure was simple and effective with the primary goal to compress the uterus without occluding the uterine arteries or uterine cavity [5]. Since this publication, more than 10 variants of uterine compressing sutured have been reported [6–10]. If this method fails, the next step will be vascular ligation (uterine, ovarian and hypogastric) or hysterectomy as a last resort [5]. In the current literature, a new modified technique of classic B-Lynch has been reported that has more effective compression on the uterus with added hemostatic effect of uterine artery ligation. BODY.METHOD: This prospective clinical trial was performed in the Department of Obstetrics and Gynecology, Ain Shams University Maternity Hospital, Cairo, Egypt, between January 2013 and October 2015. A total of 160 women who suffered from PPH during lower segment cesarean section (LSCS) and were not responding to uterotonics were operated upon by the modified technique or the classic one after counseling of the patient and a written consent given by the patient. BODY.METHOD.PATIENT GROUPS: 160 women with PPH refractory to uterotonics were divided into:Group I (modified technique): 80 patients were operated upon by the new techniqueGroup II (classic technique): 80 cases were operated upon by the classical technique of B-Lynch. BODY.METHOD.PATIENT SELECTION: Patients under general anesthesia with atonic postpartum hemorrhage refractory to uterine massage, ecbolics (oxytocin 30 units IM, methergine 0.25 mg, misoprostol 1000 μg rectal) and bimanual compression. If these measures failed, the modified or classic technique was done randomly. 5 min later, the patient is observed abdominally and vaginally for bleeding. If bleeding had stopped, closure of the abdomen with intraperitoneal drain is done, but if bleeding still continued, this represents a failure of the compression sutures thus, internal iliac artery ligation or hysterectomy will be done. BODY.METHOD.EXCLUSION CRITERIA: Patients with traumatic PPH, DIC, bleeding diathesis, retained parts of the placenta or cases with uterine anomalies. BODY.METHOD.OUTCOME: Successful: if the bleeding stopped and no need for hysterectomyUnsuccessful: if the bleeding continued and there is a need for hysterectomy BODY.METHOD.RANDOMIZATION: For allocation of the participants, a computer-generated list of random numbers was used and was kept in Ain Shams Maternity Hospital computer and with research supervisors. Participants were randomly assigned for following simple randomization procedures (computerized random numbers) to 2 treatment groups modified Lynch and classical Lynch groups. Group assignments were allocated according to a computer-generated randomized series, were kept in sealed envelopes. BODY.METHOD.POSTOPERATIVE RESULTS: Vital data, urine output, follow up of any vaginal bleeding, the output of the intraperitoneal drains, hemoglobin and hematocrit concentration. Blood transfusion was given according to patient preoperative hemoglobin and amount of blood loss intraoperative. In stable cases, observations were made for 48 h, then discharging the patient with discharge card, including all the operative details to be rechecked after one week for any abnormality (wound gaping, deep venous thrombosis, puerperal sepsis, uterine wall necrosis, vesicovaginal fistula). BODY.METHOD.STEPS OF THE NEW MODIFIED LYNCH TECHNIQUE (SEE FIG. : Exteriorize the uterus, removal of sutures of lower segment cesarean section. The assistant stretched up the uterus, the 1st stitch is placed 2 cm below the lower segment incision and 2 cm medial to the lateral angle to come on the same side on the upper flap then cross on the fundus to the contralateral side above the uterosacral then to the other uterosacral then to the contralateral flap in a figure of eight fashion then after tightening of this suture the needle is passed through avascular area in the broad ligament to the back while the tape is passed through a window on the opposite side made by an artery forceps to become on the posterior aspect of the uterus. Tightening of the transverse suture is done.Fig. 1Steps of the new modified Lynch technique BODY.METHOD.ADVANTAGES OF THE MODIFIED TECHNIQUE OVER THE CLASSICAL TECHNIQUE: The 8 shaped ligatures appear to be more hemostatic and compressing the uterus. Also, the transverse limb ligates the uterine artery with more compression on the lower segment, so it is more effective in case of placenta previa or bleeding from the lower segment in general. BODY.RESULTS: A total of 160 women was recruited in the current study. The clinic-epidemiological data of women under the study were analyzed, in group I, the mean age was 29.6 ± 4.5 years, the mean parity was 1.55 ± 1.35, the mean weight was 76 ± 13.13151 kg, the gestational age was 39.1 ± 1.1 weeks and the neonatal birth weight was 3.49 ± 0.365 kg compared to group II in whom the mean age was 29.3 ± 5.1 years, the mean parity was 1.6 ± 1.21, the mean weight was 79 ± 12.325 kg, the gestational age was 38.7 ± 1.8 weeks and the neonatal birth weight was 3.49 ± 0.365 kg with no significant difference between the two groups (P < 0.001). The modified new technique was done in 80 patients with atonic postpartum hemorrhage refractory to usual measures and it was found to be superior to the classic technique with a success rate 95 % (4 cases needed hysterectomy as a lifesaving measure) compared to 85 % with the classic technique (in 12 cases, a life-saving hysterectomy was done) as shown in Table 1.Table 1Shows the clinic-epidemiological, operative and postoperative data of women under the studyParameterG1G2 P-valueAge (year) Mean ± SD29.6 ± 4.529.3 ± 5.10.694Parity1.55 ± 1.351.61 ± 1.210.431Weight76 ± 13.15179 ± 12.3250.139Gestational age39.1 ± 1.138.7 ± 1.80.092Neonatal birth weight3.496 ± 0.3653.397 ± 0.3890.099Postoperative blood loss324 ± 105568 ± 2090.0001 (HS)Need for blood transfusion2.8 ± 0.54.2 ± 0.80.0001 (HS)Need for hysterectomy4120.0001 (HS)Hospital stay2.8 ± 1.52.7 ± 1.60.684Duration of procedure6 ± 0.953 ± 1.30.0001 (HS)Pre-operative Hb%9.8 ± 1.210.1 ± 1.30.131Post-operative Hb%8.7 ± 1.18.9 ± 1.80.398Bleeding from multiple bites7130.152Hematometra150.212Wound hematoma441.00Wound infection441.00Fever580.563Table (1) shows that there was no significant difference between the two groups as regards the clinic-epidemiological characters of the study groups and the postoperative data, but the modified new technique was found to be superior to the classic technique with a success rate 95 % compared to 85 % with the classic technique The two groups were compared as regards the complications of the conservative operative intervention, as regards the bleeding from multiple bites; it was clear that it was lower in group I when compared to group II which appears due to the hemostatic added effect of uterine artery ligation but the difference was not statistically significant, the same with the other postoperative parameters as development of hematometra, wound infection or post-operative fever, there was no significant difference between the two groups (Table 1). BODY.DISCUSSION: Postpartum hemorrhage is a potentially life-threatening complication of fetal delivery [11]. It may occur after vaginal delivery (4 %) or caesarean births (6 %) [12, 13]. The most important step in the management of PPH is to identify and correct the underlying cause [14]. Most of the cases of PPH can be controlled by traditional treatment modalities like uterotonics, uterine massage, bimanual compression and balloon tamponade [14]. Uncontrolled PPH is usually managed by different uterine suture techniques (modified B-Lynch, and square suture) or with stepwise devascularization surgical procedures. These techniques have reported variable outcomes and many of the patients finally require emergency hysterectomy [5, 7, 14]. A review of peripartum or cesarean hysterectomy reported an average mortality rate of 4.8 % [15]. This does not mean that hysterectomy caused this high maternal mortality, but critically ill situations requiring this surgery may eventually cause it. Indeed, cesarean hysterectomy is one of the most difficult obstetric surgeries and is always challenging [16]. The surgical method of controlling uterine bleeding by inserting B-Lynch suture has been developed to reduce the incidence of emergency hysterectomy and to preserve fertility in these patients. Because of simplicity of application and less time taken to put the modified B-Lynch stitch, it should be the preferred choice [6]. BODY.DISCUSSION.VARIOUS PARAMETERS IN THE CURRENT STUDY ARE COMPARED AS FOLLOWS: In Khatoon et al. study [17], B-Lynch stitch was applied in 9 cases, i.e. 60 % after vaginal delivery and on 6 cases i.e. 40 % during cesarean section. In the current study the new technique of the B-Lynch stitch was taken on 160 cases during cesarean section, but actually, in our institute the new technique is now being performed on cases also after vaginal delivery and on the closed uterus to avoid the additional bleeding on incision of the uterus but this is still under trial. In a prospective study of Hackenthal et al. [18], Hb difference is 3gm% after using the modified B-Lynch technique and in the current study, the mean intraoperative blood loss was 568 ± 209 ml after classical technique and 324 ± 105 ml after the new technique with a highly significant difference between the two groups. After the blood transfusion according to the clinical condition, there was no significant difference between the two groups as regards the postoperative hemoglobin. In Koh et al. study [19], 4 patients required more than 3 units of blood transfusion and 2 patients did not require any blood transfusion while in the current study the mean units of blood required was 4.2 ± 0.8 in the group who was operated upon by the classical technique and was 2.8 ± 0.5 in the group who was operated upon by the new technique with highly significant difference between the two groups, reflecting the effectiveness of new modified B-Lynch stitch in the control of atonic postpartum hemorrhage. In a study of Anamika et al. [20], time was taken to put stitch was 11 to 20 min in 35 patients and less than 10 min in 3 patients, more than 20 min in 5 patients. In Our study, time taken to put stitch of the classic technique was 3 ± 1.3 while the time taken to put stitches of the new technique was 6 ± 0.95 with highly significant difference between the two groups, but this factor may be changed with more training and familiarity with the new technique and must be kept in mind the effectiveness of the new method. Study conducted by Hackenthal et al. [18] and Anamika et al. [20] had a success rate of 100 %, thus proving that this technique was highly effective and 1 patient died on the 21st postpartum day due to Acute Respiratory Distress Syndrome and Septicaemia In Our study success rate was 85 % with the classic technique and 95 % with the new technique with highly significant difference between the two groups with no mortalities.. In a prospective study conducted by Ghodake et al. [21], 31 patients underwent B-Lynch stitch, out of which 5 patients had a post-operative fever, 3 patients had surgical wound gaping. In our study, 8 patients had a post-operative pyrexia in the group operated upon by the classic technique and 5 patients in the group operated upon by the new technique with a highly significant difference between the two groups. But as regards wound infection and gaping there was no significant difference between the two groups. In Our study, there were no major complications. BODY.CONCLUSION: The new technique of the B-Lynch is highly effective in controlling an atonic postpartum hemorrhage so we suggest strongly this technique as an alternative safe option to stop an atonic postpartum hemorrhage. There was no adverse effect on the fertility potential for the observed 2 years; however, a long-term follow-up is required to comment on its actual rate. All pertinent study information was explained to them and they were informed that rejection or withdrawal from the study will not affect any medical service provided. A summarized study information sheet was shown to all cases before obtaining their verbal agreement. Finally, an informed verbal consent was obtained and witnessed by the attending nurse. A log book was created including the participant's study number and the date of consent. The IRB waived the requirement for taking a written consent as the research had minimal risk of harm to subjects and involved no risky procedures for which written consent is required.

TITLE: Effects of Adding Sodium Nitroprusside to Semen Diluents on Motility, Viability and Lipid Peroxidation of Sperm in Holstein Bulls ABSTRACT.BACKGROUND: Nitric oxide (NO) that plays important role in all sexual activities of animals is made from the amino acid L-arginine by the enzymatic action of NO synthase (NOS). NO makes a band with sulfur-iron complexes, but due to production of steroid sexual hormones related to the enzymes involved in this complex, NO can change the activity of these enzymes. NO affects many cells including vein endothelial cells, macrophages and mast cells. These cells are also found in Leydig cells; therefore, they are important source of NO in testis tissue. Therefore, minimizing damages to sperm at the time of freezing thawing process are really important. The aim of this study was to determine the appropriate NO concentration to be added to the freezing extender to improve the quality of thawed sperm. ABSTRACT.MATERIALS AND METHODS: In this experimental randomized study, sperms of four Holstein bulls with an average age of 4 were collected twice a week for 3 weeks. They received sodium nitroprusside (SNP) in concentrations of 0, 10, 50 and 100 nmol/ml. Data analysis was performed using the special issue and static (SAS) 98 software. Also, mean comparison was done using Duncan's multiple ranges test (P<0.05).This research was conducted at the laboratory of Science and Research Branch, Islamic Azad University, Tehran at spring and summer of 2013. ABSTRACT.RESULTS: All concentrations of SNP used was found to increase motility and viability of spermatozoa at 1, 2 and 3 hours after thawing, significantly (P<0.05), but there was no significant difference at zero time. Different concentrations of SNP reduced the membrane lipid peroxidation level of sperm and increased acrosome membranes integrity, implying that SNP generally improved samples membranes, especially in 50 and 100 nmol/ml concentrations. ABSTRACT.CONCLUSION: According to the obtained results, addition of SNP to semen diluents increases motility and viability of spermatozoa. Also, it reduces membrane lipid peroxidation level that leads to improved sperm function. BODY.INTRODUCTION: Fertility is very important in livestock, especially in bulls, because obtained sperm are used in insemination of other cows. Freezing thawing process causes sperm damage, like loss of lipid membrane integrity, mitochondria activity, and acrosome membrane integrity, leading to a reduction in motility, viability and fertility of sperm (1). Therefore, the most important factor in freezing thawing process is to minimize the sperm damages. Freezing-thawing process causes physiochemical stresses on sperm membrane, leading to decrease quality, motility, viability and fertility of sperm (2). Two types of damages are as follows: Production of lots of free radicals and occurrence of peroxidation of phospholipids in sperm membrane which increases the level of fatty acid oxidation including malondiealdehyde (MDA) (3-5). Nitric oxide (NO) as an active non organic molecule is spreadable and free and non stable which is considered as endothelium-derived relaxing factor in veins. It is made in body from the amino acid L-arginine by the enzymatic action of NO synthase (NOS). After making band with sulfur-iron complexes NO changes the activity of these enzymes. NO is an important transmitter molecule in mammalian cells including human and plays a main role in physiological and pathological processes (6, 7). Effect of NO has been observed on many physiological activities of organs, especially male sexual system, like sperm motility, acrosomic reaction, chemotaxis, ability of sperm to bind to the egg, spermatogenesis and balancing the action of hypothalamic-pituitary-gonadal axis (8). There are few studies about the effects of NO on quality of bulls' sperm, especially on measurement of membrane damage via measuring MDA level. The aim of this study was to determine the appropriate NO concentration to be added to the freezing extender in order to improve the quality of thawed sperm. BODY.MATERIALS AND METHODS: This research was conducted at the laboratory of Science and Research Branch, Islamic Azad University, Tehran at spring and summer of 2013. In this experimental randomized study, four Holstein bulls with a mean age of four years and appropriate quantitative and qualitative characteristics of sperm were selected one month before the test. Bulls (jahed research center) were kept in separate boxes and fed according to the National Research Council (NRC, 2000) (9). BODY.MATERIALS AND METHODS.SPERM METHOD: Sperm was collected twice a week for 3 weeks. Then samples were transferred to laboratory rapidly and incubated at 37 ̊C in a bain-marie. After semen volume, population and sperm motility of each bull were determined, samples were mixed, divided in four aliquots, and diluted using Bioxell (Bioxell Inc., USA) containing various concentrations of sodium nitroprusside (SNP). So, four SNP treatments including 0 (control), 10, 50 and 100 nmol/ml were studied for 6 weeks (replications). After dilution and packing in 0.5 cc pivots, 2 pivots of each sample were transferred to laboratory, and quality and quantity of sperms were analyzed using computer-aided system analysis (CASA). Samples were then frozen using digital freezer and kept in nitrogen tanks. At least after 24 hours, thawed samples were incubated and motility percentage, viability, acrosome status and MDA level were analyzed at 0, 1, 2, and 3 hours being kept at 37 ̊C. Freezing or cry therapy is a process of longterm preservation of cells and tissues at very low temperatures. To perform the insemination, the frozen sperms should reach the proper temperature. Payout melting process was performed in a water bath of 32-35 ̊C. BODY.MATERIALS AND METHODS.MEASURING THE MOTILITY OF SPERMATOZOA: Samples were placed in warm water bath at 37 ̊C for 3 minutes, 5 μl was transferred on a slide, and covered with a covers lip of 18×18 mm. Sperm analysis was done using CASA at 0, 1, 2 and 3 hours after thawing. BODY.MATERIALS AND METHODS.MEASURING VIABILITY PERCENTAGE OF SPERM: This was carried out using eosin staining method, while nigrosin was used here as background color. BODY.MATERIALS AND METHODS.MEASURING THE LEVEL OF MEMBRANE LIPID PEROXIDATION: MDA level was measured by Esterbauer and Cheeseman method (1990) using a spectrophotometer (spectrophotometer uv-varian-CARY50 scan visible, Thermo, USA) at 532 nm wavelength. This method is carried out on the bases of reaction of MDA with thiobarbituric acid (TBA), leading to elimination of double water molecules. BODY.MATERIALS AND METHODS.MEASURING ACROSOME INTEGRITY OF SPERM: In fertility, determining the acrosome percentage is a morphological method for measuring viability of sperm after thawing. For this, sperm motility is inhibited initially by mixing semen with Glutaraldehyde 2% in phosphate buffer (NJ, USA). This buffer fixes the membrane and prohibits its deterioration. About 0.5 ml of semen sample was placed on a slide and was mixed with one drop of buffer. This mixture was scattered on slide, and slide was observed using a contrast phase microscope with ×1000 to ×3000 magnifications. BODY.MATERIALS AND METHODS.MEASURING SPERM MEMBRANE FUNCTIONALITY: About 250 ml of diluted semen was incubated in 1 ml of hypo-osmotic swelling test (HOST) solution with osmolality of 100 m Osm/kg for 40 to 60 minutes and analyzed around 400 spermatozoa using a contrast phase microscope with ×1000 to ×3000 magnifications. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Data were analyzed using the special issue and static (SAS) 98 software. ANOVA was used to compare the mean values, while Duncan's multiple range test was carried out to analyze the difference between control and treatment groups. The significance level was set at P<0.05. BODY.RESULTS.TOTAL MOTILITY AND PROGRESSIVE MOTILITY OF SPERMATOZOA: According to results, SNP increased total motility of sperms significantly (P<0.05) in comparison with control group (Table 1). It increased progressive motility in 1, 2 and 3 hours after thawing, but at zero hour (immediately after thawing), motility increased only by 100 nmol/ml, indicating that it is not significant (P>0.05). Increase in mobility by SNP was also not significant before freezing (P>0.05). Our findings showed that there is no significant difference in 100-nmol treatment regarding total motility of spermatozoa (P>0.05) (Table 1). In case of progressive motility of spermatozoa, the first and second hours after thawing showed the best results, while among SNP concentrations, 100 nmol showed a significant difference compared to the control (Table 2). Table 1 The effects of different concentration of SNP on total motility of spermatozoa at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 74.08 ± 1.08a 77.08 ± 1.80a 76.5 ± 1.08 a 79.75 ± 1.85 a Immediately after thawing 57.00 ± 2.06 a 50.16 ± 1.08 a 49.25 ± 1.08 b 56.08 ± 2.02 a 1 hour after thawing 45.33 ± 1.09 a 54.08 ± 2.8 b 53.75 ± 2.8 b 58.41 ± 2.10 b 2 hours after thawing 41.42 ± 1.02 a 48.33 ± 2.8 b 46.83 ± 2.3 ab 50.66 ± 2.10 b 3 hours after thawing 33.83 ± 1.03 a 41.66 ± 1.06 b 39.58 ± 1.05 b 45.08 ± 1.09 b The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05). Table 2 The effects of different concentration of SNP on progressive motility of spermatozoa at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 39.75 ± 2.07 a 42.41 ± 2.6 a 45.91 ± 2.73 ab 47.25 ± 2.8 b Immediately after thawing 37.08 ± 1.93 a 34.25 ± 1.09 a 34.75 ± 1.09 a 40.08 ± 2.1 a 1 hour after thawing 27.41 ± 1.07 a 36.41 ± 1.7 b 35.83 ± 1.7 b 40.33 ± 2.5 b 2 hours after thawing 24.66 ± 1.04 a 29.91 ± 1.35 b 30.08 ± 1.35 b 35.33 ± 1.49 c 3 hours after thawing 20.91 ± 1.03 a 26.08 ± 1.30 b 27 ± 1.32 b 27.58 ± 1.36 b The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05). BODY.RESULTS.SPERM VIABILITY: SNP affected sperm viability and improved this parameter 1 hour after thawing. All concentrations of SNP increased viability significantly (<0.05). This increase was observed in second hour only in 10 and 50 nmol/ml concentrations. Three hour after thawing, all concentrations increased spermatozoa viability significantly (P<0.05). However, none of the used treatments could increase viability in zero treatment (Table 3). BODY.RESULTS.LIPID PEROXIDATION: Different treatments of SNP reduced this parameter significantly (P<0.05) and damaged sperm membrane at 1 and 2 hours after thawing, but in zero time only 50 nmol caused a significant (P<0.05) reduction (Table 4). BODY.RESULTS.ACROSOME INTEGRITY OF SPERM: Variance analysis of data showed that before freezing, SNP increased this parameter significantly (P<0.05) only in 100 nmol/ml treatment. Also immediately after thawing, 100 nmol treatment increased acrosome integrity significantly (P<0.05). In first hour after thawing, 50- and 100- nmol treatments increased this parameter, while all concentrations at second and third hours increased this parameter compared to the control group. The highest ratio of healthy spermatozoa was observed in 100-nmol treatment (Table 5). BODY.RESULTS.SPERM MEMBRANE FUNCTIONALITY: SNP at all different hours after thawing and even before freezing could reduce the membrane damage significantly (P<0.05) in all treatments, while 100 nmol/ml treatment demonstrated the best results during the first hour after thawing (Table 6). Table 3 The effects of different concentration of SNP on viability of spermatozoa at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 79.66 ± 1.07 a 82.75 ± 1.5 a 82.08 ± 1.09 a 84.66 ± 1.72 a Immediately after thawing 65.00 ± 2.4 a 59.50 ± 2.08 a 59.08 ± 2.04 a 64.58 ± 2.4 a 1 hour after thawing 52.50 ± 1.07 a 60.08 ± 1.09 b 60.25 ± 1.6 b 64.75 ± 1.77 b 2 hours after thawing 48.91 ± 1.06 a 55.33 ± 1.6 b 54.33 ± 1.5 b 57.33 ± 1.69 a 3 hours after thawing 40.75 ± 1.03 a 48 ± 1.38 ab 46.5 ±1.3 b 51.33 ± 2.01 c The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05). Table 4 The effects of different concentration of SNP on level of membrane lipid peroxidation at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 0.20 ± 0.05 a 0.19 ± 0.05 a 0.15 ± 0.01 b 0.16 ± 0.01 ab Immediately after thawing 0.17 ± 0.008 a 0.15 ± 0.005 ab 0.11 ± 0.001 b 0.14 ± 0.005 c 1 hour after thawing 0.21 ± 0.009 a 0.14 ± 0.002 b 0.14 ± 0.002 b 0.12 ± 0.001 b The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05). Table 5 The effects of different concentrations of SNP on acrosome integrity of spermatozoa at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 75. 5 ± 0.09 a 74.16 ± 0.09 b 72.83 ± 0.08 c 78.66 ± 1.05 d Immediately after thawing 55.66 ± 0.01 a 50.00 ± 0.09 b 47.16 ± 0.08 c 56.83 ± 0.02 d 1 hour after thawing 38.33 ± 0.05 a 38.16 ± 0.04 a 39.33 ± 0.05 b 46.33 ± 0.08 c 2 hours after thawing 29.08 ± 0.04 a 33.00 ± 0.06 b 38.33 ± 0.08 c 38.33 ± 0.08 c 3 hours after thawing 20.25 ± 0.04 a 22.16 ± 0.06 b 25.16 ± 0.09 c 27.00 ± 0.2 d The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05). Table 6 The effects of different concentrations of SNP on membrane functionality at various thawing times Treatment (SNP) 0 nmol/ml 10 nmol/ml 50 nmol/ml 100 nmol/ml Before freezing 74.79 ± 0.06 a 80.79 ± 0.42 b 83.87 ± 0.5 c 87.83 ± 0.61 d Immediately after thawing 68.54 ± 0.07 a 76.45 ± 0.5 b 79.54 ± 0.7 c 82.25 ± 0.75 d 1 hour after thawing 67.29 ± 0.05 a 74.37 ± 0.3 b 79.75 ± 0.5 c 84.08 ± 0.57 d 2 hours after thawing 69.37 ± 0.07 a 75.00 ± 0.43 b 79.37 ± 0.6 c 83.66 ± 0.73 d 3 hours after thawing 72.00 ± 0.07 a 75.83 ± 0.18 b 78.12 ± 0.3 c 83.95 ± 0.57 d The data are presented as mean ± SD. SNP; Sodium nitroprusside. Common letters in each row indicate no significant difference (P>0.05). BODY.DISCUSSION: NO is an important biological molecule which plays critical role in sperm physiology like sperm chemotaxis, sperm motility, and spermatogenesis (8). Several studies have reported sperm motility in low concentrations of NO in mice (10), sheep and human (11, 12). Direct and significant correlation was seen between NO concentrations and sedentary sperm numbers (13, 14). Some researchers have shown that NO is the main activator of guanylate cyclase (ubiquitous enzymes). It is noteworthy to mention that considerable amount of cyclic guanosine monophosphate (cGMP) obtained from this enzyme causes acrosome reaction, chemotaxis and sperm-egg reaction (8, 15). In this study, increasing effects of SNP on some parameters like total motility and progressive motility were in agreement with some studies (15-17), whereas some other studies have reported different results which is due to various SNP concentrations (11, 18-20). Previous studies have shown that concentrations higher than nmol per liter can reduce sperm activity due to toxicity of NO, but NO in low concentrations increases quality parameters of sperm. The concentrations of 50 and 100 nmol/ml are ideal which are reported by Sharma and Aqarwal (16). SNP increased sperm viability and improved sperm parameters which are consistent with some studies (16, 17) and not consistent with others (11, 19, 20). Membrane lipid peroxidation and acrosome integrity are considered as the best indications of a healthy sperm membrane. Our findings showed that different concentrations of SNP increased HOST and incubation times, while reduced MDA level in MDA test indicating controlling effect of NO on lipid per oxidation. Other studies also confirmed these results (17, 21). By studying lipid per oxidation process, we found that membrane injures resulted in reduction in motility and death of sperm. Disorders in balance of oxygen free radicals and in difference mechanism eliminating these radicals lead to reactive oxygen species (ROS) accumulation and induction of oxidative stress that results in damage of proteins, membrane lipids and other cell components (22). It has been reported that role of NO in prohibition of lipid per oxidation is associated with its ability in reaction with alkoxy lipid radicals (LX), lipid peroxyl (LOO) and chain-breaking oxidation (23, 24), which is in agreement with results of this study. Furthermore our results indicated that SNP improved the health of acrosome integrity compared to control, while the best effect was achieved for 100 nmol/ml treatment, which was in agreement with other studies (15). Researchers have emphasized mostly on morphological changes of sperm like twisted tail, injured membranes and injured acrosomes (25). Apparently, injured sperm in freezing process increases ROS production which causes damage to other normal sperms, but SNP reduces acrosome injury via decreasing oxidative stress. BODY.CONCLUSION: Based on the obtained results, SNP may improve motility, viability, acrosome and plasma membrane integrity during freezing-thawing process. Addition of SNP before freezing provides better results for bovine semen cryopreservation than its inclusion in the thawing extender.

TITLE: Intravitreal versus Posterior Subtenon Injection of Triamcinolone Acetonide for Diabetic Macular Edema ABSTRACT.PURPOSE: To compare the short-term effects of intravitreal versus posterior subtenon injection of triamcinolone acetonide for diabetic macular edema. ABSTRACT.METHODS: This is a prospective and interventional study. Sixty eyes of 60 patients who had diffuse diabetic macular edema were assigned to receive a single intravitreal injection (4 mg) or a single posterior subtenon injection (40 mg) of triamcinolone acetonide. The central retinal thickness was measured using optical coherent tomography before injection and at 1 and 3 months after injection. Visual acuity and intraocular pressure (IOP) were also measured. ABSTRACT.RESULTS: Both intravitreal and posterior subtenon injections of triamcinolone acetonide resulted in significant improvements in visual acuity at 1 month and 3 months after injection. Both groups resulted in a significant decrease in central macular thickness (CMT) at 1 month and 3 months post-injection. IOP in the intravitreal injection group was significantly higher than in the posterior subtenon injection group at 3 months after injection. ABSTRACT.CONCLUSIONS: The posterior subtenon injection of triamcinolone acetonide had a comparable effect to the intravitreal triamcinolone injection and showed a lower risk of elevated IOP. Posterior subtenon injection of triamcinolone acetonide may be a good alternative for the treatment of diffuse diabetic macular edema. BODY: Diabetic macular edema is a common cause of visual loss in patients with diabetic retinopathy. It may occur by focal leakage from microaneurysms, which is often associated with intraretinal lipid deposition in a circinate pattern, and by diffuse leakage from the perifoveal retinal capillaries. In the Early Treatment Diabetic Retinopathy Study (ETDRS), focal laser photocoagulation was applied for leakages from the microaneurysms and grid laser photocoagulation was applied for areas of diffuse capillary leakage.1,2 However, several studies have shown that eyes with diffuse macular edema carry a particularly poor prognosis despite laser photocoagulation.3,4 In the ETDRS,1 only 17% of the eyes had any improvement in visual acuity and less than 3% had a visual improvement of 3 or more ETDRS lines. In recent years, the intravitreal administration of triamcinolone acetonide has provided promising results for the treatment of diffuse macular edema.5-9 However, the risk of ocular complications such as intraocular pressure (IOP) elevation,10-12 endophthalmitis,13 retinal detachment and glaucoma was reported. Posterior subtenon injection of a steroid is an alternative method with proven effectiveness in various ocular diseases like cystoid macular edema and intermediate uveitis. Posterior subtenon injection of triamcinolone has also been reported to be an effective and safe treatment for diffuse diabetic macular edema.14 We performed a prospective study to compare effectiveness and safety between intravitreal and posterior subtenon injection of triamcinolone acetonide for the treatment of diffuse macular edema. BODY.MATERIALS AND METHODS: Sixty eyes of 60 patients with macular edema involving the fovea were enrolled in this study. Diffuse macular edema was defined by central thickening on a biomicroscopy using a 90-diopter noncontact lens and by diffuse fluorescein leakage on fluorescein angiography. Central macular thickness (CMT) was required to be >250 μm on an optical coherent tomography (OCT). Patients with other pathologies of macula or optic disc such as glaucoma or ocular hypertension were excluded. Patients with extensive foveal ischemia with more than one disc diameter of capillary closure on fluorescein angiography were excluded. Patients who had undergone intraocular surgery or macular grid laser photocoagulation within 3 months prior to the injection were excluded. One of the authors (K.H.) performed intravitreal injection and another (J.O.) performed posterior subtenon injection during the same period. The physicians who checked visual acuity, IOP and CMT had not been informed of the purpose of this study or the assignment schedule. Intravitreal injection was done under sterile conditions in the operating room. Eyes receiving the intravitreal injection were anesthetized by topical instillation of 0.5% proparacaine hydrochloride. Under the operating microscope, 0.1 ml of triamcinolone acetonide (4 mg) was injected slowly via a 30-gauge needle through the pars plana, 3.0 mm posterior to the limbus in the pseudopakic eyes and 4.0 mm posterior to the limbus in phakic eyes. For the posterior subtenon injection, under topical anesthesia (0.5% proparacaine hydrochloride), a small incision (8 mm posterior to the limbus) was made through the superotemporal forniceal conjunctiva and tenon's capsule to bare the sclera using Westcott scissors. The curved portion of the pinpoint cannula was inserted and a volume of 1 ml containing 40 mg triamcinolone was slowly injected behind the eye through the incision site. After the triamcinolone application was completed, the cannula was withdrawn slowly, with gentle pressure maintained by a sterile swab along the path of the cannula. No drug reflux was observed. The patients were evaluated on the basis of central retinal thickness from OCT, visual acuity and IOP. All the patients underwent these examinations before injection and at 1 week, 1 month and 3 months post-injection. During the OCT procedure, each eye underwent six radial scans centered on the fovea. The value which printed on the OCT automated mode determined the central retinal thickness. The best-corrected visual acuity on the decimal charts was examined at each visit and was converted to the logarithm of minimal angle of resolution (logMAR) scale for statistical analysis. The IOP was measured using Goldmann applanation tonometry and the number of glaucoma medications used was recorded. The temporal changes in logMAR visual acuity, central retinal thickness and IOP were compared using the Wilcoxon signed rank test. The differences between groups in logMAR visual acuity, central retinal thickness, IOP and other continuous variables were compared using the Mann-Whitney U test. BODY.RESULTS: The mean age of the patients (±SD) was 60.69±10.8 years, with a range of 46 to 70 years. The mean (±SD) duration of diabetes was 16.3±8.1 years (range, 4-30 years). Patient demographics are shown in Table 1. No statistically significant differences were found between groups regarding age, gender, the ratio of left to right eyes, the ratio of PDR to NPDR or DM duration. The mean (±SD) visual acuity before triamcinolone acetonide injection and at 1 week, 1 month and 3 months thereafter is shown in Table 2 and Fig. 1. The mean baseline visual acuity was not significantly different between the 2 groups (p=0.338). In both groups, the visual acuity significantly improved throughout the study. In the intravitreal injection group, the mean visual acuity at 1 month (0.571±0.393; p=0.001) and 3 months (0.535±0.382; p=0.001) after the injection were significantly better than baseline measurements. In the posterior subtenon injection group, the mean visual acuity at 1 month (0.650±0.281; p=0.011) and 3 months (0.623±0.264; p=0.007) after the injection was also significantly better than the baseline measurements. Between the 2 groups, there were no significant differences in the mean visual acuity changes before injection or at 1 month and 3 months after injection. The mean baseline CMT was not significantly different between the 2 groups (p=0.461). In both the intravitreal and posterior subtenon injection group, the CMT was significantly decreased through the study (Table 3, Fig. 2). At 3 months after injection, OCT demonstrated a reduction of the mean CMT (intravitreal group: 46.2%, subtenon group: 43.5%). Between the 2 groups, there was no significant difference in the mean CMT change before injection or at 1 month and 3 months after injection. Fig. 3 illustrates the changes in the OCT images of a representative patient in the intravitreal injection group and of a patient in the posterior subtenon injection group. The mean baseline IOP did not show differences between the 2 groups (p=0.524). In the intravitreal injection group, IOP tended to rise after the injection although the change was not statistically significant (Table 4, Fig. 4). Ten eyes (33%) experienced an IOP elevation to 21 mmHg or higher during the follow-up period. In the posterior subtenon injection group, a temporal change of IOP was not found. Only 1 eye (3.3%) experienced an IOP of 21 mmHg or higher during the follow up period. At 3 months after injection, the IOP change of the intravitreal injected eyes was greater than that of the posterior subtenon capsule injected eyes (p=0.026). BODY.DISCUSSION: This study demonstrates that intravitreal injection or posterior subtenon injection has a beneficial effect in reducing the diabetic macular edema. Two groups did not show any significant difference in visual acuity or mean CMT thickness improvement after injection. In this study, the change of mean visual acuity (LogMAR) after intravitreal injection was 0.160 (21.8%) at 1 month and 0.196 (26.8%) at 3 months after the injection. The change of mean CMT was 171.6 μm (40.1%) at 1 month and 197.7 μm (46.2%) at 3 months after intravitreal injection. These results were similar to previous studies. Martidis et al.5 reported that CMT decreased by 55% and 57.5% at 1 and 3 months after intravitreal trimcinolone injection, respectively. Ciardella et al.8 reported decreases of 42% and 46.4%. Jonas et al.9 reported that visual acuity(LogMAR) improved by 0.15 (15.3%) and 0.19 (19.3%) at 1 and 3 months after intravitreal trimcinolone injection, respectively. Posterior subtenon injection of triamcinolone has been used with proven effectiveness in conditions with a breakdown in the blood-retinal barrier, such as cystoid macular edema and intermediate uveitis. Recently, Ohguro et al.14 reported an observational case series indicating the effectiveness of posterior subtenon triamcinolone infusion in diffuse diabetic macular edema in eyes that had not responded to vitrectomy. Bakri et al.15 also reported that visual acuities remained stable or improved over a 12-month period after posterior subtenon triamcinolone injections for refractory diabetic macular edema. These recent results have a general connection with our study. Freeman et al.16 have shown by ultrasound B-scan that the superotemporal placement technique results in more accurate placement of steroids near the macula. Geroski et al.17 reported the usefulness of the transscleral pathway in delivering the drug to the retina. Weijtens et al.18 reported that the intravitreal concentration of the steroid increased after its peribulbar injection. On the basis of these reports, it can be summarized that the injected triamcinolone is located on the subtenon macular area and its therapeutic concentration on the choroids or retina can be obtained through the transscleral pathway. Cardillo et al.19 compared intravitreal injection with posterior subtenon injection of triamcinolone in diabetic macular edema. They concluded that the intravitreal injection was more favorable than the posterior subtenon injection for the anatomic and functional aspects of improvement. Bonini-Filho et al.20 also suggested that intravitreal injection may be more effective than posterior subtenon injection for the management of refractive diffuse diabetic macular edema. These two studies differ from our results in that our study show that both intravitreal and posterior subtenon injections may be equally tolerated with a short-term performance. The study of Cardillo et al was informative in that two approaches were performed to each eye of the same patient with bilateral symmetric diffuse macular edema. However one limitation of the study was the relatively small sample size (12 patients). In Bonini-Filho et al's study, the patients had refractive diabetic macular edema. Thus, their results cannot directly be compared to our results. One advantages of posterior subtenon administration is a lower risk of complication. IOP elevation is the most common complication after intravitreal triamcinolone injection.10-12 Although not statistically significant, IOP after intravitreal injection tended to rise in our study. At 3 months after injection, the change of IOP in the intravitreal injection group was greater than that of the posterior subtenon injection group. Other complications such as endophthalmitis and retinal detachment were also reported following intravitreal injection in other studies. In conclusion, the short-term efficacy of the intravitreal injection and of the posterior subtenon injection of triamcinolone in diffuse diabetic macular edema was similar. The posterior subtenon injection was less invasive and safer than the intravitreal injection. Therefore, posterior subtenon injection of triamcinolone acetonide may be a good option for the treatment of diffuse diabetic macular edema.

TITLE: The comparative effectiveness of Integrated treatment for Substance abuse and Partner violence (I-StoP) and substance abuse treatment alone: a randomized controlled trial ABSTRACT.BACKGROUND: Research has shown that treatments that solely addressed intimate partner violence (IPV) perpetration were not very effective in reducing IPV, possibly due to neglecting individual differences between IPV perpetrators. A large proportion of IPV perpetrators is diagnosed with co-occurring substance use disorders and it has been demonstrated that successful treatment of alcohol dependence among alcohol dependent IPV perpetrators also led to less IPV. The current study investigated the relative effectiveness of Integrated treatment for Substance abuse and Partner violence (I-StoP) to cognitive behavioral treatment addressing substance use disorders including only one session addressing partner violence (CBT-SUD+) among patients in substance abuse treatment who repeatedly committed IPV. Substance use and IPV perpetration were primary outcome measures. ABSTRACT.METHOD: Patients who entered substance abuse treatment were screened for IPV. Patients who disclosed at least 7 acts of physical IPV in the past year (N = 52) were randomly assigned to either I-StoP or CBT-SUD+. Patients in both conditions received 16 treatment sessions. Substance use and IPV perpetration were assessed at pretreatment, halfway treatment and posttreatment in blocks of 8 weeks. Both completers and intention-to-treat (ITT) analyses were performed. ABSTRACT.RESULTS: Patients (completers and ITT) in both conditions significantly improved regarding substance use and IPV perpetration at posttreatment compared with pretreatment. There were no differences in outcome between conditions. Completers in both conditions almost fully abstained from IPV in 8 weeks before the end of treatment. ABSTRACT.CONCLUSIONS: Both I-StoP and CBT-SUD+ were effective in reducing substance use and IPV perpetration among patients in substance abuse treatment who repeatedly committed IPV and self-disclosed IPV perpetration. Since it is more cost and time-effective to implement CBT-SUD+ than I-StoP, it is suggested to treat IPV perpetrators in substance abuse treatment with CBT-SUD+. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov: NCT00847873 BODY.BACKGROUND: Intimate partner violence (IPV) perpetration is highly prevalent among substance abusers. Several studies demonstrated that up to more than half of male and female patients in substance abuse treatments committed at least one act of physical IPV in the past year (e.g., [1-5]; Kraanen, Vedel, Scholing, Emmelkamp: Do specific substance use disorders or combinations of specific substance use disorders predict past year intimate partner violence in patients referred to substance abuse treatment?, submitted). These figures are substantially higher than in the normal population, where about 20% of the population committed IPV in the past year [6]. Also, substance abuse is overrepresented in male and female IPV perpetrators in batterers' treatment [7-13]. The relationship between substance use (particularly alcohol use) and IPV received increased attention over the past years. Several reviews confirmed the association between alcohol use and IPV (e.g., [14-16]). In addition, a number of studies demonstrated that IPV perpetration in alcohol dependent patients decreased substantially after successful treatment for substance abuse (for reviews, see: [17,18]). These results were found when substance abusers were treated individually [4,19] as well as when participants had received couples therapy [20-22]. Moreover, patients who relapsed to alcohol use after successful alcohol use disorder treatment were much more likely to relapse to IPV perpetration than patients who abstained from alcohol [23,24]. Also, the use of illicit drugs (particularly cocaine and cannabis) is connected to IPV perpetration (for reviews, see: [15,25-27]). Presumably, different routes lead from cocaine use and cannabis use to IPV perpetration [25]. For cocaine it is hypothesized that its psychopharmacological effects directly increase aggressive behavior (e.g., [28,29]), whereas for cannabis it is assumed that irritability as a result of withdrawal may lead to committing IPV [30]. To our knowledge, it has not been studied yet whether successful treatment of cannabis and cocaine use disorders in patients who are referred for substance use treatment and commit IPV, also reduces IPV perpetration. Results described above indicate that successful treatment of alcohol dependence in substance abusing IPV perpetrators leads to reduced IPV perpetration. Particularly since there are no evidence-based treatments for IPV perpetrators, these results are valuable. Several meta-analyses examined the effectiveness of IPV treatment; feminist psychoeducation (Duluth model) as well as cognitive behavioral interventions were found to have effect-sizes near zero [31,32] and were thus hardly effective in reducing IPV. There are some indications that couples therapy aiming to reduce IPV may be promising for specific couples and types of IPV [33,34], but it is too early to draw firm conclusions. One major problem of the early treatment studies on IPV is that most treatments were offered in a group format, followed a one-size-fits-all approach, and did not take individual characteristics of patients (such as substance use) into account. Recently, several authors (e.g., [35-37]) claimed that treatments for IPV should be more effectively tailored to specific patient and couple characteristics. Because treatment for alcohol use disorders is effective in reducing IPV perpetration in alcohol abusing patients referred for substance use treatment whereas IPV treatment alone is not, several researchers called for integrating IPV and substance abuse treatment (e.g., [38-41]). However, to date, only one randomized controlled trial (RCT) has been carried out on the effectiveness of such an integrated treatment. [42] conducted a pilot study that compared the effectiveness of a combined alcohol dependence / domestic violence group therapy based on cognitive behavioral therapy (CBT) to a 12-step facilitation group that did not address partner violence. Participants who received the combined treatment abstained significantly more days from alcohol than participants in the 12-step facilitation group and there was a trend for participants in the combined treatment to engage in less frequent IPV than participants in the 12-step group. However, since there were differences in days of abstinence between both conditions and alcohol use is possibly causally related to IPV perpetration, it is necessary to control for days of abstinence when assessing differences between treatments in IPV perpetration. Further, in [42]'s study the treatments for substance abuse in both conditions differed from one another (i.e., CBT vs. 12-step approach). Therefore, it was not possible to determine whether reductions in IPV were attributable to the different treatment of substance abuse or to the focus on IPV in the combined treatment. Also, some participants had no actual intimate relationship. Although these participants may benefit from the treatment in future relationships, it is not possible to measure reductions in IPV perpetration when no partner is present. In addition, only participants who were (also) diagnosed with alcohol dependence were included, whereas research has demonstrated a relationship between use of cocaine and cannabis and IPV perpetration as well. Besides, only men were included whereas a substantial proportion of the IPV perpetrators consist of women [43,44]. The current study compared CBT addressing both substance use disorders and IPV (Integrated treatment for Substance abuse and Partner violence; I-STOP; [45,46]) to CBT addressing only substance use disorders plus one session addressing IPV for enhancing safety (CBT-SUD+). The interventions addressing substance use were equal in both conditions; CBT-SUD+ contained the same topics as I-StoP regarding substance use but had more sessions to address these topics. Further, only participants who actually were in an intimate relationship with the victim at the start of treatment were included. In addition, participants with other (primary) substance use disorders than alcohol dependence could participate. The study was open for male and female patients, and treatments were conducted individually. Aim of this study was to compare the effectiveness of IPV perpetration between I-StoP and CBT-SUD+ on IPV perpetration and reduction of substance abuse among patients in substance abuse treatment who were involved in a pattern of IPV perpetration. It was hypothesized that 1) participants' substance use in both conditions would be significantly reduced at posttreatment compared to pretreatment, 2) there would be no significant differences between conditions regarding substance use at posttreatment, 3) physical IPV perpetration would be significantly reduced at posttreatment compared to pretreatment in participants in both conditions, and 4) patients receiving I-StoP would engage in less frequent IPV at posttreatment compared to patients allocated to CBT-SUD+. Effects on secondary outcome measures (verbal IPV, inflicted injuries, general mental health, marital satisfaction, and treatment satisfaction) were studied exploratory. BODY.METHODS.PARTICIPANTS: Participants were recruited from patients who sought treatment at a substance abuse treatment facility (Jellinek) in Amsterdam, the Netherlands, between August 1, 2009 and June 1, 2012. Patients were included if they 1) disclosed 7 or more acts of physical IPV in the past year at intake, 2) were diagnosed with abuse and / or dependence of alcohol, cannabis and / or cocaine, 3) were in an intimate relationship with the partner against whom they committed IPV, and 4) were triaged to outpatient treatment. We aimed to include patients who committed at least 7 acts of IPV in the past year since the goal of the treatment was to break a pattern of IPV perpetration in an enduring relationship. Therefore, we intended not to include patients to whom IPV perpetration was an incident. Patients who were involved in incidental IPV typically reported 2 or 3 acts of physical IPV (for example, they reported to have pushed, grabbed, and slapped their partner). To select patients who were involved of a pattern of IPV perpetration we decided to select a cutoff that was well above 3 and settled for a cutoff of 7. In addition, patients were recruited on the basis of past year IPV, but IPV as treatment outcome was assessed in blocks of 8 weeks. Another reason to include only patients who were involved in a pattern of IPV perpetration was to minimize the chance that patients would report no acts of physical IPV at pretreatment and could thus not improve during treatment. Finally, only patients who were able to follow outpatient treatment were invited to participate because inpatients were offered various other treatment modules besides substance abuse treatment, including social skills training and emotion regulation treatment, which would overlap with I-StoP. Patients were excluded if they 1) were diagnosed with crack cocaine or heroin abuse or dependence, 2) currently received treatment for other mental health problems, 3) had insufficient knowledge of the Dutch language to complete questionnaires, and 4) in case of severe mental health problems (e.g., psychosis, suicidal ideation) or cognitive disorders (e.g., Korsakoff's syndrome). Patients were excluded in case of crack cocaine and/or heroin use disorders because these patients usually need more intensive treatment than outpatient treatment. Initially it was aimed to include 100 participants in the current study. BODY.METHODS.TREATMENTS: Two treatment protocols were developed for this study, i.e., I-StoP and CBT-SUD+. Both treatments consisted of 16 sessions of 45 minutes and were ideally delivered weekly to the participants. Treatments were conducted individually, but the partner was invited to attend the first session of both I-StoP and CBT-SUD+ in order to check and enhance safety. I-StoP and CBT-SUD+ were flexible treatment protocols, i.e., treatment could be adjusted according to motivation and / or level of functioning of the patient. Furthermore, sessions could be modified in case of crisis, such as relapse to substance use or IPV, or topics could be treated in different order if relevant. BODY.METHODS.TREATMENTS.I-STOP: I-STOP concurrently addressed both substance abuse and IPV perpetration. Substance abuse interventions were based on evidence-based CBT protocols addressing substance abuse adapted for use in The Netherlands [47-50]; for a description of interventions, see [51]). Interventions targeting IPV comprised CBT and were based on the work of Dutton [52,53]. Motivational interviewing techniques [54] were included to increase participant's motivation to change substance abuse and stop IPV perpetration. Sessions primarily targeting IPV and substance use were alternated. In addition, participants received a workbook containing psychoeducation, weekly assignments that correspond to the central topic of a session, and diary cards to daily register substance abuse / craving and anger / perpetration of IPV. Therapists were instructed to address both IPV and substance abuse in each session by emphasizing anger / IPV registrations if the central theme of a session was related to substance abuse, and vice versa. Each session followed the same structure: 1) addressing completed diary cards and assignments, 2) discussing the main topic of the session, and 3) explaining the assignment for the next session. I-StoP contained the following interventions. First, the 'Cycle of Violence' [55] was discussed with the patient and partner and the couple was taught to take time-outs in case of high-risk situations for IPV. It was important that the partner was present in order to maximize the chance that the time out was going to be successful and to minimize the risk that the patient would use the tool abusively, for example, to control the interaction with the partner (for a more detailed description of the negotiated time-out procedure, see [56]). In addition, the following topics and interventions were addressed: explanation of the treatment rationale, assessment of types of IPV that took place in the relationship, assessment of pros and cons of IPV and substance abuse, formulating treatment goals regarding IPV (abstaining from IPV was the only adequate treatment goal) and substance use (preferable abstinence, but controlled substance use was also an accepted treatment goal; see [51]), identifying self-control measures to prevent substance use, making functional analyses of substance use, anger management, coping with craving and emotions that may lead to substance use, the association between thoughts, feelings, and behavior in relation to substance use and IPV, communication skills, and relapse prevention for IPV and substance use. BODY.METHODS.TREATMENTS.CBT-SUD+: CBT-SUD+ is a manualized, cognitive behavioral treatment that can be considered treatment-as-usual for substance use treatment in The Netherlands. For ethical reasons (checking and promoting safety), the first session of CBT-SUD+ is the same as the first session of I-STOP (i.e., a session addressing the 'Cycle of Violence' and time-out procedure with the partner). Sessions followed the same structure as I-StoP-sessions. Further, topics and interventions of CBT-SUD+ were the same as I-STOP interventions addressing substance abuse. However, since CBT-SUD+ treatment consisted of 16 sessions as well, there was twice as much time to discuss these topics. BODY.METHODS.TREATMENTS.THERAPISTS AND TREATMENT ADHERENCE: Five female social workers, who had received formal training in CBT and motivational interviewing and who had extensive experience in substance abuse counseling, were trained in both treatment protocols. PE and FK supervised therapists once every two weeks during supervision sessions lasting 90 minutes. All patients were discussed comprehensively during supervision and meetings focused on adherence to treatment manuals and preparation of future sessions. BODY.METHODS.TREATMENTS.ETHICS AND RANDOMIZATION: The study was registered at the clinical trials registry (http://www.clinicaltrials.gov) (# NCT00847873) and was ap-proved by the Ethics Review Board of the University of Amsterdam (2008-KP-466). Participants were randomly assigned to either I-STOP or CBT-SUD+ using http://www.randomization.com. Outcome of randomization was written on cards and put in a closed envelope containing participant numbers. Envelopes were handed to participants after completing pretreatment assessment. BODY.METHODS.MEASURES.OUTCOME MEASURES.IPV: The Revised Conflict Tactics Scales (CTS2; [57]) was used to assess frequency and prevalence of IPV. The CTS2 consists of 39 item pairs addressing both perpetration and victimization of a specific act of violence. An example is: 'I pushed my partner' and 'My partner pushed me'. Answers are scored on a 7-point scale: 0 = never; 1 = once; 2 = twice; 3 = 3–5 times; 4 = 6–10 times; 5 = 11–20 times and 6 = more than 20 times. The CTS2 comprises of 5 scales measuring different aspects of handling conflicts between partners: 1) physical violence, 2) verbal violence, 3) sexual violence, 4) negotiation, and 5) injuries resulting from IPV. Frequency scores of violent acts were calculated by taking the average of the frequency range (e.g., 3–5 times = 4), as recommended by [57]. The CTS2 is worldwide the scale that is most often used to measure the frequency, type, and gravity of IPV. Previous research showed that the CTS2 is reliable and valid for this goal (e.g., [57-61]). As primary outcome measure, the physical violence subscale was used; the verbal violence and injuries scales were used to assess secondary treatment outcomes. The CTS2 was administered pretreatment, halfway treatment (after session 8), and posttreatment. The partner was invited to complete the CTS2 pretreatment and posttreatment as well. On every occasion IPV was assessed with reference to the past 8 weeks. BODY.METHODS.MEASURES.OUTCOME MEASURES.SUBSTANCE USE: To assess substance use, the Timeline Follow Back Interview (TLFB; [62]) and the Quick Drinking Screen (QDS; [63]) were used. The TLFB is a calendar-based method to assess frequency and quantity of substance use and days of abstinence [64]. The TLFB is often studied and most studies found the TLFB to be a highly valid method to assess alcohol (e.g., [65,66]) and drug use (for a review, see [67]). The instrument was administered pre- en posttreatment to assess substance use in 8 weeks prior to treatment and 8 weeks before the end of treatment. In addition, the QDS was administered. The QDS is a self-report instrument that contains 5 aggregate summary questions regarding alcohol use. Data obtained by the QDS were found to be very similar to data obtained by the TLFB [63,68,69]. The QDS was modified to assess drug use as well. Participants were asked about substance use in the past 8 weeks. The modified QDS was administered to the participant pretreatment, halfway treatment (after session 8), and posttreatment, and assessed substance use in the past 8 weeks. BODY.METHODS.MEASURES.OUTCOME MEASURES.GENERAL PSYCHOPATHOLOGY: To assess general psychopathology, the Brief Symptom Inventory (BSI; [70]; Dutch translation: [71]) was used. The total BSI score provides an overall measure of severity of psychopathology. Psychometric qualities of the BSI are good [72]. Participants completed the BSI at pre- and posttreatment; the BSI assessed symptoms of psychopathology during the past week. BODY.METHODS.MEASURES.OUTCOME MEASURES.MARITAL SATISFACTION: The marital maladjustment-scale of the Dutch version of the Maudsley Marital Questionnaire (MMQ; [73]) was used to assess marital satisfaction. Psychometric qualities are good (e.g., [73,74]). Participants completed the MMQ at pre- and posttreatment. BODY.METHODS.MEASURES.TREATMENT SATISFACTION: Participants were asked at posttreatment to rate treatment satisfaction on a scale from one to ten. BODY.METHODS.CLINICAL DIAGNOSES AND ASSESSMENT OF ELIGIBILITY.PATIENT CHARACTERISTICS: The Measurements in the Addictions for Triage and Evaluation (MATE; [75]) was used to assess patient characteristics and to guide treatment allocation (i.e., allocation to inpatient or outpatient treatment). BODY.METHODS.CLINICAL DIAGNOSES AND ASSESSMENT OF ELIGIBILITY.IPV: The Jellinek Inventory for assessing Partner Violence (J-IPV; [76]) was used to screen patients for past year IPV perpetration. The J-IPV consists of 4 items; a positive answer to at least one item is indicative for IPV perpetration. The J-IPV possesses good psychometric properties to screen for IPV perpetration. In addition, the CTS2 (further named CTS2-screen to distinguish its purpose here from the CTS2 when used as outcome measure) [56] was administered to assess type and frequency of acts of past year IPV perpetration; patients had to have engaged in at least 7 acts of IPV in the past year to be included in the study. BODY.METHODS.CLINICAL DIAGNOSES AND ASSESSMENT OF ELIGIBILITY.AXIS-I DISORDERS: Axis-I disorders were classified using the Structured Clinical Interview for DSM-IV Axis-I Disorders (SCID-I; [77]; Dutch translation: [78]). BODY.METHODS.PROCEDURE: At the intake, the MATE was administered, followed by the J-IPV. Patients who answered positive to one or more J-IPV items were invited for a second intake, which involved administration of the SCID-I and CTS2-screen. Participants who met inclusion criteria and were willing to participate were scheduled for pretreatment assessment. During this meeting, first, the purpose of the study was explained and informed consent was obtained. Then, the TLFB was administered and patients consequently completed the BSI, the QDS, the CTS2, and the MMQ. After that, participants were handed an envelope containing the treatment condition they were randomly assigned to (either I-StoP or CBT-SUD+) and an appointment for participant and partner was scheduled with one of the therapists. After that, treatment started. After session 8, the therapist asked participants to complete the modified QDS and the CTS2. After session 16, an appointment was scheduled for posttreatment assessment, during which, consequently, the TLFB, the BSI, the QDS, the CTS2, and the MMQ were completed. There was no compensation for treatment participation or completion of assessments by patients who completed treatment; patients who dropped out from treatment received 15 euro (about 20 US dollar) if they completed posttreatment assessment. BODY.METHODS.STATISTICAL ANALYSES.PARTICIPANT CHARACTERISTICS: Demographics, current Axis-I disorder diagnoses, frequency of past year IPV and number of dropouts were compared between conditions using chi-square-tests for dichotomous variables, and t-tests and Mann–Whitney tests for normally and nonnormally distributed continuous variables, respectively. BODY.METHODS.STATISTICAL ANALYSES.TREATMENT EFFECTS: For the analysis of treatment effects, both completers and intention-to-treat (ITT) analyses were conducted. For ITT analyses, the last observation carried forward (LOCF) method was used. Patients were classified as completer if they attended at least 75% of treatment sessions. Patients were categorized as ITT if at least one treatment session was attended. Two measures were used to assess changes in substance use: days of abstinence and the average quantity of substances that were weekly used (since controlled substance use was also an accepted treatment goal). Since participants used different substances (measured on different scales, e.g. units or grams) and -in several cases- more than one substance, it was necessary to calculate new (standardized) Z-scores for substance use. First, pre- and posttreatment quantities of 1) alcohol (mean standard units per week), 2) cannabis (mean grams per week), and 3) cocaine (mean grams per week) that had been used by all patients in the preceding 8 weeks were each displayed in a separate column. Then, the data in each column were transformed to Z-scores, after which the Z-scores in each column were again split in the pre- and posttreatment scores. Finally, for pretreatment and posttreatment separately, the Z-scores for alcohol, cannabis, and cocaine were added up in order to obtain a quantity-frequency summary measure of the combined use of different substances at both pretreatment and posttreatment. Days of abstinence and Z-scores were calculated on the basis of the QDS (instead of the TLFB) because QDS scores obtained halfway treatment were of use for the LOCF procedure (the TLFB was administered only at pre- and posttreatment). TLFB outcomes were not further used. IPV outcomes at pretreatment, halfway treatment and posttreatment were assessed by calculating frequency scores of physical IPV (primary outcome measure), verbal IPV, and injuries inflicted to the partner within the preceding 8 weeks. Pre- and posttreatment measures of substance use and IPV were compared within treatments using one-tailed paired samples t-tests for normally distributed values and one-tailed Wilcoxon signed rank tests for non-normally distributed values. ANCOVA's using pretreatment substance use / IPV as covariate were carried out to assess between-treatment differences because initial analyses showed a correlation between the pre- and posttreatment scores on both substance use and IPV, respectively. For a few ANCOVA's the assumption of normality was violated. However, since sample sizes were roughly equal it was assumed that the F-statistic would be relatively robust against violations of normality [79]. Further, Levene's test was used to assess homogeneity of variance. Finally, homogeneity of regression slopes was assessed; for some analyses for the ITT sample this assumption was violated. However, ANCOVA results are relatively unaffected by violations of the assumption of homogeneity of regression when group sizes are equal [80-82]. Based on the above, there was no need to refrain from using ANCOVA's. BODY.METHODS.STATISTICAL ANALYSES.TREATMENT EFFECTS–SECONDARY OUTCOME MEASURES: Pre- and posttreatment BSI and MMQ scores to assess overall psychopathology and marital satisfaction, respectively, were compared within treatments using paired samples t-tests and Wilcoxon signed rank tests; ANCOVA's with pretreatment outcomes as covariate were performed to assess differences in posttreatment BSI and MMQ scores between treatments. An independent samples Mann–Whitney test was used to compare treatment satisfaction across both treatments. BODY.METHODS.STATISTICAL ANALYSES.POWER ANALYSES: We argued that to be clinically meaningful, the difference between pre- and posttreatment IPV should be large. For example, a small decrease in IPV might be statistically significant, but if an IPV perpetrator abuses a partner 3 times instead of 4 times in 8 weeks we did not consider this of clinical importance. The same applied to the difference between treatments. If, for example, IPV would decrease only slightly more in the I-StoP condition than in the CBT-SUD+ condition, these results would not match the effort needed to implement I-StoP in routine clinical care. The number of participants that were needed for a power of .80 was determined using G*Power [83]. The analyses demonstrated that 12 participants per condition were necessary to detect a large difference within conditions (Cohen's d > .80; [84]) at α = .05, one-sided testing; 21 participants per condition were needed to detect a large difference between conditions including one covariate (i.e., a difference between patients who received I-StoP and patients who received CBT-IPV+ after controlling for pretreatment values; Cohen's f > .40; [84]) at α = .05, one-sided testing. BODY.RESULTS.PARTICIPANT CHARACTERISTICS: Figure 1 displays the CONSORT 2010 flow diagram of participants from IPV screening through treatment completion. The final sample consisted of 52 patients who were included in the treatment study; 27 and 25 participants were randomly assigned to I-StoP and CBT-SUD+ respectively. Table 1 displays demographics and clinical characteristics of participants. There were no statistically significant differences between participants receiving I-StoP and participants receiving CBT-SUD+. Figure 1CONSORT 2010 flow chart of participant enrollment, randomization, and retention of the current study. Table 1 Demographics and clinical characteristics of patients at pretreatment   I-StoP CBT-SUD+ Overall   (N = 27) (N = 25) (N = 52) Between group analyses   N (%) N (%) N (%)   Gender            Male 19 (70.4) 17 (68.0) 36(69.2) X 2 (1) = 0.03; p = .85    Female 8 (29.6) 8 (32.0) 16 (30.8)   Age (M, SD) 34.85 (9.87) 37.08 (8.87) 36.18 (9.29) t (50) = 0.43; p = .51 Relationship length (years) (M, SD) 5.08 (4.59) 7.94 (8.59) 6.48 (6.93) U = 270.00; p = .55 Current alcohol use disorder 22 (81.5) 19 (76.0) 41 (78.8) X 2 (1) = 0.23; p = .63 Current cannabis use disorder 13 (48.1) 8 (32.0) 21 (40.4) X 2 (1) = 1.41; p = .24 Current cocaine use disorder 6 (22.2) 9 (36.0) 15 (28.8) X 2 (1) = 2.02; p = .16 Number of SUDs (M, SD) 1.48 (0.64) 1.44 (0.58) 1.46 (0.61) U = 330.50; p = .88 Current major depressive episode 4 (15.4) 6 (24.0) 10 (19.6) X 2 (1) = 0.23; p = .63 Current panic disorder 1 (3.8) 0 (0.0) 1 (2.0) X 2 (1) = 0.98; p = .32 Current social phobia 2 (7.7) 5 (20.0) 7 (13.7) X 2 (1) = 1.63; p = .20 Current OCD 0 (0.0) 2 (8.0) 2 (3.9) X 2 (1) = 2.17; p = .14 Current PTSD 1 (3.8) 4 (16.0) 5 (9.8) X 2 (1) = 2.13; p = .15 Current GAD 1 (3.8) 0 (0.0) 1 (2.0) X 2 (1) = 0.98; p = .32 Past year physical IPV perpetration 32.37 (47.62) 23.00 (23.20) 27.87 (37.84) U = 334.50; p = .96 Past year verbal IPV perpetration 69.19 (43.58) 52.24 (27.54) 61.04 (37.39) U = 2.96.00; p = .21 Past year sexual IPV perpetration (n = 51) 2.65 (9.90) 0.72 (2.51) 1.71 (7.28) U = 301.50; p = .48 Past year inflicted injuries (n = 51) 3.50 (3.96) 4.72 (6.63) 4.10 (5.42) U = 296.50; p = .59 Underwent clinical detoxification 1 6 (24.0) 6 (25.0) 12 (24.5) X 2 (1) = 0.01; p = .94 1  = clinical detoxification took place during the course of treatment if it appeared that patients could not obtain their treatment goal at home and lasted 1 week; I-StoP = Integrated treatment for Substance abuse and Partner violence; CBT-SUD+ = substance abuse treatment; SUD = substance use disorder; OCD = obsessive compulsive disorder; PTSD = posttraumatic stress disorder; GAD = generalized anxiety disorder; IPV = Intimate partner violence. BODY.RESULTS.RETENTION: A total of 19 participants (36.5%) completed at least 75% of treatment sessions (I-StoP: N = 11 (40.7%); CBT-SUD+: N = 8 (32.0%)). Dropout rates did not differ significantly between I-StoP and CBT-SUD+ (p = .51). Two out of 52 participants (1 in each condition) completed the pretreatment assessment but never started treatment and were excluded from further analyses. Reasons for dropout and other information regarding treatment completion/dropout are displayed in Table 2. There were no significant differences between conditions. Table 2 Reasons for dropout and completion rates per condition   I-StoP CBT-SUD+ Overall   N = 25 N = 24 N = 49 Between group analyses   N (%) N (%) N (%)   Reasons for drop-out         Patients could not be contacted 9 (36.0) 10 (41.7) 19 (38.8) X 2 (4) = 6.18; p = .19 Agreement treatment was no longer needed 3 (12.0) 0 (0.0) 3 (6.1)   Did not want treatment anymore 2 (8.0) 4 (16.7) 6 (12.2)   Needed different treatment 0 (0.0) 2 (8.3) 2 (4.1)   No. of sessions attended–ITT M (S.D.) 9.25 (6.54) 8.68 (5.59) 8.96 (6.02) U = 293.50; p = .89 No. of sessions attended–completers M (S.D.) 16.00 (0.00) 15.50 (1.41) 15.79 (0.92) U = 38.50; p = .68 No. of partners that attended first session 15 (60.0) 12 (50.0) 27 (55.1) X 2 (1) = 0.50; p = .48 Relationship intact at posttreatment (completers) 24 (96.0) 24 (100.0) 48 (98.0) X 2 (1) = .98; p = .32 Treatment duration–ITT (weeks) M (S.D.) 22.19 (14.43) 20.21 (12.44) 21.22 (13.39) t (47) = 0.51; p = .61 Treatment duration–completers (weeks) M (S.D.) 32.31 (12.53) 33.93 (6.80) 32.99 (10.22) U = 30.00; p = .25 Completed pretreatment assessment (t1) 25 (100.0) 24 (100%) 49 (100%) - Completed halfway treatment assessment (t2) 11 (44.0%) 13 (54.2%) 24 (49.0%) X 2 (1) = 0.51; p = .48 Completed posttreatment assessment (t3) 11 (44.0%) 8 (33.3%) 19 (38.8%) X 2 (1) = 0.59; p = .44 No. = number; I-StoP = Integrated treatment for Substance abuse and Partner violence; CBT-SUD+ = substance abuse treatment. BODY.RESULTS.PRIMARY OUTCOMES.SUBSTANCE USE: Days of abstinence and substance use Z-scores of completers and ITT sample are displayed in Table 3. Table 3 Pre- and posttreatment substance use (QDS) per condition   Completers Intention-to-treat Variable I-StoP CBT-SUD+ I-StoP CBT-SUD+   M (SD) M (SD) M (SD) M (SD) Days abstinence 8 weeks pretreatment 20.36 (23.27) 17.50 (16.55) 16.48 (10.14) 11.67 (14.96) Days abstinence 8 weeks posttreatment 35.27 (20.54) 40.00 (18.27) 25.28 (22.23) 23.50 (20.54) Z-scores 8 weeks pretreatment 1.55 (3.81) 0.13 (1.06) 0.34 (2.18) 0.33 (1.57) Z-scores 8 weeks posttreatment −0.73 (1.19) −1.19 (0.58) −0.43 (1.45) −0.26 (1.76) I-StoP = Integrated treatment for Substance abuse and Partner violence; CBT-SUD+ = substance abuse treatment. In both treatment conditions the completers had been significantly more days abstinent at posttreatment than at pretreatment (I-StoP: Z (10) = −1.99; p = .02 (1-tailed); SA: (t (7) = −4.17; p = .00). Comparable findings were found on substance use Z-scores at posttreatment versus pretreatment (I-StoP: Z (10) = −2.05; p = .02 (1-tailed); CBT-SUD+: Z (7) = −2.37; p = .01 (1-tailed)). There were no statistically significant differences between I-StoP and SA in posttreatment days of abstinence and substance use Z-scores after controlling for pretreatment values. ITT analyses demonstrated similar results. Participants receiving I-StoP (Z = −2.13; p = .02; 1-tailed) as well as CBT-SUD+ (Z = −3.08; p = .00; 1-tailed) had been significantly more days abstinent at posttreatment than at pretreatment. Also, participants allocated to I-StoP (Z = −1.97; p = .02; 1-tailed) as well as CBT-SUD+ (Z = −2.72; p = .00; 1-tailed) had significantly lower substance use Z-scores at posttreatment than at pretreatment. Again, there were no differences between both conditions in days of abstinence and substance use Z-scores after controlling for pretreatment values. BODY.RESULTS.PRIMARY OUTCOMES.IPV: Table 4 displays physical and verbal IPV perpetration and injuries inflicted to the partner for both treatment groups, at pretreatment and posttreatment and for completers and ITT sample. Table 4 IPV perpetration 8 weeks before pretreatment assessment and 8 weeks before posttreatment assessment of completers and ITT sample per condition   Completers Intention-to-treat sample   I-StoP CBT-SUD+ I-StoP CBT-SUD+ Variable M (SD) M (SD) M (SD) M (SD) Acts of physical IPV perpetration pretreatment 6.91 (6.61) 18.75 (34.87) 12.12 (17.41) 9.91 (20.65) Acts of physical IPV perpetration posttreatment 0.82 (1.40) 0.38 (0.74) 7.84 (14.23) 3.17 (4.78) Acts of verbal IPV perpetration pretreatment 24.27 (18.15) 47.75 (33.49) 37.00 (31.09) 29.41 (26.55) Acts of verbal IPV perpetration posttreatment 12.27 (13.85) 8.62 (6.36) 34.67 (38.68) 14.71 (14.03) Inflicted injuries pretreatment 2.91 (5.63) 1.75 (1.98) 1.32 (1.91) 2.54 (4.05) Inflicted injuries posttreatment 0.09 (0.30) 0.00 (0.00) 1.00 (1.80) 1.21 (3.37) IPV = intimate partner violence; I-StoP = Integrated treatment for Substance abuse and Partner violence; CBT-SUD+ = substance abuse treatment. In both treatment conditions, the completers had committed significantly less physical IPV at posttreatment than at pretreatment (I-StoP: Z (10) = −2.68; p = .00 (1-tailed); CBT-SUD+: Z (7) = −2.37; p = .01 (1-tailed)). There were no significant differences between both conditions regarding posttreatment physical IPV perpetration after controlling for pretreatment physical IPV. ITT analyses yielded similar results. I-StoP patients and CBT-SUD+ patients both had committed significantly less acts of physical IPV at posttreatment (i.e., LOCF for dropouts) than at pretreatment (I-StoP: Z = −2.32; p = .01 (1-tailed); CBT-SUD+: Z = −2.87; p = .00 (1-tailed)). There were no differences between the treatments after controlling for pretreatment physical IPV perpetration. Further, both treatments were effective in reducing verbal IPV perpetration among the completers (I-StoP: t (10) = 1.99; p = .04 (1-tailed); CBT-SUD+: t (7) = 3.31; p = .01 (1-tailed)), with no differences between the treatments after controlling for pretreatment verbal IPV perpetration. ITT analyses demonstrated that patients receiving I-StoP did not commit significantly less verbal IPV at posttreatment than at pretreatment (Z = −0.78; p = .19; 1-tailed), whereas participants receiving CBT-SUD+ did (Z = −2.76; p = .00; 1-tailed). However, after controlling for pretreatment verbal IPV perpetration, no differences between both treatments remained. Finally, there was a non-significant trend that completers in both treatments had less often injured their partner at posttreatment than at pretreatment (I-StoP: Z = −1.83; p = .06 (1-tailed); CBT-SUD+: Z = −1.89; p = .06 (1-tailed)); with no significant posttreatment difference between both groups. ITT analyses demonstrated that participants allocated to I-StoP had not injured their partner significantly less often at posttreatment than at pretreatment (Z = −1.11; p = .13; 1-tailed), whereas patients receiving CBT-SUD+ did show a significant decrease in injuries inflicted (Z = −2.38; p = .01; 1-tailed). However, after controlling for pretreatment inflicted injuries, no differences between treatments remained. BODY.RESULTS.PRIMARY OUTCOMES.SECONDARY OUTCOME MEASURES: Secondary outcome measures (general psychopathology, marital satisfaction and treatment satisfaction) of completers and ITT sample are displayed in Table 5. Table 5 Secondary outcome measures of completers and ITT in both treatment conditions   Completers Intention-to-treat sample   I-StoP CBT-SUD+ I-StoP CBT-SUD+ Variable M (SD) M (SD) M (SD) M (SD) BSI score–pretreatment 55.40 (29.91) 55.75 (33.39) 55.96 (37.91) 56.33 (34.01) BSI score–posttreatment 26.10 (20.78) 25.38 (24.51) 41.08 (33.53) 46.00 (35.04) MMQ score 1 –pretreatment 29.88 (18.36) 26.29 (12.65) 24.09 (16.98) 21.32 (12.39) MMQ score–posttreatment 24.75 (15.68) 20.43 (23.22) 22.95 (15.55) 19.86 (15.89) Treatment satisfaction–rating 2 8.38 (1.16) 8.19 (0.65) - - 1  = the first five participants did not complete the MMQ since the instrument was added to the assessment in a later phase of the study; 2  = in both conditions, 8 participants evaluated treatment; BSI = brief symptom inventory; MMQ = Maudsley marital questionnaire; I-StoP = Integrated treatment for Substance abuse and Partner violence; CBT-SUD+ = substance abuse treatment. BSI scores reflecting psychopathology of participants in both conditions were significantly lower at posttreatment than at pretreatment for completers (I-StoP: t (9) = 2.92; p = .01 (1-sided); CBT-SUD+: t (7) = 3.97; p = .00)) as well as ITT (I-StoP: t (24) = 2.69; p = .01 (1-sided); CBT-SUD+: t (23) = 2.69; p = .01 (1-sided)). There were no differences between both conditions. MMQ scores, reflecting marital satisfaction, had not significantly decreased (lower MMQ scores are associated with higher marital satisfaction) within conditions, nor were there any differences between the conditions for completers as well as ITT analyses. Completers in both conditions reported high rates of treatment satisfaction (on average over 8 out of ten). There were no differences in treatment satisfaction between conditions. BODY.DISCUSSION: This is the first study that compared individual integrated CBT addressing both substance abuse and partner violence (I-StoP) to a bona fide substance use treatment as usual (i.e., CBT addressing substance abuse). Primary aim of the present study was to compare the effectiveness of I-StoP to substance use treatment as usual (with one session addressing IPV added; CBT-SUD+) in reducing physical IPV among patients referred for substance abuse treatment who were involved in a pattern of IPV perpetration. Analyses were conducted both on completers only and on all included patients (ITT sample). As expected, both I-StoP and CBT-SUD+ were effective in reducing substance use and no significant differences between both conditions were found; completers and ITT analyses yielded similar results. Further, in accordance with expectations, both treatments led to a significant decrease in physical IPV perpetration from pretreatment to posttreatment. However, contrary to expectations, I-StoP had not been superior to CBT-SUD+ in decreasing IPV. Again, the results on decrease in IPV were similar in completers and ITT analyses. Regarding secondary outcome measures, completers and ITT analyses showed slightly different outcomes. Completers analyses revealed a significant decrease in verbal IPV and inflicted injuries and a significant improvement in general psychopathology, with no differences between both treatments. In contrast, ITT analyses demonstrated that I-StoP had not led to less verbal IPV at posttreatment compared to pretreatment, whereas CBT-SUD+ had. This difference between conditions was significant. In addition, there was a non-significant trend that CBT-SUD+ had been effective in decreasing inflicted injuries, whereas I-StoP had not. Further, even though marital satisfaction at pretreatment was close to the maritally distressed range [85], both treatments (completers and ITT) did not improve marital satisfaction. Finally, completers in both conditions reported high rates of treatment satisfaction. Although I-StoP was not more effective in reducing IPV than CBT-SUD+, the results of the current study are promising. Patients who completed treatment hardly engaged in IPV perpetration in the 8 weeks before treatment completion (I-StoP: M = 0.82 and CBT-SUD+: M = 0.38 acts of physical IPV perpetration at posttreatment). Therefore, the decrease of IPV perpetration after treatment completion was not only statistically significant but also clinically relevant. Moreover, the current study demonstrated that, on case level, patients with cannabis and/or cocaine use disorders also decreased IPV perpetration after substance abuse treatment. Since CBT-SUD+ is easier to implement in substance abuse treatment centers than I-StoP, it may be concluded from the above that CBT-SUD+, a CBT treatment addressing substance use disorders with the inclusion of one session addressing IPV, is a sufficient treatment to treat IPV perpetration in patients in substance abuse treatment. Despite the favorable outcome, the results should be interpreted considering several limitations of the study. The foremost shortcoming is the high dropout rate (61.2%), which resulted in a completers sample of only 19 participants. Even though high dropout is common for patients in substance abuse treatment (a review by [86] described that the majority of studies reported dropout rates over 50% in the first month of treatment) and IPV treatment (ranging from about 20 to 70% [87]), dropout rates in this study were in the top range. Presumably, patients in whom both substance abuse and IPV perpetration are present are even more difficult to retain in treatment than patients with either problem alone. Also, dropout rates are comparable to dropout rates in a similar (pilot) study comparing the effectiveness of I-StoP to CBT addressing IPV alone among substance abusing IPV perpetrators referred to outpatient forensic treatment after committing IPV [Kraanen, Scholing, Emmelkamp: The comparative effectiveness of a combined substance abuse–partner violence treatment to partner violence treatment alone among patients in forensic outpatient treatment: A pilot study, submitted]. We argue however that because this study was conducted in routine clinical care, these dropout rates accurately reflect clinical practice, and that the external validity of this study is probably high. Further, it is also noticeable that low numbers of dropouts completed questionnaires (49% completed assessment halfway treatment, 39% completed posttreatment assessment), even though it has been tried repeatedly to obtain data by mailing questionnaires to patients and phoning them in order to remind participants to complete the forms. Unfortunately, it is common for patients in substance abuse treatment that they are difficult to contact after dropout, probably because they often suffer from various psychosocial problems as well. Further, due to high dropout rates, statistical power for the completers sample (but not the ITT sample) was low. As described in the Methods section, 21 participants per condition were needed to detect a large difference between groups, tested 1-sided against alpha = .05 for a power of .80. For the ITT analyses, this number of 21 participants per condition was met, but not for the completers sample. Therefore, it is possible that true differences among completers (within groups as well as between groups) were not demonstrated. However, the main finding of the study is that the frequency of IPV had dropped to about zero in both treatment conditions for the patients who finished the treatment, independent of pretreatment severity of IPV. Also, dropouts and completers were compared and were found not to differ regarding pretreatment physical IPV perpetration, verbal IPV perpetration, inflicted injuries, days of abstinence, substance use Z-scores, and pretreatment Axis-I disorders, except for major depressive episode. Chi-square tests demonstrated that dropouts were more often diagnosed with major depressive episode (N = 9; 29.0%) than completers (N = 1; 5.0%) (X2 (1) = 4.45; p = .04). This is in accordance with previous research; several studies have demonstrated that depression is linked to early treatment attrition (e.g., [88-90]). However, the fact that almost no differences were found between dropouts and completers might also be attributable to low statistical power (i.e., at least 64 or 21 participants per condition were necessary to demonstrate (according to [84]) medium or large effect sizes of Cohen's f = .25 and .40, respectively; [83]). For example, the pretreatment physical IPV scores of completers who received I-StoP (M = 6.91) were obviously lower than ITT allocated to I-StoP (M = 12.12). In addition, since there were fewer patients who reported at least 7 acts of physical IPV in the past year than we had expected, we were unable to reach the target of 100 participants within the period that patients were recruited. Yet, despite the relatively small number of completers, IPV decreased significantly in the completers as well as the ITT sample. Nonetheless, future research should focus on finding approaches, apart from using motivational interviewing techniques, to increase treatment adherence and include more participants. For example, by targeting depressive symptoms from the beginning of treatment, as suggested by [90]. Also, expectancies regarding treatment should be assessed at the beginning of and during the course of treatment to examine whether treatments correspond with patients' expectations and whether incorrect expectancies may be a reason for dropout. Related to the high dropout and low retention rates is the fact that no 'state of the art' analyses could be used to analyze the results, such as multiple imputation (MI). MI performs well with dropout rates of at most 50% but power decreases fast as dropout rates get higher [91]. Further, MI demands that data are missing at random (MAR) (e.g., [92-94]). Many dropouts could not be contacted, but we assumed that they had probably relapsed and thus were not missing at random (NMAR). Moreover, the variables to be imputed exceeded the number of participants and imputing scale scores would lead to increased standard errors [91]. Since the data were probably NMAR, multilevel analysis could not be performed as well [79]. Another point of discussion is that the results were based on self-report, which may have been influenced by patients' tendency to underreport IPV perpetration. Alternative methods would have been using police records and/or partners reports as outcome. However, police records to verify IPV have a similar problem since only a small proportion of IPV is reported to the police (e.g., [95,96]). On the other hand, partner reports are also subject to limitations. [97] for example even demonstrated that victims reported less IPV than perpetrators. We have two reasons to assume that the findings in this study are fairly reliable and valid. 1) We tried to obtain pre- and posttreatment measures of partner on IPV by administering the CTS2. At pretreatment, 40.4% of partners completed the CTS2; at posttreatment this was only 7.7% of the total sample. Using these questionnaires as primary outcome measure would result in even lower completion rates. However, when comparing CTS2 physical IPV outcomes of both partners, we found no significant differences between participant and partner reports (Z = −0.82; p = .41), with partners reporting less acts of physical IPV (M = 6.5) than participants (M = 9.0). The second is that not only treatment progress but also study inclusion was based on self-disclosure of IPV. It is probable that some patients were not included at all because they underreported IPV, but the group who was finally included reported at least enough IPV to meet inclusion criteria. Another limitation is that the treatments in general lasted significantly longer than intended (on average 33 instead of 16 weeks). Reasons for this delay were, for example, that therapist and patient could not schedule sessions during certainweeks, were on vacation, or because patients were doing so well that they, in agreement with their therapist, did not need to come to the institution every week and instead preferred to spread treatment sessions over a longer period. This last point possibly implies that a shorter treatment might also be effective in reducing both substance use and partner violence. Further, results of the present study appear promising compared to the results of the meta-analyses by [31] and [32] that demonstrated that IPV treatment alone was hardly effective in reducing IPV. However, the comparison between the current study and these meta-analyses is difficult; the meta-analyses included follow-up data (6 months or more) whereas the current study only reported posttreatment results. Moreover, the meta-analyses included only police and partner reports compared to self-report in the present study. Further, although lacking follow-up results are also a limitation of the study, we argue that posttreatment and follow-up results are two different research topics that each have merits of their own. Finally, due to the small completers sample, it was not possible to add covariates to the analyses and control, for example, for participants' own substance use, partner substance use and IPV victimization since statistical power would be too low. The limitations described above lead to the following suggestions for future research. In the first place, it would be interesting to study whether a shorter treat-ment (of, for example, 10 sessions) would be as effective in reducing IPV perpetration as the 16 sessions counting CBT-SUD+ treatment. Further, since CBT-SUD+ contains 1 session addressing IPV, a prospective study could compare CBT-SUD+ to substance abuse treatment without addressing IPV to investigate whether it is indeed necessary to address IPV to reduce IPV perpetration or that substance abuse treatment alone is sufficient. Further, patients receiving inpatient treatment were excluded from the study. Future research should investigate whether CBT-SUD+ is as effective in reducing IPV in inpatients with more severe substance use disorders than in outpatients. In addition, predictors of treatment success should be studied. For example, there is substantial evidence that three or four subtypes of IPV perpetrators could be distinguished, i.e., the family only perpetrator who is only violent towards the partner, the borderline / dysphoric IPV perpetrator, who is characterized by psychic distress and is sometimes violent outside the family, and the antisocial IPV perpetrator (sometimes divided in two subtypes with different severity) who commits most extrafamilial violence of the subtypes [98,99]. It would be relevant to assess whether subtypes (or other patient characteristics) predict treatment success and/or dropout rates. Also, it should be examined whether type of substance use disorder predicts treatment outcome, whereas previous research only demonstrated that successfully treating alcohol dependence led to reductions in IPV perpetration. Although individual patients with alcohol, cannabis and/or cocaine use disorders who were included in the present study had improved with regard to IPV perpetration at posttreatment, unfortunately, the number of participants in this study was too low to analyze patients with different substance use disorders separately. Finally, it should be studied whether I-StoP is effective in reducing IPV perpetration in substance abusing patients who are referred to IPV treatment. Despite its limitations, the study has several strengths. In the first place, it was demonstrated among patients entering substance abuse treatment that both treatments were successful in reducing IPV perpetration. Completers in both conditions almost did not perpetrate any IPV 8 weeks before the end of treatment compared to (on average) about 12 acts of physical IPV in the 8 weeks before starting treatment. Moreover, results are directly applicable to clinical practice. BODY.CONCLUSIONS: Based on the outcome of the present study we advise to routinely screen for IPV perpetration at intake, for example by using the J-IPV. After screening positive, IPV should be further assessed with, for instance, the CTS2. If IPV perpetration is confirmed, patients should be allocated to CBT-SUD+, since CBT-SUD+ is easier to implement in a substance abuse treatment center and therapists only need a limited amount of additional training to carry out the protocol. Moreover, since results indicated that it is not necessary to refer IPV perpetrating patients in substance abuse treatment to IPV treatment, CBT-SUD+ is a cost- and time-effective approach and it saves patients the inconvenience from attending treatment at two separate institutions. However, since this is the posttreatment report and effects of I-StoP may be delayed, follow-up results should be awaited to draw more firm conclusions regarding the comparative effectiveness of I-StoP and CBT-SUD+ and effectiveness of both treatments on the long term. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: FLK, EV, AS, and PMGE contributed to the design of the study and development of the treatment protocols. EV contributed to implementation of the study. PMGE and FLK supervised therapists. FLK drafted the manuscript and EV, AS, and PMGE edited and added to the manuscript. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-244X/13/189/prepub

TITLE: Comparison of Efficacy and Safety of Lispro and Aspart Evaluated by Continuous Glucose Monitoring System in Patients with Newly Diagnosed Type 2 Diabetes ABSTRACT.OBJECTIVE: To compare the effect of the rapid-acting insulin analogues (RAIAs) aspart (NovoRapid) and lispro (Prandilin) on glycemic variations by continuous glucose monitoring system (CGMS) in patients within newly diagnosed type 2 diabetes mellitus (T2DM) receiving continuous subcutaneous insulin infusion (CSII) and metformin intensive therapy. ABSTRACT.METHODS: This is a single-blind randomized controlled trial. A total of 110 patients with newly diagnosed T2DM and with hemoglobin A1c (HbA1c%) above 9% was hospitalized and randomly divided into two groups: group Asp (NovoRapid group) and group Lis (Prandilin group). They all received CSII and metformin therapy. Treatments were maintained for 2-3 weeks after the glycaemic target was reached. C-peptide and insulin and fructosamine were determined. CGMS was continuously applied for 4 days after reaching the glycemic target. ABSTRACT.RESULTS: There were no significant differences in daily dosages of insulin, fasting plasma C-P and 2 h postprandial C-P and insulin, and fructosamine at the baseline and endpoint between the groups Asp and Lis. No significant differences were seen in the 24 h mean amplitude of glycemic excursions (MAGE), 24 h mean blood glucose (MBG), the standard deviation of the MBG (SDBG), fasting blood glucose, number of glycemic excursion (NGE), and the incidence of hypoglycemia between the two groups. Similarly, no significant differences were found in areas under the curve (AUC) of glucose above 10.0 mmol/L or the decremental area over the curve (AOC) of glucose below 3.9 mmol/L between the two groups. ABSTRACT.CONCLUSIONS: Lispro and aspart had the similar ability to control the glycemic variations in patients with newly diagnosed T2DM. This study was registered with ClinicalTrials.gov, number ChiCTR-IPR-17010338. BODY.1. INTRODUCTION: The continuous subcutaneous insulin infusion (CSII) therapy more closely mimics physiological insulin release with short-acting insulin analogues. Insulin lispro or insulin aspart, as the common rapidly absorbed insulin analogues, offers an advantage over regular human insulin used in insulin pumps to achieve better glucose control and quality of life. Most studies tested the effect of insulin lispro versus regular insulin [1–4]. Few clinical trials compared the efficacy and safety of lispro with aspart in type 2 diabetes mellitus (T2DM) [5, 6]. It was showed that insulin lispro or insulin aspart may have different pharmacological mechanism [7–9]. Compared with insulin aspart, subcutaneous injection of lispro has a faster absorption rate, the earlier plasma insulin peak time, and the faster rate of decline [7]. Some studies showed that insulin aspart has better chemical and physical stability than insulin lispro in insulin pumps [10]. Currently, the results on the effective and safety of insulin lispro compared with insulin aspart in type 1 diabetes mellitus (T1DM) remain controversial [7, 10–12]. Few studies have investigated the value and safety of insulin lispro compared with insulin aspart in patients with newly diagnosed T2DM by continuous glucose monitoring system (CGMS) in the Chinese population. Metformin was recommended for the treatment of type 2 diabetes as a first-line medication by the American Diabetes Association (ADA) and Chinese Diabetes Society (CDS) [13]. Recently, we reported that newly diagnosed T2DM patients who are receiving CSII therapy achieved glycemic control monitored in terms to significant improvement in the 24 h mean amplitude of glycemic excursions (MAGE) compared to those with multiple daily injections (MDI) therapy [14]. Using CGMS, we further identified that young-onset type 2 diabetes, defined as age less than 40-yrs old, received a metformin combination with CSII therapy which required significantly lower insulin doses to maintain glycemic control compared to the late onset diabetes patients [15]. Thus, the purpose of this study is to compare the efficacy and safety of insulin lispro with insulin aspart by CGMS in patients with newly diagnosed T2DM and with HbA1c more than 9%. BODY.2. METHODS.2.1. SUBJECTS: This is a single-blind randomized controlled trial and lasted for 2-3 weeks. The study protocol and informed consent document were approved by the Institutional Ethics Committee, Nanjing First Hospital, Nanjing, China. All patients gave written informed consent. 110 patients with newly diagnosed T2DM and with HbAlc ≥ 9.0% were enrolled between February 2015 and May 2016, from Nanjing First Hospital, Nanjing, China. Patients were excluded if they were positive for antiglutamic acid decarboxylase antibodies, had severely impaired liver and kidney function and psychiatric disorders, or were pregnant or planning to become pregnant. Patients with maturity-onset diabetes in youth and mitochondria diabetes mellitus, with cognitive disorder, or abuse of alcohol or drugs were also excluded [16]. BODY.2. METHODS.2.2. STUDY DESIGN: All patients were admitted to the hospital. Three days after euglycemic control, fasting and 2 h postprandial blood samples were collected for measuring blood glucose, C-peptide, insulin, HbA1c%, and fructosamine in all patients before and after treatment. Then, subjects underwent oral glucose tolerance tests (OGTTs) using 75 g of glucose (dissolved in 200 ml water) [17]. CGMS data were obtained with Medtronic Minimed CGMS Gold (Medtronic Incorporated, Northridge, California, USA) for 3 days after 3 euglycemic control. All subjects received the same energy intake during the CGM periods. All patients were instructed to maintain their usual physical activity. The received meals for patients consisted of a total daily caloric intake of 25 kcal/kg/day, and the ratio of carbohydrate, proteins, and fats was 55%, 17%, and 28%, respectively. After completing OGTTS, enrolled subjects (new diagnosed T2DM) were randomly assigned into two groups: group Asp (Novo Nordisk, Bagsvaerd, Denmark) and group Lis (Gan & Lee Pharmaceuticals, Beijing, China) in CSII, combined with metformin (Bristol-Myers Squibb, USA) therapy. During insulin intensive therapy, every patient generally uses 1.5 g metformin per day. If the patient is unable to tolerate the side effects of metformin, such as diarrhea, nausea, vomiting, and allergies, the daily dose of metformin is reduced to 1.0 g. If the patients are still unable to tolerate the daily 1.0 g of metformin, patient will be excluded from this study. Computer-generated random order was prepared by the data-coordinating center and distributed to each patient to determine the patient's treatment assignments. The total daily dose of insulin was calculated in international units (IU). Intensive insulin treatment by CSII was initiated with an insulin pump (H-Tron Plus V100; Disetronic Medical System, Burgdorf, Switzerland). Starting insulin doses were 0.1–0.3 IU/kg, with 50% provided as bolus (premeal) injection and 50% provided as basal injection. Insulin doses were subsequently adjusted according to blood glucose values obtained by self-monitoring. The finger point blood glucose was monitored at 7 time-points: 0700, 0900, 1100, 1300, 1700, 1900, and 2200. Insulin doses were then titrated on an individual-patient basis using the algorithm (if the fasting blood glucose (0700) level was less than 4.4 mmol/L, the nocturnal basal insulin dose was reduced 0.2 units per hour from 1900 to 0600 by slowing the infusion speed; if the fasting blood glucose level was within 4.4 to 7.0 mmol/L, the nocturnal basal insulin dose would be unchanged; if the fasting blood glucose level was from 7.0 to 7.8 and 7.9 to 10.0 and >10.0 mmol/L, the nocturnal basal insulin dose would be increased subsequently by 0.2, 0.4, and 0.6 units per hour from 1900 to 0600 by increasing the infusion speed, resp.). If the prelunch blood glucose level (1100) was less than 4.4 mmol/L, the forenoon basal insulin dose was reduced 0.2 units per hour from 0600 to 1100 by slowing the infusion speed; if the prelunch blood glucose level (1100) was within 4.4 to 7.0 mmol/L, the forenoon basal insulin dose would be unchanged; if the prelunch blood glucose level was from 7.0 to 7.8 and 7.9 to 10.0 and >10.0 mmol/L, the forenoon basal insulin dose would be increased subsequently by 0.2, 0.4, and 0.6 units per hour from 0600 to 1100 by increasing the infusion speed, respectively. If the predinner blood glucose level (1700) was less than 4.4 mmol/L, the afternoon basal insulin dose was reduced 0.2 units per hour from 1100 to 1900 by slowing the infusion speed; if the predinner blood glucose level (1700) was within 4.4 to 7.0 mmol/L, the afternoon basal insulin dose would be unchanged; if the predinner blood glucose level was from 7.0 to 7.8 and 7.9 to 10.0 and >10.0 mmol/L, the afternoon basal insulin dose would be increased subsequently by 0.2, 0.4, and 0.6 units per hour from 1100 to 1900 by increasing the infusion speed, respectively [15]. Premeal insulin was divided evenly into units and given premeal. Premeal insulin doses were adjusted according to 2 h postprandial glucose levels (0900, 1300, and 1900) to achieve the target of glucose ≤10.0 mmol/L. The investigator may also terminate the adjustment of insulin dose to improve glycaemic control according to investigator's discretion and the changes should be documented in the CRF (case report form). Treatments were maintained for 1-2 weeks. The 3-day CGMS was performed after 3 days when the glycemic control with short-term intensive insulin therapy added on metformin. Glycemic control would be considered as achieved if the fasting capillary blood glucose was less than 7.0 mmol/L and 2 h postprandial blood glucose was less than 10.0 mmol/L [16, 18], meanwhile the insulin doses were unchanged during the 3-day CGMS. No antidiabetic agents were used during the intensive insulin therapy period other than metformin. The primary endpoint was the difference of the 24 h mean amplitude of glycemic excursions (MAGE). Secondary endpoints were 24 h mean blood glucose (MBG), the standard deviation of the MBG (SDBG), number of glycemic excursion (NGE), the percentage time duration (%) of hyperglycemia (glucose > 10.0 mmol/L) and hypoglycemia (glucose < 3.9 mmol/L), the incremental area under curve (AUC) of blood glucose above 10.0 mmol/L, the decremental area over the curve (AOC) of blood glucose below 3.9 mmol/L, and hypoglycemia episodes and the effect of different interventions on insulin dose and β-cell function in these patients, between the two groups. Symptomatic hypoglycemic episodes and symptom-free hypoglycemia (detected with CGMS of glucose < 3.9 mmol/L) were recorded. The β-cell function and insulin resistance were assessed by the homoeostasis model assessment-B (HOMA-B) and HOMA-IR, calculated as previously described [16, 19]. The incremental area under curve (AUC) of blood glucose, insulin, and C-peptide were calculated using trapezoidal estimation. Insulin and HbA1c were measured centrally at the Department of Endocrinology, Nanjing First Hospital, Nanjing Medical University. Plasma glucose was measured using the glucose oxidase method. Insulin and C-peptide were measured by chemiluminescent immunometric assay on the Modular Analytics E170 (Roche® Diagnostics GmbH, Mannheim, Germany). HbA1c was measured by high-performance liquid chromatography (HPLC) assay (Bio-Rad Laboratories Inc., California, USA). Fructosamine was determined by Glamour 2000 automatic biochemical analyzer (MD Inc., California, USA). BODY.2. METHODS.2.3. STATISTICAL ANALYSIS: Data were analyzed with the SPSS PASW Statistics 18 Package. For normally distributed data, the means (± standard error (SE)) of the two groups were compared using t-test. The rates between two groups were compared using the chi-square test. A two-way ANOVA was used in comparing the hourly glucose concentrations between the two groups. A P value < 0.05 was considered as statistically significant. BODY.3. RESULTS.3.1. CHARACTERISTICS OF PATIENTS: Eleven patients were excluded from this study: 4 were from the NovoRapid group (group Asp) and 7 from Prandilin group (group Lis). The excluded eleven patients either were unable to tolerate the 1000 mg/day dose of metformin or did not reach the glycaemic control target. Thus, there were 51 patients with newly diagnosed T2DM (36 men and 15 women, mean age 51.08 ± 1.59 years, body mass index 25.78 ± 0.41 kg/m2) in the group Asp and 48 (37 men and 11 women, mean age 48.94 ± 1.68 years, body mass index 24.82 ± 0.54 kg/m2) in the group Lis. There are no significant differences in the gender composition ratio, BMI, and age between the two groups (Table 1). A total of 9 subjects were using 1000 mg/day metformin in the group Asp (5) and group Lis (4). There were no statistically significant differences in the mean dose of metformin between the group Asp (1458.33 ± 139.66 mg/day) and the group Lis (1450.98 ± 150.16 mg/day). BODY.3. RESULTS.3.2. THE EFFECT OF TRANSIENT INSULIN INTENSIVE THERAPY WITH METFORMIN ON GLYCEMIC CONTROL AND : Oral glucose tolerance test was performed and the function of islet function, HbA1c, fructosamine, fasting plasma glucose, 2 h postprandial glucose, fasting plasma C-peptide, 2 h postprandial C-peptide, fasting plasma insulin, and 2 h postprandial insulin were measured before and after intensive treatment with insulin pump combined with metformin. These indices were similar between the two groups (Table 1). The HbA1c decreased by 8.14% and in group Asp versus 8.50% in group Lis, while fructosamine decreased by 21.56% in group Asp versus 20.66% in group Lis from baseline to endpoint. The differences in the reduction of HbA1c and fructosamine levels were not statistically significant between the two groups. The insulin doses were almost the same in the two groups after blood glucose target was reached. Between the group Asp and group Lis, the total insulin doses (0.26 ± 0.02 versus 0.29 ± 0.02 IU/kg), the premeal insulin dose (0.11 ± 0.01 versus 0.12 ± 0.01 IU/kg), the basal insulin dose (0.16 ± 0.01 versus 0.18 ± 0.01 IU/kg), and the intensive treatment days (10.04 ± 0.22 versus9.45 ± 0.31 days) found no significant differences. There were no statistically significant differences in the dose of metformin between the two groups. BODY.3. RESULTS.3.3. THE EFFECT OF TRANSIENT INSULIN INTENSIVE THERAPY WITH METFORMIN ON CONTROL OF BLOOD GLUCOSE FLUCTUATION: We collected CGMS data at least 3 days on 2 days after glycemic control treatment by insulin pump combined with metformin. Since the continuous blood glucose monitoring data of the second day was relatively stable, we selected 24 h continuous blood glucose data of the second day to further analyze the fluctuation of blood glucose. When the patients reached the glycaemic target, the 24 h mean glucose concentration showed no significant differences between the group Asp and group Lis (6.49 ± 0.10 versus 6.49 ± 0.15 mmol/L P > 0.05, Table 2). The glucose concentration per hour for 24 h was similar between the group Asp and group Lis (P > 0.05, Figure 1). The glucose concentration per 5 minutes for 24 h was mostly similar to the group Asp and group Lis, with the exception of before breakfast (from 0630 to 0710) (Figure 2). The glucose levels before breakfast in the aspart group were transiently lower than those in the lispro group from 0630 to 0710 (P < 0.05), and after 07:10, there were no significant difference of glucose levels between both groups. The prebreakfast insulin doses were similar between the group Asp and group Lis (4.27 ± 0.25 versus 4.53 ± 0.44 IU, P = 0.608). We calculated the mean glucose before breakfast (from 0630 to 0700, 5.66 ± 0.12 versus 6.33 ± 0.15 mmol/L, P < 0.05) as the premeal glucose and the peak postprandial glucose after breakfast (from 0700 to 0900, 8.84 ± 0.33 versus 8.96 ± 0.24 mmol/L, P > 0.05) in the group Asp and group Lis. The spikes following breakfast calculated as the difference between the premeal glucose and the peak postprandial glucose found no significant differences between the group Asp and group Lis (3.18 ± 0.34 versus 2.66 ± 0.22 mmol/L, P > 0.05). The postprandial 2 h incremental area under the curve of the breakfast meal using the trapezoidal method found no significant differences between the group Asp and group Lis (198.78 ± 27.27 versus 142.08 ± 128.98 mmol/L × 120 min, P = 0.095). The MAGE in group Asp had no significant differences as compared with group Lis (group Asp 3.33 ± 0.27 mmol/L, group Lis 3.18 ± 0.19 mmol/L). There was no significant difference of NGE and SDBG between the two groups (Table 2). We calculated the AUC and the time spent in 10.0 mmol/L and the AOC and the time spent in 3.9 mmol/L as the cut-off point in the two groups (Table 2). The incremental AUC (>10 mmol/L) detected by CGMS did not significantly decrease (0.04 ± 0.01 mmol/L per day) in group Asp as compared with group Lis (0.04 ± 0.01 mmol/L per day). The time in normal glycemia (between 3.9 and 10.0 mmol/L) in group Asp did not significantly increase compared to group Lis (97.59 ± 2.48% in group Asp versus 94.81 ± 1.05% in group Lis). BODY.3. RESULTS.3.4. THE HYPOGLYCEMIC EPISODES IN GROUP ASP VERSUS GROUP LIS: No severe hypoglycemic episodes, defined as an event requiring the assistance of another person or other resuscitative treatments, were reported in any treatment group. The number of hypoglycemia events (<3.9 mmol/L) in group Asp was 22 and it was 15 in group Lis. There were no statistical differences found between the two groups by chi-square analysis (chi square = 1.493, P = 0.222). The decline AOC and the time when blood glucose < 3.9 mmol/L as detected by CGMS had no significant differences between the two groups. BODY.4. DISCUSSION: In the present study, CGMS data showed that lispro had a similar effect as aspart on blood glucose fluctuation and hypoglycemia. There were no significant differences seen in MAGE, 24 h MBG, SDBG, fasting blood glucose, and NGE between two groups in patients with new diagnosis of T2DM. The results indicated that the blood glucose fluctuation were similar in group aspart and group lispro. Meanwhile, the AOC of 3.9 mmol/L as the cut-off point was similar between the two groups, suggesting that the patients with newly diagnosed T2DM would have the similar risk of hypoglycemia when using aspart or lispro. The rapid-acting insulin analogues (RAIAs) aspart and lispro have been approved for use in CSII therapy in patients with diabetes. Some studies have demonstrated that aspart has greater compatibility for use in insulin pumps in comparison with lispro or glulisine [13]. Studies demonstrate that aspart has a more rapid rate of absorption [20–24], more stable postprandial control [10, 11, 25–27], and lower rates of fibrillation and occlusion [10, 28–31] for use in insulin pumps compared with lispro. These qualities make aspart compatible for use in CSII therapy and perhaps a better choice of insulin to ensure improved outcomes, which in turn may result in improved treatment adherence. Meanwhile, the study by Tamborlane et al. showed lispro had a decreased rate of hypoglycemia compared with aspart in type 1 diabetes [12]. Our data was partially agreed with the study reported in CSII in adult patients with noninitial diagnosis of T2DM [6]. Insulin lispro was not inferior to insulin aspart in HbAlc, total daily insulin dose, weight change, and incidence and rates of hypoglycemia [6]. In that study, the patients operated the insulin pumps by themselves at home, which may cause operation errors. Our study selected patients with newly diagnosed T2DM, and insulin pump and CGM were uniformly operated by trained nurses to avoid the operation errors. The patients' diet had a unified diet in the hospital to avoid interference by casual dining outside the hospital. We found that there were no significant differences in daily dosages of insulin, C-P 0, C-P 2 h, Ins 0, Ins 2 h, HbA1c, and fructosamine between group Asp and group Lis before and after treatment. The glucose concentration per hour for 24 h was similar between the group Asp and group Lis, but the glucose concentration per 5 minutes had transient differences from 0630 to 0710 (before breakfast). These differences may be related to the moderate sample size. The spikes following breakfast and the postprandial 2 h incremental area under the curve of the breakfast meal found no statistical differences between the group Asp and group Lis. These data suggested that the effects of the two kinds of insulin were similar in reducing postprandial glucose for breakfast. More currently, clinical trial results, comparing the effectiveness and safety of insulin lispro and insulin aspart, were controversial in patients with T1DM [12]. Thus, the aim of the current study is to investigate the efficacy and safety of aspart and lispro delivery by insulin pump combined with metformin using CGMS in patients with newly diagnosed T2DM. Compared with the conventional 7-point peripheral blood glucose monitoring, CGMS can record the blood glucose automatically every 5 minutes. CGMS can monitor the dynamic changes of blood glucose within 72 hours to show the daily change of blood glucose curve accurately [16]. Our present CGMS data showed no statistical significances in 24 h MBG (6.49 ± 0.10 versus 6.49 ± 0.15 mmol/L), the glucose concentration per hour, MAGE (3.33 ± 0.27 versus 3.18 ± 0.19 mmol/L), SDBG (1.30 ± 0.08 versus 1.28 ± 0.06 mmol/L), NGE (4.54 ± 0.27 versus 4.60 ± 0.21 times), and 10.0 mmol/L as the cut-off point calculation with the blood glucose area under the curve (0.04 ± 0.01 versus 0.04 ± 0.01 mmol/L per day) and the duration of blood glucose (3.00 ± 0.86 versus 3.00 ± 0.78%) between the group Asp and group Lis in patients with newly diagnosed T2DM. The data showed that the two RAIAs had the similar function in controlling the level of blood glucose and blood glucose fluctuation. The current study also analyzed the hypoglycemia episode in the two groups. There was no severe hypoglycemia event and symptomatic hypoglycemia found during the study period between the two groups. The number of blood glucose less than 3.9 mmol/L and the AOC of glucose below 3.9 mmol/L was similar between the two groups. The data indicated that aspart and lispro had no difference in hypoglycemic events. In summary, aspart and lispro had similar effects on blood glucose fluctuation, 24 h average blood glucose, fasting blood glucose, and the incidence of hypoglycemia in new diagnosis of T2DM. Because of the considerable effect of aspart and lispro, they can both be regarded as suitable insulin intensive treatment options.

TITLE: Evaluation of a novel method to assess corticosteroid responsiveness in chronic obstructive pulmonary disease ABSTRACT.BACKGROUND:: Some patients with chronic obstructive pulmonary disease (COPD) may benefit from oral steroid therapy. These steroid-responsive patients are diagnosed based on laboratory spirometry. We hypothesize that daily, home-based spirometry is a better tool. ABSTRACT.METHODS:: Thirty patients with COPD underwent a single-blinded study, with a crossover design. They received 2 weeks of placebo followed by 2 weeks of prednisone therapy (40 mg/day). Laboratory spirometry was done at the beginning and end of the study and daily home-based spirometry was done twice a day. ABSTRACT.RESULTS:: Analysis of variance model was used. The variability of the median day-to-day forced expiratory volume in 1 s (FEV1) was 72.5 mL (25th percentile of 40 mL and 75th percentile of 130 mL). The daily FEV1 variation was 70 mL (25th percentile of 50 mL and 75th percentile of 100 mL). The overall laboratory FEV1 variability was larger after the steroid course (P < 0.001), but not clinically significant. The variability was not significant postplacebo treatment compared with the baseline values. For home-based spirometry, steroid treatment was not significantly different. The majority (97%) completed more than 80% of the measurements. Ninety percent of the performed tests were considered acceptable. Only 53% of the tests were considered accurate. Overall both laboratory and home-based measurements did not show significant association between airway responsiveness and dyspnea or exercise capacity. ABSTRACT.CONCLUSION:: Twice-daily home measurements of FEV1 might be better than the conventional approach to identify steroid responsive COPD patients. However, this finding was only statistically but not clinically significant. Therefore, we would not recommend this approach to identify COPD patients with steroid responsiveness. BODY: Chronic obstructive pulmonary disease (COPD) based on the definition by the American Thoracic Society (ATS)[1] is a preventable and treatable disease state characterized by airflow limitation that is not fully reversible. At present, the guidelines for the treatment of COPD recommend detecting individuals who would benefit from steroids by conducting an oral steroid trial.[1] In their simplest form, physicians have been advised to administer an oral steroid trial daily for 2 weeks comparing laboratory-based measurements of breathing (spirometry) that are done in the beginning and the end of the trial, to determine whether there has been a significant response. This is defined in terms of change in the forced expiratory volume in 1 s (FEV1). A steroid responsiveness is characterized by an increase in the FEV1 by a certain level. This positive response suggests continuing the use of steroids, in the form of inhaled corticosteroids (ICS) or less likely oral steroids. Recent research has cast doubt on the strategy of using an oral steroid trial to guide the management of individual patients with COPD. There have been more than 30 trials examining the effect of oral steroids in COPD. Methodologic concerns were noted in some of these trials. For example, some did not include a control group,[2–4] and others did not define the inclusion criteria adequately,[35] with the risk that patients with asthma may have been included. Later, better-designed trials showed a significant treatment effect with steroids. This effect was usually due to a marked improvement, for example, more than 50% increase in the FEV1, in a small group of patients, rather than a moderate improvement in the group overall.[6–10] In these trials with positive results, investigators have been unable to identify patient characteristics that would predict steroid responsiveness reliably. In a meta-analysis by Calahan and colleagues, 10 studies of oral steroids in stable COPD were examined.[11] There was evidence that oral steroids reduced spirometric improvement in the FEV1 in approximately 10% of patients with stable COPD. Although such a finding might encourage physicians to look for steroid responsiveness in their patients with COPD, the results of the meta-analysis also suggest that the findings should be extrapolated to individual patients cautiously. There was a marked variability among the trials in the degree of steroid-responsiveness. To some degree, this may be accounted for by the differences in the patients recruited, disease definition, steroid dosing, and the study methodology. Some small trials suggest that a few patients with stable COPD experience an unusually large FEV1 improvement following a 1- or 2-week course of systemic corticosteroids. Reference is sometimes made to COPD patients as being either "steroid-responsive" or "steroid-nonresponsive," and the existence of these subtypes is implied in published guidelines for outpatient treatment of COPD. However, the ISOLDE study[12] failed to show a positive predictive value for the steroid trial in a large group of patients subsequently randomized to inhaled fluticasone in high dosage or inhaled placebo. Therefore, the use of steroids in COPD might be controversial. Random fluctuations in breathing measurements could be mistaken for a positive response to therapy. Evidence of previous research indicates that steroid overuse may result from such random fluctuation being overinterpreted by physicians. The accuracy of the steroid trial might be improved by the use of repeated measurements throughout the period of steroid administration. In the past, it has been impractical and expensive to make twice or even daily measurement of lung function in the pulmonary function testing (PFT) laboratories. However, new handheld portable electronic devices can allow patients to perform this test themselves, at home, as frequently as needed to gauge responsiveness accurately. The Canadian guidelines for the assessment and management of COPD[13] recommend assessing each patient's response to steroids if airway obstruction and symptoms persist despite smoking cessation and optimum bronchodilator (BD) therapy. The steroid trial must be undertaken only at a time of clinical stability. During a maximum BD therapy, the patient will be given prednisone with a dose of 0.5 mg/kg for 2 or 3 weeks, after which PFT will be repeated. An improvement in the FEV1 of at least 20% and 200 mL will be regarded as steroid responsiveness. The ATS recommends in COPD patients with suboptimal control of symptoms that the physician should consider an oral steroid trial, for example, prednisone, up to 40 mg/day for 10–14 days.[114] If improvement occurs, the dosage should be lowered or alternate-day dose should be followed. If no improvement occurs, the steroid administration should be stopped. The positive steroid response is defined as an increase in the FEV1 ≥ 10% of the predicted value and/or >200 mL. The trial should be done in an exacerbation-free period, using a dose of 0.4-0.6 mg/kg for a duration of 2–4 weeks. The present study is designed to test the usefulness of repeated home-based measurements to detect clinically important steroid responses among patients with stable COPD. The study also compares this method of treatment assessment with the current clinical method of measuring FEV1 before and after, pre and post, respectively, treatment. We hypothesize that twice-daily home measurements of FEV1 will be better than conventional clinic-based measurement of FEV1 to distinguish steroid responsive from steroid nonresponsive COPD patients. BODY.METHODS.OBJECTIVES: In this study, we are interested to determine the spirometric variability of patients with clinically stable COPD and to obtain the measurements using a hand-held electronic spirometer. We are also testing the hypothesis that stable COPD patients will be deemed steroid nonresponsive more often using daily FEV1 measurements than with laboratory's pre- and post-BD FEV1 measurements alone. Assessing the association of variability with subjective and objective measurements of improvements is considered as well. In addition, we would like to explore the rate of adherence and technical inadequacy at home in using the home spirometer by the included patients. BODY.METHODS.STUDY POPULATION: For this study, participants were drawn from outpatients attending the respiratory clinics at the Toronto Western Hospital (a division of the University Health Network, Ontario, Canada) between 2003 and 2004. Patients were included in the study if they had an established diagnosis of COPD according to the ATS criteria.[1] Each patient had to be an exsmoker for at least 1 year and with greater than 20 pack-year history of tobacco smoking. The participants also had to be clinically stable, which was defined as the absence of exacerbations within 3 months of enrolment. Finally, the participants were required to be symptomatic despite optimal BD therapy, to be included in the study. Exclusion criteria included patients with major nonrespiratory diseases, such as congestive heart failure; chronic renal failure; diabetes mellitus or glucose intolerance; history of tuberculosis; use of steroid medication, oral or inhaled, 1 month prior to enrolment; or any known contraindication to the use of oral steroids. All the participants signed a written consent prior to their enrolment in the study. After obtaining the research ethics board approval, the participants were identified and recruited for the study. The study protocol and rationale were explained to all of them, both verbally and in writing. BODY.METHODS.THE TRIAL: The study has a single-blinded prospective placebo-control design of 30 stable COPD patients who received 2 weeks of placebo medication followed by 2 weeks of prednisone at a dose of 40 mg/day. To maintain blinding, the order of the placebo medication first and the oral steroid second was not outlined for the cardiopulmonary technologist performing the spirometry. Spirometries pre- and post-BD (standard procedure) were performed in a laboratory setting before and after each treatment period. The participants were asked to abstain from inhaled BDs for 6 h and from sustained release theophylline for 48 h before the baseline spirometry. For spirometry repeated at the end of the placebo and prednisone treatment periods, only short-acting inhaled BDs were to be withheld before testing, and long-acting preparations were continued if they were part of the patients' usual maintenance regimen. Post-BD testing was done 30 min after the administration of 4 puffs of inhaled salbutamol/ipratropium combination (Combivent, Boehringer Ingelheim Ltd, Canada) administered via adult aerochamber. The participants were instructed by a research physician[Appendix A] about the use of a hand-held turbine-style electronic Diary Card® (MicroMedical Diary Card, MicroMedical Ltd., Rochester, UK). This is a portable spirometer, which allows the patients to perform spirometry at home or anywhere outside the hospital. The device's control unit has memory for several hundred spirometric recordings, including graphical storage of flow–volume curves (FVCs). The participants were instructed about spirometry for about 1 h, and they received written information on how to perform spirometry at home. After a learning period of 1 week, all the patients were able to produce technically acceptable flow–volume loops. Appendix A Instructions for the usage of Diary Card 1 Take a deep breath in. 2 Put the mouthpiece of the Diary Card into your mouth and make sure:  You do not stick your tongue into the mouthpiece.  Close your mouth tight so that air will not escape.  Blow as hard as you can till you see 3 marks on the Diary Card. 3 Pull out the mouthpiece after the above procedure. 4 Read the instructions on the Diary Card and follow them. 5 Repeat the same procedure as above. 6 Blow 3 times at each occasion. 7 Repeat it 30 min after BD. 8 Repeat the same procedure in the evening twice (before and 30 min after the BD). BODY.METHODS.MONITORING AND FOLLOW-UP: All the 30 patients were asked to use the Diary Cards 4 times a day at home during the 2 treatment periods of the trial. The expected number of spirometric measurements for a patient was 112, as 4 spirograms were done per day for a total duration of 28 days. The participants had to complete 4 follow-up visits. They were seen in the clinic at the beginning of week 1 (1st visit for recruitment), beginning of week 2 (2nd visit to start placebo), beginning of week 4 (3rd visit to start prednisone), and at the end of the study after week 5. BODY.METHODS.OUTCOME MEASURES: In the 2nd visit, baseline PFT took place in a hospital laboratory. The PFT included the measurements of height and weight, to calculate the body mass index. The participants were also instructed about using the Borg Scale[15] to report their symptom perception. In addition, assessment of the baseline dyspnea index (BDI)[16] and 6-min walk test (6MWT)[17] took place during the visit. Home-based spirometry was to be performed before (pre-BD) and 30 min after inhalation of BD (post-BD) every morning (AM) and evening (PM) using the Diary Card. The results reported are the highest FEV1 and forced vital capacity (FVC). To be considered technically acceptable, the test had to be performed correctly by inspection of the curve, and the chosen values should not exceed the next highest by more than 5%. Each participant used the same Diary Card for both periods of treatment. To obtain a true measure of the baseline pulmonary function, only the pre-BD home-based measurements of FEV1 during the placebo treatment period were used. The day-to-day variability was obtained by calculating the difference in home-based FEV1 (only AM measurements) compared with the first day value. The daily FEV1 variation was obtained by comparing the differences of FEV1 AM–PM measurements with the one presented on the first day of the placebo treatment. The participants were re-assessed to measure their transitional dyspnea index (TDI)[16] at the end of both placebo and steroid trials. The 6MWT was also measured at the end of each study trial. In the last visit, another hospital laboratory PFT was done. The participants' reports of the Borg Scale were collected at the end of the study period. BODY.METHODS.ADHERENCE, ACCEPTABILITY, AND ADEQUACY: The subjects' adherence with the use of the Diary Card was calculated using the formula: Adherence = Number of Tests Performed × 100/Total Expected Number The FEV1 readings obtained from the Dairy Card measurements were analyzed for quality control according to 2 criteria: the appearance of the graphic record of the FVC and the arithmetic reproducibility criteria. A spirogram was considered acceptable if it was free from artifacts (see Data handling section), and had a good start and a satisfactory exhalation. The percentage of acceptable spirometric recordings was calculated using the formula: Percentage of acceptable spirograms = Number of Acceptable Spirograms � 100/Total Number of Performed Spirograms A spirogram was considered adequate if it was considered technically acceptable and the chosen values did not exceed the next highest by more than 5%. The percentage of adequate spirometric recordings was calculated using the formula: Percentage of adequate spirograms = Number of Adequate Spirograms × 100/Total Number of Performed Spirograms BODY.METHODS.DATA HANDLING: Patient-generated data (results of home spirometry) were analyzed for quality control before statistical analysis. Quality control of the Diary Card took place in the first 4 visits. All the flow–volume loops recorded were assessed. Using customary criteria, the loops were excluded if the graphic record suggested inadequate effort or improper technique. BODY.METHODS.SAMPLE SIZE: We assumed a variance of 0.3, that is, a standard deviation (SD) of 0.55, based on the findings of several clinical trials.[46718] Thirty participants were required to achieve an 80% power and a 20% difference in the FEV1 using this design. The latter percentage was considered the minimal clinically significant difference in the FEV1 for a responder to steroid treatment.[1] Only a change in the post-BD FEV1 was examined for this difference. BODY.METHODS.STATISTICAL TECHNIQUES: The data were tested for normality and equal variance. For normally distributed data, paired t test was performed, reporting the mean and SD. For nonnormally distributed data, nonparametric Wilcoxon signed rank test was used, reporting the median and the 25th and 75th percentile points. Comparisons were made with a significance level of 0.05. The analysis of variance was used to assess the reliability of the FEV1 measurements. General linear regression model was built to assess the association between the variability of FEV subjectively (using Borg Scale and dyspnea index measurements) and objectively (using 6MWT). All data were analyzed with a database and statistical package (SigmaStat; Jandel Scientific, San Raphael, CA). BODY.RESULTS: Thirty participants were recruited for the study, 14 males and 16 females. The mean age of the men and women was 70.0 and 68.3 years, respectively. The mean initial FEV1 in the male group was 1.15 L (37.7% of the predicted value), and it was 1.15 L (54.7% of the predicted value) in the female group. The variability of the median day-to-day FEV1 was 72.5 mL (3.0% of the predicted value). The 25th and 75th percentiles were 40 and 130 mL, respectively. The daily FEV1 variation was 70 mL (4.1% of the predicted value) with a 25th percentile of 50 mL and 75th percentile of 100 mL [Figure 1]. Figure 1Box plot showing data distribution of both day-to-day (Day2Day) and daily variability of FEV1 during spirometric measurements The overall variability in spirometric measurements was found to be larger than expected from random variability in the treatment, and therefore the treatments had different effects than would be expected by chance (P < 0.001). Furthermore, there was a statistically significant difference between the FEV1 values poststeroid treatment compared with both placebo treatment and baseline. A nonstatistically significant difference was found for the FEV1 values postplacebo treatment compared with the baseline values. For the laboratory testing, FEV1 measurements poststeroid treatment were compared with those of postplacebo treatment when evaluating the efficacy of the oral steroid trial [Table 1]. There was a statistically significant difference for laboratory measurements after the 2 types of treatment. Table 1 Results of the comparison of post-BD FEV 1 laboratory measurements Group Median 25% 75% Post-placebo 1.13 0.90 1.54 Post-steroid 1.21 0.99 1.76 The change that occurred with the treatment is greater than that would be expected by chance; there is a statistically significant difference ( P < 0.001) For home-based spirometry, steroid treatment was not significantly different [Table 2] for Diary Card measurements of FEV1. There was no significant difference between the responsive and nonresponsive groups. Table 2 Results of the comparison of post-BD FEV 1 Diary Card measurements Group Median 25% 75% Post-placebo 1.08 0.79 1.30 Post-steroid 1.06 0.76 1.33 The change that occurred with the treatment is not great enough to exclude the possibility that it is due to chance ( P = 0.86) Both conventional and Diary Card measurements did not show significant association between airway responsiveness to steroids and symptoms of dyspnea assessed by Borg Scale, BDI, and dyspnea index measurements. The Borg Scale percentage of decrease in the mean of the baseline rating, after oral steroid or placebo trial, for responders was 22.51%, whereas it was 18.02% for nonresponders (P = 0.71). The BDI/TDI ratios for responders and nonresponders were 1.12 and 0.7, respectively (P = 0.09). However, 6MWT distance was associated significantly with airway responsiveness in participants using the Diary Card measurements with a mean percentage of increase in the walk distance, after oral steroid or placebo trial, of 13.3% among responders and 5.74% among nonresponders (P < 0.01) [Table 3]. Table 3 Comparison of responders and nonresponders, according to the Diary Card Measurements Outcome Mean % responders Mean % Nonresponders Mean % Nonresponders P value Borg scale 22,52 28.02 t test 0.71 BDI/TDI 1.12 0.7 Wilcoxon signed rank test 0.09 6MWT 13.3 5.74 t test < 0.01 BDI = Baseline dyspnea index, TDI = Transitional dyspnea index, BDI/TDI = Ratio between BDI and TDI, 6MWT = 6-min walk test, *For normally distributed data, paired t test was performed. For nonnormally distributed data, nonparametric Wilcoxon signed rank test was used (see Statistical Techniques section) The percentage of performed tests was calculated for each participant. The majority (29 participants, 97%) completed more than 80% of the measurements. Ninety percent of performed tests were considered acceptable. However, only 16 participants (53%) met the criteria for adequacy according to their test performance. BODY.DISCUSSION: In this study, a new method of assessing the response in stable COPD patients to a short trial of oral steroid was assessed. Our hypothesis was that twice-daily home measurements of FEV1 would be better than conventional clinic-based measurements of FEV1 to distinguish steroid responsive COPD patients from steroid nonresponsive COPD patients. The design of the study and the criteria used to assess the responsiveness to steroids are according to the existing ATS guidelines at the time when the study began. The inclusion criteria limited the possibility of confounders. All the patients had a smoking history of at least 20 pack-years, were exsmokers for at least 1 year, had a stable disease, and their FEV1 was less than 80% of the predicted value. Therefore, we specifically designed the study so no asthmatic patients would be included as possible. In addition, the inclusion criteria were chosen to eliminate potential biases related to smoking status and degree. By excluding recent exacerbations, we intended to prevent a potential bias as such patients would be more susceptible to respond to steroids than patients with a stable disease. A major advantage of crossover studies is that they usually require smaller sample size than parallel group trials, as patients form their own controls. The reason for not randomizing the order of treatment was the uncertainty regarding the "wash-out" duration and the risk of steroids' carried-over effect. A 2-week treatment period was used as it is felt that maximum improvement in airway obstruction, treated with steroids is achieved in approximately 8 days.[19] It was known from other studies that the variability in the FEV1 is increased in patients with obstructive lung disease. Since this study is the first one to use repeated measurements of FEV1 in COPD patients, one of the objectives was to determine the spirometric variability in these participants while they have a stable disease. In our study participants, the day-to-day and daily variability of FEV1 was much lower than the values mentioned in other studies conducted on COPD patients. We showed that laboratory measurements have a statistically significant response to steroids, whereas the daily home-based measurements did not. However, the size of improvement in the FEV1 represents a lower value than the clinically significant, since the difference found was less than 200 mL. This could be explained by the differences in the variability of FEV1 calculated using laboratory or home (Diary Card) measurements. To detect a smaller change in the FEV1 than planned, a bigger sample size would be needed given the assumed variance in the sample size formula. It is probable, however, that the real variance is smaller than the one chosen for the sample size. If the variance used for the sample size calculation was the same as the one found in our data, there would be no statistically significant difference. However, one of the objectives was to prove that FEV1 would be less variable using the Diary Card measurements. Higher variability of FEV1 measurements in the laboratory could appear to have a statistically significant difference after the steroid treatment. If the Diary Card measurements were more consistent, there would be less variation of FEV1, and so less frequent false-positive responses to steroids could be detected. The hypothesis that twice-daily home measurements of FEV1 is better than the conventional clinic-based measurement of FEV1 to distinguish steroid responsive from steroid nonresponsive COPD patients was validated only from the statistical point of view (P < 0.005). The differences in the FEV1 were not clinically significant as the absolute difference was less than what was considered a priori a significant threshold for an acceptable steroid responsiveness, that is, 200 mL. Eighty-three percent of the enrolled participants performed more than 91% of the required 112 home spirometry measurements. This means that they were adherent to the study intervention. Previous data[20] concluded that Diary Card machine could be used to monitor asthmatic patients. Ninety-two percent of our subjects had performed correct FEV1 home measurements according to the aspect of the graphic record criteria. This proves that the Diary Card machine could be used for monitoring PFTs for COPD patients as well. A much smaller number of corrected recordings according to both criteria (graphic and 5% value) could be explained by the absence of the technicians and their coaching ability during the home spirometry performance. With more training and may be frequent check-up of the quality of the recordings, the percentage of correct recordings could be improved. This might be achieved by increasing the frequency of clinic visits to once a week for each treatment period instead of only at the beginning and the end of treatment. We recognize some limitations in our study. The study has a "fixed" crossover design and therefore was not randomized. This may limit the internal validity of the trial. However, we attempted to improve this limitation by blinding both participants and outcome assessors. Another limitation, also related to the study design, is the small sample size. In order to improve inferences and the external validity of the study to be more generalizable, a larger sample size might be required. BODY.FUTURE RESEARCH: Many existing COPD guidelines[113] still recommend the use of a short course (2 weeks) of oral steroids to identify patients who might benefit from long-term treatment with inhaled or even oral steroids. On the other hand, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines[21] recommend a trial of 6 weeks to 3 months with ICS, to identify COPD patients who may benefit from long-term inhaled steroid therapy. In addition, one of the GOLD new directions for future research is to develop and evaluate in clinical practice other measures than laboratory spirometry to assess and monitor COPD. Since 2 of the goals of effective COPD management are to relieve the symptoms and to improve exercise tolerance, the repeated measurements of FEV1 using Diary Card at home could be used in a future study. This could be done to assess the response to inhaled steroids. Since the GOLD guidelines use 15% increase in the FEV1 above baseline as a criterion for steroid response instead of 20% used in our study, a bigger sample size would be needed for a future study. The results of our study allow calculating an estimate of the variance of the variability of single measurements on these participants. This estimate may be used to determine the required sample size in the subsequent study. Our method to assess steroid responsiveness that may lead to a reduction in prescribing excessive steroids in COPD could have significant impact on provincial and national health care costs. This also needs to be explored in further research. Finally, since inhaled steroids have fewer contraindications than oral steroids, the inclusion criteria for a following study should not be strict as the ones used in this study. This means that more eligible participants for the study could be allocated in a limited timeframe. In this situation, a crossover design with a randomization of 2 treatments (placebo and inhaled steroids) could be more feasible than in the present study. BODY.CONCLUSION: This is the first study demonstrating that twice-daily home measurements of FEV1 are better than the conventional clinic-based measurement of FEV1 to distinguish steroid responsive from steroid nonresponsive COPD patients. However, this finding was statistically but not clinically significant. Therefore, we would not recommend this approach to identify COPD patients with steroid responsiveness. Further research is needed in this area to explore further aspects of this interesting clinical problem.

TITLE: Medical Abortion Provided by Nurse-Midwives or Physicians in a High Resource Setting: A Cost-Effectiveness Analysis ABSTRACT.OBJECTIVE: The objective of the present study is to calculate the cost-effectiveness of early medical abortion performed by nurse-midwifes in comparison to physicians in a high resource setting where ultrasound dating is part of the protocol. Non-physician health care professionals have previously been shown to provide medical abortion as effectively and safely as physicians, but the cost-effectiveness of such task shifting remains to be established. ABSTRACT.STUDY DESIGN: A cost effectiveness analysis was conducted based on data from a previously published randomized-controlled equivalence study including 1180 healthy women randomized to the standard procedure, early medical abortion provided by physicians, or the intervention, provision by nurse-midwifes. A 1.6% risk difference for efficacy defined as complete abortion without surgical interventions in favor of midwife provision was established which means that for every 100 procedures, the intervention treatment resulted in 1.6 fewer incomplete abortions needing surgical intervention than the standard treatment. The average direct and indirect costs and the incremental cost-effectiveness ratio (ICER) were calculated. The study was conducted at a university hospital in Stockholm, Sweden. ABSTRACT.RESULTS: The average direct costs per procedure were EUR 45 for the intervention compared to EUR 58.3 for the standard procedure. Both the cost and the efficacy of the intervention were superior to the standard treatment resulting in a negative ICER at EUR -831 based on direct costs and EUR -1769 considering total costs per surgical intervention avoided. ABSTRACT.CONCLUSION: Early medical abortion provided by nurse-midwives is more cost-effective than provision by physicians. This evidence provides clinicians and decision makers with an important tool that may influence policy and clinical practice and eventually increase numbers of abortion providers and reduce one barrier to women's access to safe abortion. BODY.INTRODUCTION: Lack of trained providers is a common barrier to safe provision of abortion. Shortage of physicians as well as physicians' unwillingness to provide abortion is common in rural areas, but is also becoming an increasing problem in high resource environments and in settings where abortion is legal [1] [2]. Unsafe abortion is estimated to cause eight percent of global maternal mortality, implying that 23000 preventable deaths occur each year [3]. Thus eliminating barriers to women's access to safe abortion is an important tool to reduce maternal mortality. The number of providers of safe abortion can be increased, by task-shifting abortion provision from physicians to health care providers with adequate but less training [4, 5]. Non-physician providers have been shown to provide medical abortion as effectively and safely as physicians in different settings[6–8]. Medical abortion using mifepristone and misoprostol is highly effective, and is recommended by the World Health Organization (WHO) [9]. Health care budgets are limited worldwide and determining the cost effectiveness of an intervention is, together with aspects such as efficacy, safety and accessibility, important in order to influence policy and clinical practice. One previous study from a high resource setting using a mifepristone-misoprostol regimen found medical abortion to be less costly than surgical methods [10]. The cost-effectiveness of medical abortion performed by non-physician providers compared to provision by physicians using a mifepristone and misoprostol regimen with WHO recommended dosages, remains to be evaluated. The objective of our study was to conduct a cost-effectiveness analysis of medical abortion provided by nurse-midwifes or physicians in a high resource setting where ultrasound examination and dating of pregnancy is part of the protocol. BODY.MATERIAL AND METHODS.STUDY BACKGROUND: This study is based on a previously published randomized two-sided equivalence study by Kopp Kallner et al including 1180 healthy women seeking treatment for abortion at an out-patient clinic of a university hospital in Sweden between February 2011 and July 2012 [8]. Eligible participants willing to undergo medical abortion were randomized to the intervention where a nurse-midwife counseled, examined, informed, and treated the woman (n = 597), or the standard treatment, in which counselling and physical examination was provided by a physician, and a nurse-midwife gave additional information and medication (n = 583). Contraceptive counselling and prescription was provided by the allocated care-giver. All women were treated with mifepristone 200mg orally at the clinic on day one, and misoprostol 800mcg vaginally at home or in the clinic 24–48 hours after the mifepristone administration. Follow up was provided by a nurse-midwife not participating in the study approximately three weeks after the mifepristone administration using a low sensitivity urinary human chorionic gonadotropin (u-hCG) test (cut-off 500 IU/ml). Outcome measures in the parent study were efficacy defined as complete abortion without need for surgical intervention, safety defined as no complication, and acceptability defined as preferred provider, should the woman have an abortion in the future. Complication was defined as the need for causal treatment at an unscheduled visit within 6 weeks after the abortion. Efficacy and safety were assessed using electronic patient records and self-administered questionnaires that were also used for measuring acceptability. The duration of the patients' initial visit to the clinic, as well as the allocated provider's need for a second opinion from a doctor, was recorded in the study protocol. Statistical calculations were made using SPSS 20 (IBM Corporation, Somers, NY, USA) except the generalized estimating equation performed with SAS 9.3 (SAS Institute, Gary, NC, USA). BODY.MATERIAL AND METHODS.DETAILS OF ETHICAL APPROVAL: Permission was granted by the National Board of Health and Welfare and by the Ethical Review Board of Stockholm (permission number 2010/1828-31/3, 23 December 2010) to allow midwifes to independently provide medical TOP, according to the study protocol. After approval by the regional ethics committee at Karolinska Institutet all applications were publicly available. The study was registered with Clinicaltrials.gov NCT01612923. BODY.MATERIAL AND METHODS.COST-EFFECTIVENESS ANALYSIS: The main goal of the present study is to examine the cost-effectiveness of the standard versus the intervention treatment for medical abortion using direct costs. The secondary aims are to evaluate indirect costs, the costs of the subsequent patient's waiting time and the cost of complications. Direct costs were calculated for the woman's first visit to the clinic where she was treated according to the standard or intervention arms. Treatment on day 2 (24–48 hours after mifepristone) and follow-up did not differ between groups. We calculated the incremental cost effectiveness ratio (ICER) to determine the cost for achieving a complete abortion without surgical intervention when adopting the intervention as compared to the standard treatment. The ICER considers changes in effectiveness as well as cost of treatment and was established using the formula: [Cost of Intervention–Cost of Standard treatment]/ [Effectiveness Intervention–Effectiveness of Standard treatment] where Intervention is nurse-midwife provision of medical abortion, and the Standard treatment is abortion provision by physicians. The Effectiveness is the difference in numbers of complete abortions without surgical intervention between the groups, which was measured as 1.6 fewer abortions requiring vacuum aspiration for completion per 100 patients in the Intervention group in the parent study. It is important to conduct a cost-effectiveness analysis even though the effectiveness of the Intervention treatment has already been established. The costs of the two treatments could still be significantly different. If the Intervention treatment is substantially more expensive than the Standard treatment, the difference in efficacy might not be enough to justify its wide-spread adoption. A model taking into account the cost of midwives and physicians (based on salaries, payroll tax, and time spent with the patients), usage of surgery rooms, consultation time for second opinion with a physician or senior physician, cost of the treated women's time, and training of participating midwives was constructed. Direct costs of salaries and examination room rent were derived from the department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden in 2011. The cost of consultations was calculated based on the average consultation time and participating physicians' average salary. The Statistics Sweden (Statistiska Centralbyrån, SCB) report"Average income among women per age group in Stockholm County 2011" was used to estimate the cost of the treated women's time. Searches in the SCB database were conducted in February and November 2014 [11]. The costs of training the nurse-midwifes were assessed based on the cost of a subsequently developed ultrasound course for midwifes providing medical abortion, including the salary costs of the participating midwifes and the cost of physician supervision. The cost of complications was calculated based on recorded unscheduled visits using the registered diagnosis and treatment according to the diagnostic-related group-coding system, a weighted average of costs per diagnostic related group (DRG) (www.socialstyrelsen.se). There was no difference in costs of disposables, ultrasound, or medication. The comparison was made per procedure. To derive a conservative estimate of the cost of the intervention the reduced waiting time of the subsequent patient was added. All costs were calculated in Swedish Krona (SEK) and converted to 2011 Euro (EUR) (average exchange rate 2011; 1 Euro € = SEK 9.0298). BODY.RESULTS: The randomized-controlled equivalence trial showed that provision of medical abortion by nurse-midwifes was superior to provision by physicians, with a risk difference for effectiveness, complete abortion without surgical intervention, of 1.6% (95% CI; 0.2–3.6%, p = 0.027). This means that for every 100 patients (procedures), the Intervention treatment resulted in 1.6 fewer follow-up surgical abortions than the Standard treatment. In per-procedure terms, this translates into 0.016 fewer surgical interventions per treatment in the Intervention arm. The woman's initial visit to the clinic was significantly less time consuming in the intervention arm (average 42 minutes) than the standard treatment (60 minutes) (P<0.001), which affects most direct cost parameters. Patients allocated to the standard treatment spent, on average, the same amount of time (30 minutes) with each provider. The direct costs of the woman's first visit to the clinic of the standard treatment was EUR 58.3 per procedure and the cost of the intervention treatment was EUR 45, see table 1. 10.1371/journal.pone.0158645.t001 Table 1 Direct costs of standard and intervention treatment, per procedure, 2011 EUR. Cost item Cost Standard n = 533 Cost Intervention n = 535 Salary midwife 18 25 Salary physician 21 0 Examination room 5 4 Consultation w physicians 0.3 1 Patient’s time 14 10 Training of midwives 0 5 Total cost per procedure EUR 58.3 45 The direct costs are dependent on treatment time mean 60 minutes (SD18.3) in the standard care group, and 42 minutes (SD 24.1) in the intervention group(P<0.001) Training costs are based on the total cost of training for the 2 participating midwives and include the cost of a 2-day course, salary and physician supervision of 50 ultrasounds. Total EUR 2774, 535 procedures were performed during study Consultations, when the caregiver obtained a second opinion from another physician, occurred in 4% of the cases for doctors and in 26% for nurse-midwifes (95%CI 18–26%, P<0.001). The physician providers' consultations lasted 14 minutes on average, 17% of the consultations were ultrasound queries. Less experienced physicians consulted more often than more senior physicians. Nurse- midwifes consultation lasted six minutes on average and 42% the consultations were ultrasound queries, which decreased over time. Costs of the time women spent at the clinic were assessed using the time women spent with their allocated provider and the average income among women per age group in Stockholm County, adjusted to participating women per age group. Women's travel time was not included. The nurse-midwives participating in the study were highly experienced in all aspects of abortion care, including contraceptive counselling and insertion of IUDs and implants, but had not previously performed ultrasounds. In the study setting, participating nurse-midwives were introduced to the concepts of ultrasound by a senior consultant, and coupled with physicians performing ultrasounds. No additional cost was associated with training of the physicians. In the Intervention arm the waiting time for future abortion seeking women was reduced by 0.3 hours, which further reduces the cost of the intervention by EUR 4. This is assuming that these women have the same age distribution and the same average income as the women included in the study. The overall complication rate, defined as an unscheduled visit for symptoms that lead to further treatment was 4.1% (20/493) in the nurse-midwife group and 6.1% (29/472) in the standard care group (95%CI; -0.7–5%, p = 0.14). There were no significant differences in safety parameters, and costs for complications were thus not included in the ICER calculations. In the intervention group, 13 (2.6%) women were treated as outpatients and 6 (1.2%) admitted to the hospital. In the standard care group, 21 women (4.4%) were treated at the outpatient clinic and 7 patients admitted (1.5%). Secondary costs of unscheduled visits, complications and surgery were derived from the hospital files for each patient. Costs of complications are shown in table 2. 10.1371/journal.pone.0158645.t002 Table 2 Cost of complications 2011 EUR. Treatment Standard (n = 533) * Intervention (n = 535) ** Outpatient 10122 (n = 21) 5306 (n = 13) Clinic 15362 (n = 7) 14273 (n = 6) Total 25485 19579 Per procedure 48 37 * 2 missing, 1 patient had 2 complications. One of the women in the physician group was treated as an outpatient and later admitted to the hospital. **1 missing A woman in the intervention group sought care outside of Stockholm county thus treatment costs could not be tracked. The previous study has established that nurse-midwife provision of medical abortion to healthy women in a high resource setting where ultrasound dating of the gestational length is part of the protocol is more efficacious than provision by physicians. Calculation of direct and indirect costs show that the intervention is also cheaper. Based on these findings a negative ICER ranging from -831.25 EUR when only direct costs are considered to -17500 EUR evaluating total costs was calculated. This means that by implementing the intervention a saving in the range of 831 to 1768.75 EUR is obtained for each avoided surgical intervention, see table 3. 10.1371/journal.pone.0158645.t003 Table 3 Incremental cost effectiveness ((ICER) of different measures of costs, EUR. Item Difference in costs per case Difference in efficacy per case ICER Direct cost per woman treated 45–58.3 0.016 -831.2 Direct costs including waiting time for the consecutive patient 41–58.3 0.016 -1081.2 Total direct and indirect costs 78–106.3 0.016 -1768.8 The parent study found that women in the nurse-midwife group had long-acting reversible contraceptives (LARCs) inserted within 3 weeks of the TOP significantly more often than women counselled by physicians (95% CI 3.2–15.2, P = 0.005). Calculations of direct savings due to this finding have not been performed within the scope of the present study but its implication is further discussed below. BODY.DISCUSSION: We have shown that provision of medical abortion by nurse-midwives is cost-saving and equally effective as provision by physicians in a high resource setting. These findings are important as the evidence can influence policy-makers' decisions, change clinical practice in settings where health care budgets are limited, and eventually contribute to increase numbers of abortion providers that are needed in both high and low resource settings. Previous studies have demonstrated that task-shifting of different health care services, such as treatment of orthopedic conditions in low income environment, is cost effective, but this is the first study showing that task-shifting is cost effective in provision of medical abortion [12]. In our study, task shifting of medical abortion generated direct economic benefits associated with the shorter time spent in the clinic by providers and patients and nurse-midwives' lower salaries. Costs were also reduced due to shorter waiting time for subsequent patients and a possible lower cost for the treatment of complications. The incremental cost effectiveness was negative, which occurs when the intervention is superior in efficacy and cheaper than the standard treatment. In addition, the costs of the intervention are expected to decrease further over time as the cost of training nurse-midwives is disseminated on more procedures and the number of consultations with physicians' decline further. Medical abortion has been shown to enhance access to safe abortion and to be cost-effective[13]. It is preferred by women who want to avoid surgery and find the method natural and private. Some previous studies comparing medical and surgical methods of abortion provision and treatment of miscarriage have argued that the cost effectiveness of medical methods is reduced due to complications and method failure[14, 15]. However, complication rates of both medical and surgical termination of pregnancy are low, and dependent on the choice of treatment regimens as well as method of follow-up. Mifepristone-misoprostol regimens are more effective than misoprostol alone and ultrasound can be difficult to interpret leading to unnecessary surgical interventions [16]. Our study using a mifepristone and misoprostol regimen and a 3-week follow up shows an overall efficacy of 98.2%, which is in line with WHO guidelines. The women in the nurse-midwife group had long-acting reversible contraceptives (LARCs, implants and intrauterine devices and systems) inserted within 3 weeks of the abortion significantly more often than women counseled by physicians (95% CI 3.2–15.2, P = 0.004). It has previously been shown that LARC use decreases numbers of repeat abortions [17]. The type of abortion provider as well as their training in insertion and counseling has previously been shown to be an important factor influencing women's uptake of LARCs [18, 19] [20]. Approximately 90% of all mortality and morbidity related to abortion could be averted by the use of effective contraception [21]. LARCs are more effective in preventing unintended pregnancies than oral contraceptives and even modest increases in the uptake of LARCs in women of fertile age have been shown to generate cost-savings for societies. A study from the United States shows that if 10% of women aged 20–29 years switched from oral contraception to LARC, total costs for unintended pregnancies would be reduced by USD 288 million per year[22] and a recent study from Norway estimated cost-savings generated from a 5% increased LARC use in women 15–24 years to be 7.2 million NOK or almost 800000 Euro. Rates of unintended pregnancies in the United States have decreased from 51% to 45% between 2008 and 2011, most likely due to increased use of long acting reversible contraception [23]. A recent study showed that a high proportion (24%) of women sought a repeat abortion within four years after a first abortion, fewer repeat abortions were seen among women who started long acting contraception after the index abortion[24]. Repeat abortions and unintended pregnancies that are continued soon after a first abortion, particularly among teenaged and young women, could be significantly reduced by of LARCs BODY.DISCUSSION.STRENGTHS AND LIMITATIONS: To our knowledge, this is the first study to evaluate the cost effectiveness of first trimester medical abortion provided by nurse-midwifes or physicians in a high resource setting where ultrasound dating of gestational age is part of the protocol. The study is a straightforward analysis of actual direct costs based on institutional prices derived from the outpatient clinic, where the previous equivalence trial was carried out. Possible limitations associated with the parent study include that only healthy women were randomized and that the study was not blinded. The participating nurse-midwives were highly experienced and motivated, which might have affected outcomes such as acceptability and higher prescription and provision of LARCs. Neither the parent study, nor the present one provide conclusive answers on why nurse-midwives provide abortion more effectively than physicians. As suggested in the parent study a possible reason women prefer nurse-midwifes may be that abortion is not seen as a medical condition. The shorter time for the first visit in the intervention arm may be associated with women seeing only one provider which reduces waiting times at the medical office. Implementing task-shifting in abortion provision it is important to consider that non-physician providers' willingness to participate in abortion provision vary between individuals and settings. Two large surveys from California and India respectively indicate that mid-level providers are willing to be trained to perform medical abortions [25, 26], on the other hand a systematic review covering health care providers attitudes toward induced abortion in sub-Saharan Africa and South-East Asia found that nurses and midwives disliked being involved in abortion provision[27]. Savings following implementation of the intervention might be underestimated as we did not consider the opportunity cost due to released physician time. Neither did we assess the gain from increasing the total number of abortions provided. Reduced quality of life and societal costs related to delaying the procedure for those waiting to undergo an abortion are difficult to quantify and beyond the scope the current analysis. As patients are free to seek care at their own discretion it was not possible to trace the costs for all unscheduled visits to clinics outside the Karolinska University Hospital which might have occurred after the follow-up visit. BODY.CONCLUSION: Provision of medical abortion by nurse-midwives is more cost effective than the standard treatment, provision by physicians, in a high resource setting where ultrasound dating is part of the protocol. This finding supports previous evidence of the efficacy and acceptability or task shifting in medical abortion, and provides decision-makers and clinicians with an important tool when assuring increased access to safe abortion services.

TITLE: Aquatic therapy for boys with Duchenne muscular dystrophy (DMD): an external pilot randomised controlled trial ABSTRACT.BACKGROUND: Standard treatment of Duchenne muscular dystrophy (DMD) includes regular physiotherapy. There are no data to show whether adding aquatic therapy (AT) to land-based exercises helps maintain motor function. We assessed the feasibility of recruiting and collecting data from boys with DMD in a parallel-group pilot randomised trial (primary objective), also assessing how intervention and trial procedures work. ABSTRACT.METHODS: Ambulant boys with DMD aged 7–16 years established on steroids, with North Star Ambulatory Assessment (NSAA) score ≥8, who were able to complete a 10-m walk test without aids or assistance, were randomly allocated (1:1) to 6 months of either optimised land-based exercises 4 to 6 days/week, defined by local community physiotherapists, or the same 4 days/week plus AT 2 days/week. Those unable to commit to a programme, with >20% variation between NSAA scores 4 weeks apart, or contraindications to AT were excluded. The main outcome measures included feasibility of recruiting 40 participants in 6 months from six UK centres, clinical outcomes including NSAA, independent assessment of treatment optimisation, participant/therapist views on acceptability of intervention and research protocols, value of information (VoI) analysis and cost-impact analysis. ABSTRACT.RESULTS: Over 6 months, 348 boys were screened: most lived too far from centres or were enrolled in other trials; 12 (30% of the targets) were randomised to AT (n = 8) or control (n = 4). The mean change in NSAA at 6 months was −5.5 (SD 7.8) in the control arm and −2.8 (SD 4.1) in the AT arm. Harms included fatigue in two boys, pain in one. Physiotherapists and parents valued AT but believed it should be delivered in community settings. Randomisation was unattractive to families, who had already decided that AT was useful and who often preferred to enrol in drug studies. The AT prescription was considered to be optimised for three boys, with other boys given programmes that were too extensive and insufficiently focused. Recruitment was insufficient for VoI analysis. ABSTRACT.CONCLUSIONS: Neither a UK-based RCT of AT nor a twice weekly AT therapy delivered at tertiary centres is feasible. Our study will help in the optimisation of AT service provision and the design of future research. ABSTRACT.TRIAL REGISTRATION: ISRCTN41002956 ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40814-017-0132-0) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: International guidelines for the multidisciplinary management of people with Duchenne muscular dystrophy (DMD) recommend regular physical therapy, including stretching and positioning regimens 4–6 days/week to maintain joint ranges, and submaximum aerobic exercise/activity to avoid disuse atrophy. Exercises in water are highly recommended. They note that the evidence base for these recommendations is weak and do not detail specific therapy interventions or dosage, nor do they discuss aquatic therapy [1]. Aquatic therapy (AT), sometimes also called hydrotherapy, is defined by the UK Aquatic Association of Chartered Physiotherapists (ATACP) as "a therapy programme utilising the properties of water, designed by a suitability qualified physiotherapist specifically for an individual to improve function, carried out by appropriately trained personnel, ideally in a purpose built, and suitably heated hydrotherapy pool" [2]. Subsequently, hydrotherapy was specified to be a programme designed by a physiotherapist but implemented by carers or teaching assistants, while aquatic physiotherapy is delivered by a specialist physiotherapist [3]. There are limited data on the effectiveness of AT in general [4–6], and none in people with DMD. Our study addressed a 2012 commission from the UK National Institute for Health Research (NIHR) Health Technology Assessment (HTA) programme for a feasibility study (HTA 12/144/04). The specific objective was to collect data that would tell us whether it was feasible to run a full-scale trial, assessing the clinical effectiveness of AT in maintaining physical function in people with Duchenne muscular dystrophy. The principal focus of this paper is the feasibility of a full-scale research study. With the debate about terminology unresolved [7, 8], we present this data as an external pilot trial [9], supplemented by a distillation of qualitative research findings, reported more fully elsewhere, in line with guidance [10]. Although some reference to patient and professional views on the feasibility of the intervention are integral to understand the feasibility of the trial, this topic is addressed more fully elsewhere. BODY.METHODS.TRIAL DESIGN: The protocol for this pilot trial, which was developed with patient and public involvement, is available on the NIHR website [11]. The design was an external pilot trial for a parallel-group, open-labelled, individually randomised controlled trial with a 1:1 allocation ratio incorporating nested qualitative research. This report is compliant with the Consolidated Standards of Reporting Trials (CONSORT: see Additional file 1) [12]. BODY.METHODS.PARTICIPANTS: Between October 2014 and June 2015, we planned to recruit 40 boys from six UK centres with a paediatric neuro-muscular service (Birmingham, Great Ormond Street Hospital London, Leeds, Oswestry, Southampton, Sheffield). Medical notes were reviewed to select ambulant boys aged 7–16 years with genetically confirmed DMD, North Star Ambulatory Assessment (NSAA) score of ≥ 8, established on corticosteroid therapy, that is, a patient has been treated with prednisolone or deflazacort for at least 6 months with no major change in drug, dosage or frequency for at least 3 months before the initial assessment. The major changes were defined as (1) frequency of change from daily to alternate day or other non-daily regimen (or vice versa) and (2) dose increase in line with weight is acceptable. The other change is an exclusion criterion: (3) drug changes from prednisolone to deflazacort (or vice versa). Potential participants were assessed to determine if they were able to complete a 10-m walk test with no walking aids or assistance as part of the North Star Ambulatory Assessment (Item 2). We excluded those who were involved in another clinical trial; had more than 20% variation between screening and baseline NSAA scores; were unable to commit to the programme of twice weekly AT for 6 months; and/or had any absolute contraindications or precautions to AT listed in the study protocol [11]. Potentially eligible boys and their carers were invited to an initial consent appointment. After assessment, written assent from the boys and consent from their carers, they were randomly allocated in a 1:1 ratio by a centralised web-based randomisation system provided by the Sheffield Clinical Trials Research Unit (CTRU) to AT plus land-based therapy (LBT) or LBT alone. Allocation concealment was achieved by ensuring the participant identifier was entered by the physiotherapist, following which the allocation was revealed; no member of the study team had access to the randomisation schedule during recruitment. BODY.METHODS.INTERVENTIONS: Standardised AT was prescribed and delivered by an AT-trained physiotherapist (with specialist knowledge of DMD), based on the study protocols detailed in a manual, in 30-min sessions twice weekly in an NHS pool with a temperature of 34–36 °C (Additional file 2). Standardised LBT stretches and exercises were prescribed by a specialist physiotherapist at the baseline appointment, based on the study protocols detailed in a manual (Additional file 3). With the aim of reducing missing data, the research physiotherapists sent reminders to participants who had not sent back the LBT proforma. A maximum of three reminder letters were sent to each participant over the course of the trial. Those randomised to receive AT were asked to perform LBT on four of the other 5 days of the week, while those in the control group were asked to perform LBT 6 days/week. BODY.METHODS.OUTCOMES: The primary outcome was the feasibility of recruitment of 40 participants within 6 months from six centres. Additional feasibility outcomes were a decision on the primary endpoint for a subsequent larger trial; the number and characteristics of eligible participants who were approached for the study; the number of participants randomised, withdrawn, and lost to follow-up; the number of participants who discontinued AT and were included and excluded from analysis with reasons; the recruitment rate; reasons for refused consent; participant attrition rates and reasons; data completeness; feasibility of recruiting participating sites and estimation of the costs; participant views on acceptability of research procedures and intervention; physiotherapist views on the intervention/research protocol and perceived contamination of the control group; and intervention optimisation. The following clinical data were collected for all participants: 6-min walk distance (6MWD) [13]; North Star Ambulatory Assessment (NSAA) [14, 15]; forced vital capacity (FVC); Child Health Utility 9D Index (CHU9D)—health state utility [16]; carer quality of life (CarerQoL—carer burden) questionnaire [17]; activity limitations measure (ACTIVLIM) [18]; and a health and social care resource-use questionnaire. Parents were also requested to return, in stamped addressed envelopes, diaries recording the LBT stretches/exercises performed over the previous 4 weeks. In participants allocated to AT, the Wong-Baker visual analogue scale for pain and the Children's OMNI Scale of Perceived Exertion [19] were assessed before and after each AT session. The therapists also recorded attendance as well as the AT stretches and exercises performed. BODY.METHODS.BLINDING: The participants, physiotherapists and physicians were not blinded to treatment allocation. The statisticians and health economist were blinded to treatment allocation until the statistical analysis plan was agreed and signed. BODY.METHODS.SAMPLE SIZE: The sample size for this external pilot trial was based on a recommended minimum of 30 participants (15 per group) for feasibility objectives involving parameter estimation [20]. Assuming a drop-out rate at 6 months of 20%, we set a target of randomising at least 40 participants (20 per group). While this decision was principally informed by the need to calculate a sample size for a full-scale study [9], we believed the recruitment of 40 boys in 6 months might indicate the feasibility of a trial of n = 100 to 150 in UK centres alone, given a longer recruitment window that would still be acceptable to funding bodies (1 to 2 years). Initial sample size estimates for a full-scale study, working in a frequentist framework, centred around a total n of 610 (80% power, two-sided, to detect 5%—18 m—difference in the 6MWD with 10% drop-out at 12 months). We were confident that, by using Bayesian methods [21] and selecting an meaningful outcome measure with the right measurement properties, we could reduce this sample size considerably. BODY.METHODS.FEASIBILITY CRITERION: This pilot aimed to recruit 40 children in 6 months and deliver AT to 20 of them. If this objective success criterion was met, then we could deem a full-scale study potentially feasible. Other feasibility outcomes did not involve objective stop-go (success) criteria but provided a basis for improving the research procedures. BODY.METHODS.STATISTICAL METHODS: Quantitative analysts (SR and TY) remained blind to treatment allocation until after the statistical analysis plan was finalised, the database locked and the data review completed. The intention to treat population (ITT) included all patients who were consented and randomised. This was the primary analysis set, and unless stated otherwise, all endpoints are summarised for the ITT population. Depending on the distribution of the data, continuous variables (e.g., age) were summarised by either the mean and standard deviation or the median and interquartile range (IQR). AT adherence was assessed by the number and percentage of AT sessions attended, with mean (SD), median (IQR) and minimum–maximum numbers. LBT adherence was measured by the number of days on which the prescribed exercises were performed and the percentage of the prescribed exercises that were performed on across the total number of days on which exercise adherence was recorded. Descriptive statistics (mean differences between groups and 95% confidence intervals (CIs)) were derived for clinical outcomes. Categorical outcomes are presented as the difference between groups in the percentages in each category, together with 95% CIs. Available clinical outcomes at 6 months are presented for the ITT set, by group and overall. For continuous outcomes, we present change from baseline by group and overall. We estimated the cost of the AT intervention to families and the NHS using information from the quantitative and qualitative components of the study. Data completeness was a fidelity outcome of the study. A sensitivity analysis involving imputation for missing data was not performed. Prior to database lock, all missing data was queried with staff at centres and data management. Questionnaires were scored only when all the items that made up a domain were complete for each patient and carer questionnaires. To further assess the feasibility of a full-scale RCT evaluating the clinical effectiveness of AT, we proposed group sequential designs for such a trial that would use conventional 'frequentist' statistics to perform definitive tests of hypotheses. Testing at the 2.5% one-sided significance level, adaptive designs were calibrated to have 80% power to detect a target difference of 9 points in a primary endpoint of linearised NSAA score at 6 months, assuming scores are normally distributed with standard deviation 15 points, values which look realistic given the results in [22]. Expected and maximum sample sizes of designs were recorded. Also considered were Bayesian single-stage designs measuring a primary endpoint of change from baseline at 6 months in linearised NSAA score with prior distributions informed by pilot study data. Properties of Bayesian designs based on pragmatic sample sizes were computed via simulation, assuming that on completion the Bayesian trial would declare AT plus land-based exercises superior to land-based exercises alone if the posterior probability of a benefit of AT exceeded 0.9. BODY.METHODS.INTERVENTION OPTIMISATION: An independent rater (IR) reviewed patient records (medical, social and school history), as well as data from baseline assessments, parent-completed LBT exercise diaries, therapist-completed AT session data and attendance logs, to determine the levels of treatment optimisation. BODY.METHODS.QUALITATIVE RESEARCH: Trial management group (TMG) meeting minutes and email communications helped assess barriers to implementation of the trial and the intervention. Semi-structured interviews with participants and parents (n = 8 boys) and health professionals (n = 8) were completed to gather views on acceptability of the AT intervention and research protocols. All interviews were audio recorded and transcribed. Transcripts were coded in NVivo with analysis completed using a framework analysis. The methods and results of this qualitative research component will be reported fully in the NIHR journals library (12/144/04). BODY.METHODS.PATIENT AND PUBLIC INVOLVEMENT: Co-authors JP, a young man with DMD, and VW, his mother and former carer, were involved throughout, in the design of both the AT intervention and the study, in the qualitative research analysis and in drafting outputs. BODY.METHODS.ETHICAL APPROVAL: This trial received ethical approval from Research Ethics Committee East of England—Cambridge South (14/EE/0204). BODY.RESULTS.STUDY IMPLEMENTATION: Of the initial six partner centres, two could not participate (Liverpool, Newcastle), due to a lack of pool availability or problems accessing excess treatment costs. In the UK, treatment costs are those that fall upon healthcare commissioners rather than grant-awarding bodies, where the cost of an experimental treatment exceeds the cost of the standard care [23]. The other four centres consented a total of ten boys. In view of this, further 11 UK centres were approached of which two participated (Southampton and Oswestry), consenting an additional three boys. The other eight centres did not participate for the following reasons: inability to obtain regulatory approval in time, treatment costs, lack of eligible participants within a reasonable travelling distance, pool availability and/or lack of staff. BODY.RESULTS.RECRUITMENT AND BASELINES: Recruitment occurred between October 2014 and June 2015, and the participants were to be followed up 26 weeks (±2 weeks) from the date of randomisation. Overall, 348 boys with DMD were reviewed for potential eligibility across the six participating sites. Of these, 17 were interested and eligible after an initial telephone contact, 13 screened and consented, and 12 randomised to the study: 8 to the AT group and four to the control group (Fig. 1, Table 1). One withdrew before randomisation to participate in another clinical trial. The trial ended when the planned accrual period elapsed.Fig. 1Participant flow diagram Table 1DemographicsControlInterventionTotalAge n 4812 Mean (SD)9.8 (2.5)8.0 (0.9)8.6 (1.7) Median (IQR)9.5 (8.0, 11.5)8.0 (7.5, 8.0)8.0 (7.5, 9.5) Min, max7, 137, 107, 13Ethnicity English/Welsh/Scottish/Northern Irish/British3 (75.0%)2 (25.0%)5 (41.7%) Any other White backgrounds1 (25.0%)2 (25.0%)3 (25.0%) Indian01 (12.5%)1 (8.3%) Any other Asian backgrounds02 (25.0%)2 (16.7%) Any other mixed/multiple ethnic backgrounds01 (12.5%)1 (8.3%)Others (specify) Korean011 Filipino011 Polish123Weight (kg) n 257 Mean (SD)25.550 (2.616)26.480 (4.572)26.214 (3.910) Median (IQR)25.550 (23.700, 27.400)26.500 (23.800, 26.600)26.500 (23.700, 27.400) Min, max23.70, 27.4021.70, 33.8021.70, 33.80Height (cm) n 257 Mean (SD)117.000 (0.849)119.960 (6.280)119.114 (5.339) Median (IQR)117.000 (116.400,117.600)121.000 (114.600,121.200)117.600 (114.600,121.200) Min, max116.40,117.60113.70,129.30113.70,129.30 BODY.RESULTS.NUMBERS ANALYSED, OUTCOMES AND ESTIMATION: At the 26 week follow-up visit, eight AT and one control participants contributed 6MWT data (Fig. 2). In the control group, two participants withdrew before completing because of the burden of attending the trial procedure for child and acceptance onto another trial, respectively, while another was lost to follow-up (Table 2). NSAA data was available for one of these. Primary and secondary clinical outcomes showed a small difference favouring AT, but the numbers are too small to allow significance testing (Table 3, Figs. 3 and 4). No serious adverse events and 15 adverse events were reported: 10 falls, two influenza immunisations, and one each for chest infections, sleep hypo-ventilation and delayed onset muscle soreness. Five participants recruited to the AT group showed no pain or fatigue before or after AT on the Wong-Baker and Children's OMNI scales while the other three reported increases in one or both (Table 4).Fig. 2Number of participants randomised by months Table 2External pilot trial completion summarySiteDate initiatedConsentedRandomised6-month visit (completed)Withdrew consentLost to follow-upOther withdrawnR0124/10/2014554100R0227/11/2014222000R0411/12/2014111000R0519/11/2014211001R0628/04/2015110100R0729/04/2015221010 Table 3Summary of outcomes for primary and secondary outcomes by intervention groupControlInterventionOutcome measureFollow-up n Mean(SD)Median(IQR)Min–max n Mean(SD)Median(IQR)Min–max6 min total distanceBaseline4360(84.98)362(294.5–425.5)259–4578369.63(78.39)376.5(313.5–393)266–5256 months1255255(255–255)255–2558347.63(81.88)369.5(288–388.5)226–463NSAA scoreConsent425.75(3.4)26.5(23.5–28)21–29823.38(5.93)22.5(18.5–29)16–31Baseline426(4.55)25.5(23–29)21–32824.13(5.49)23.5(19.5–28.5)18–326 months221(15.56)21(10–32)10–32821.38(8.47)21(16–28)8–33FVC absoluteConsent41.42(0.21)1.43(1.27–1.57)1.16–1.6671.52(0.2)1.5(1.27–1.74)1.27–1.78Baseline21.29(0.21)1.29(1.14–1.43)1.14–1.4351.34(0.19)1.39(1.3–1.48)1.03–1.56 months051.33(0.42)1.52(1–1.6)0.77–1.76FVC percentConsent290.5(9.19)90.5(84–97)84–97497.25(11.62)97.5(89–105.5)83–111Baseline288.5(7.78)88.5(83–94)83–94590.8(17.09)91(79–101)70–1136 months0583.8(22.57)88(68–93)56–114CHU utility valueBaseline30.92(0.07)0.89(0.87–1)0.87–180.77(0.23)0.88(0.59–0.94)0.39–0.966 months10.950.95(0.95–0.95)0.95–0.9580.87(0.09)0.87(0.82–0.95)0.71–1CarerQol scoreBaseline331.27(10.37)26.2(24.4–43.2)24.4–43.2740.6(22.9)29.3(24.4–59.4)19.7–81.66 months150.150.1(50.1–50.1)50.1–50.1751.27(6.78)50.1(48.8–50.4)44–65.8ACTIVLIM patient scoreBaseline332.67(9.71)35(22–41)22–41830.38(7.58)32(26.5–34)17–416 months12121(21–21)21–21826.88(6.36)26(22.5–31.5)19–36ACTIVLIM patient measureBaseline32.98(2.62)4.15(−0.02–4.82)−0.02–4.8282.1(1.37)2.22(1.61–2.78)−0.6–4.156 months10.180.18(0.18–0.18)0.18–0.1881.29(1.13)1.48(0.4–2.1)−0.35–2.73NSAA is scored from 0 to 34 where higher scores represent higher function. CarerQol is scored from 0 to 100 where higher scores represent better care situation. CHU values range from 0.33(worst state) to 1 (perfect health). Higher ACTIVLIM scores represent higher activity Fig. 3North Star Ambulatory Assessment (NSAA) scores Fig. 4Change in 6mWD over 6 months Table 4Summary of clinical outcomes for the intervention group onlyBeforeAfterIncreaseIDScoringAttendance statusSession counts n Mean(SD)Median(IQR)Min–max n Mean(SD)Median(IQR)Min–max n Mean(SD)Median(IQR)Min–maxR01/002Wong–BakerFull28210.43(1.03)0(0–0)0–4210.38(0.8)0(0–0)0–221−0.05(0.5)0(0–0)−2–1Wong–BakerPartial1100(0–0)0–0100(0–0)0–0100(0–0)0–0OMNIFull28102.5(0.53)2.5(2–3)2–3272.44(0.8)2(2–3)1–4100.5(0.71)0(0–1)0–2OMNIPartial10133(3–3)3–30N/ADid not attend16R01/004Wong–BakerFull29210.1(0.44)0(0–0)0–2210.1(0.44)0(0–0)0–2210(0)0(0–0)0–0OMNIFull29102.7(0.48)3(2–3)2–3282.43(0.79)3(2–3)1–4100.3(0.48)0(0–1)0–1N/ADid not attend16R01/005Wong–BakerFull29280.04(0.19)0(0–0)0–1280.04(0.19)0(0–0)0–1280(0)0(0–0)0–0Wong–BakerPartial1000OMNIFull29100.9(1.6)0(0–1)0–5280.75(1.14)0(0–1)0–5100.1(0.32)0(0–0)0–1OMNIPartial1000N/ADid not attend15R02/001Wong–BakerFull17160.06(0.25)0(0–0)0–1160.19(0.54)0(0–0)0–2160.13(0.5)0(0–0)0–2Wong–BakerPartial660.33(0.82)0(0–0)0–260(0)0(0–0)0–06−0.33(0.82)0(0–0)−2–0OMNIFull17150.2(0.41)0(0–0)0–1163.13(0.96)3(3–4)1–5153.07(0.7)3(3–4)2–4OMNIPartial660.5(1.22)0(0–0)0–361.83(1.6)2(0–3)0–461.33(2.5)2(0–3)−3–4N/ADid not attend29R02/002Wong–BakerFull16160.25(0.68)0(0–0)0–2161.06(1.44)0(0–2)0–4160.81(1.22)0(0–2)0–4Wong–BakerPartial220(0)0(0–0)0–021(1.41)1(0–2)0–221(1.41)1(0–2)0–2OMNIFull16140.5(0.85)0(0–1)0–2161.88(1.75)2(0–2.5)0–6141.07(1.82)1.5(0–2)−2–4OMNIPartial220(0)0(0–0)0–024(0)4(4–4)4–424(0)4(4–4)4–4N/ADid not attend34R04/001Wong–BakerFull28280(0)0(0–0)0–0260(0)0(0–0)0–0260(0)0(0–0)0–0OMNIFull280280(0)0(0–0)0–00N/ADid not attend10R05/001Wong–BakerFull15150(0)0(0–0)0–0150(0)0(0–0)0–0150(0)0(0–0)0–0Wong–BakerPartial1100(0–0)0–0100(0–0)0–0100(0–0)0–0OMNIFull1520.5(0.71)0.5(0–1)0–1150.33(0.82)0(0–0)0–321(1.41)1(0–2)0–2OMNIPartial10100(0–0)0–00N/ADid not attend12R07/001Wong–BakerFull26261.65(1.65)2(0–2)0–5262.65(2)2(2–4)0–7261(1.67)0.5(0–2)−2–4Wong–BakerPartial443.5(4.12)3(0–7)0–843.75(4.35)3.5(0–7.5)0–840.25(0.5)0(0–0.5)0–1OMNIFull2684(0.76)4(3.5–4.5)3–5263.85(2.54)4(2–5)0–882.38(1.6)2(1–3.5)1–5OMNIPartial426(1.41)6(5–7)5–747.5(1.91)7(6–9)6–1022(1.41)2(1–3)1–3N/ADid not attend14Wong–Baker is scored from 0 to 10 with higher scores representing worse pain. OMNI is scored from 0 to 10 with higher scores representing more tiredness BODY.RESULTS.INTERVENTION IMPLEMENTATION: The median (range) time between randomisation and initiation of AT was 47 (7–211) days and mean 63 days. Of the 349 sessions scheduled where data were available, 203 (58.2%) of the expected sessions took place and 146 (41.8%) did not. Cancellation of AT sessions was due to healthcare provider factors in 47% and participant/family factors in 43%, with 10% unaccounted for. BODY.RESULTS.INTERVENTION OPTIMSATION: AT and LBT adherence summaries are given in Tables 5 and 6, and a summary of AT attendance by participant in Table 7. In general, there was a low return of LBT forms (n = 4/12) but in those returned, a good adherence to the prescription.Table 5Intervention adherence: aquatic therapy session summaryNSAA scoreTotal no. of sessionsTotal no. of stretches competed/total no. of stretches prescribed per session (%)RandomisationConsentBaseline6 months N MedianMinMaxMeanR01/0022527262933.3314.8148.1530.99R01/0043130302929.1712.552.1728.91R01/0051618172940.918.768.1838.63R02/0013132332392.8642.8610086.77R02/0021820171893.3346.6710090R04/0012019152841.2121.2110051.55R05/0012727251651.6726.6763.6449.76R07/001192083010036.3610091.52 Table 6Intervention adherence: land-based therapy week summaryRandomisationNSAA scoreTotal no. of sessionsTotal no. of stretches with data/total no. of stretches prescribed per session (%)IDGroupConsentBaseline6 months N MedianMinMaxMeanR01/005Research intervention1618172766.6758.3310074.69R02/001Research intervention3132332481.8730.7710072.16R02/002Research intervention1820172410087.510097.92R07/001Research intervention19208201008010093.67 Table 7AT attendance summary by participantRandomisation numberActual sessions attendedActual sessions not attendedPatient factorsPool factorsUnknownAvailable sessions*Percent attendance based on available poolR01/002291651013485R01/004291641023388R01/005301551003586R02/0012329141413762R02/0021834201403847R04/00128105503385R05/00116126602273R07/00130148603879*Due to late-starting, some participants could not have completed 52 sessions by the time of study closure. The independent reviewer (IR) judged the AT prescription as good for three participants, variable by session for two and poor for three. For 4/8, they were realistic and achievable. The number of exercises prescribed per session varied between 4 and 27. Prescription compliance varied between 20 and 40% for three participants and 70 and 90 for five. 5/8 participants completed additional non-prescribed exercises. The IR considered exercises not to be appropriately prioritised in three cases. In part, this resulted from a difference in understanding regarding whether the AT manual was to be used as a menu of possible exercises or as a whole. LBT prescriptions were considered appropriate and achievable for the four AT participants who returned the data. However, after intervention by the community physiotherapist midway through the study, the number of exercises increased and compliance decreased in three. The prescribed number of exercises ranged from 5 to 13 per day. BODY.RESULTS.FEASIBILITY OF THE INTERVENTION: In five interviews, parents or boys reported some improvement was experienced after AT; in one case, the parent's view was discordant with that of the boy and physiotherapist. Less tangible physical benefits, such as loosening of muscles, were valued by parents and most physiotherapists, many of whom also noted improvements in water confidence, social confidence or well-being. Two boys reported fatigue, one transient pain. Two parents volunteered that they had sustained back injures through delivering LBT. Parents were highly satisfied with their aquatic therapists, highlighting their skill in making exercise fun for boys. It was generally agreed that the twice weekly AT programme was too burdensome, with an average journey of 15 miles and competing pressures on time. Notwithstanding this, there was enthusiasm for continuing once weekly hydrotherapy at more convenient locations, and gaps in attendance (see above) did not present a pattern that suggested decreased commitment over time. At two trusts, the physiotherapists challenged the value of the AT, highlighting that it required diversion of scarce resources from other activities. While the training emphasised that AT prescriptions should focus on the individual's capability and needs, many therapists thought they had to prescribe stretches suitable for dry land regardless of relevance. Some were dissatisfied with the intervention as a result. NHS reorganisation, following the Health and Social Care Act 2012, made it difficult to identify/communicate with community AT/physiotherapy services [22] in order to gain the appropriate NHS R&D permissions, leading us to run the study and deliver the intervention through specialist centres. However, all physiotherapists believed that AT should be delivered in community rather than specialist settings but valued the new skills they had acquired and felt that they would advocate regular and intensive AT where resources allowed. Participating trusts discussed subcontracting out AT delivery to therapists at centres, nearer to participant homes (minutes, TMG, 7 July 2014, 19 January 2015). But, with a rare and geographically dispersed population, we would have had to seek approvals from and contracted with almost as many community trusts as we recruited participants and would have had to train as many interventionists. Over 6 months, the estimated direct NHS costs ranged from £1970 to £2734 and the societal costs ranged from £2541 to £3775, based on attendance. Financial pressures and opportunity costs were noted by the physiotherapists as making the service difficult to justify. BODY.RESULTS.FEASIBILITY OF THE TRIAL: The physiotherapists and parents believed recruitment to the trial was difficult because industry trials were more attractive to parents and co-enrolment is prohibited by UK research ethics committees. They believed that participant retention was contingent on randomisation to AT, as confirmed by the drop-out rate observed in our control arm. Recruitment was particularly difficult where access to AT was already good.That was one of the worries that by partaking in this would it stop him being eligible for another trial because, as great as this is, as a parent, you're always online seeing what NICE are going to approve. (Parent) I think any patient wanting to, to participate in this trial would basically have wanted to be in the, err in the hydro arm or not bother. (Physiotherapist) The difficulty was there's so many trials going on at the moment for Duchenne that it was difficult to get appropriate patients. And because a lot of ours get hydrotherapy anyway, yeah, it was difficult. (Physiotherapist) Another major barrier to recruitment was the distance from the pool. Partly due to therapist availability and partly due to a national reorganisation of health service provision and funding at the time, we could not easily access community pools, with the result being that many possible participants lived at least 20 miles away with round-journey times of 2 hours or more. This proved too great a burden for many families to face twice weekly for 6 months, especially given that, under the Department of Health rules for funding research, travel costs for interventions count as treatment costs and cannot be reimbursed from a research grant [23].There were several parents who... couldn't make that commitment to twice a week to driving into central [area name]. There were other parents who said that if it was more local, if they didn't have siblings, if they didn't feel the child would be tired after school, after coming all the way into central [area name]... Generally parents were quite keen on the idea... they would've [participated] had the situation been easier for them. (Physiotherapist) Where boys remembered randomisation, they were negative about it, and only two parents were positive. Some physiotherapists believed the eligibility criteria should have been relaxed to allow enrolment of younger or more disabled boys. The physiotherapists and parents found the completion of intervention documentation for fidelity purposes burdensome, and the physiotherapists would require more administrative support for any future research. They believed that a future trial would need long-term follow-up and mechanistic outcomes in order to identify a treatment effect. Three families reported the questionnaire battery as too burdensome, and two complained about the vagueness of questions on the instruments used to evaluate health-related quality of life. The 6MWD is typically the most common primary outcome in trials for ambulant boys with DMD. As in other studies, concerns about the feasibility of this assessment were raised during the study, due to difficulties finding 30-m long corridors and staff trained to complete the assessment (Minutes, Trial Management Group, 15 Sep 2014, 03 Nov 2014). Table 8 provides a data completeness summary; this shows the number of participants the data were available for per group and the percentage of data available, with 100% being all data we expected. The NSAA score was the only outcome available for all participants expected at each time point, taking into account participant withdrawals. The questionnaire completion rates are provided in Table 9 with the ACTIVLIM being the only questionnaire with missing data in the control group and the ACTIVLIM and CareQOL having missing data in the intervention group.Table 8Data completenessScoringFollow-up time pointControl (N = 4) n (col %)Intervention (N = 8) n (col %)Overall (N = 12) n (col %)NSAA scoreConsent4 (100)8 (100)12 (100)Baseline4 (100)8 (100)12 (100)6 months2 (100)8 (100)10 (100)FVC absoluteConsent4 (100)7 (88)11 (92)Baseline2 (50)5 (63)7 (58)6 months0 (0)5 (63)5 (56)FVC % predicted for heightConsent2 (50)4 (50)6 (50)Baseline2 (50)5 (63)7 (58)6 months0 (0)5 (63)5 (56)6-min total distanceConsent0 (0)0 (0)0 (0)Baseline4 (100)8 (100)12 (100)6 months1 (100)8 (100)9 (100)CHU utility valueConsent0 (0)0 (0)0 (0)Baseline3 (75)8 (100)11 (92)6 months1 (100)8 (100)9 (100)CarerQOL scoreConsent0 (0)0 (0)0 (0)Baseline3 (75)7 (88)10 (83)6 months1 (100)7 (88)8 (89)CarerQOL happy VASConsent0 (0)0 (0)0 (0)Baseline3 (75)8 (100)11 (92)6 months1 (100)8 (100)9 (100)ACTIVLIM patient scoreConsent0 (0)0 (0)0 (0)Baseline3 (75)8 (100)11 (92)6 months1 (100)8 (100)9 (100)ACTIVLIM patient measureConsent0 (0)0 (0)0 (0)Baseline3 (75)8 (100)11 (92)6 months1 (100)8 (100)9 (100) Table 9Questionnaire completion (%)ControlInterventionOverallScoringFollow-up time pointMin–maxMedianMin–maxMedianMin–maxMedianNSAA scoreConsent100–100100100–100100100–100100Baseline100–100100100–100100100–1001006 months100–100100100–100100100–100100CHUBaseline100–100100100–100100100–1001006 months100–100100100–100100100–100100ACTIVLIMBaseline81.82–10010063.64–10095.4563.64–1001006 months81.82–81.8281.8272.73–10081.8272.73–10081.82CarerQOLBaseline100–10010085.71–10010085.71–1001006 months100–10010071.43–10010071.43–100100 A full-scale RCT following a group sequential design incorporating up to two interim analyses permitting early stopping for success or futility according to the ρ-family error spending criteria (with parameter ρ = 2; see Chapter 7 of [24]) would recruit the following: on average, 74.4 patients if AT plus land-based exercises are truly superior to land-based exercises alone; on average, 62.1 patients if both interventions are equivalent; and a maximum of 93.3 patients. These sample sizes are likely to be prohibitive in light of the data from this pilot trial. Rather than make definitive comparisons between interventions, a Bayesian trial based on a pragmatic sample size would settle for the less ambitious objective of improving our understanding of the effectiveness of AT. Assuming a vague uniform (0, 100) prior for the response SD and an independent normal prior for the treatment difference with mean 5.375 and variance 213.2, Table 10 lists the probability that a full-scale Bayesian RCT would conclude declaring AT plus land-based exercises superior to land-based exercises alone, for various sample sizes and data scenarios. The prior for the treatment difference is based on pilot study data: the mean is the difference between the sample mean changes from baseline in linearised NSAA score on each trial arm; the prior SD is twice the standard error of the difference in sample means to down-weight the contribution of the pilot information.Table 10Results are based on 10,000 simulationsTrue treatment differenceProbability future RCT declares AT plus optimised land-based exercises superior to optimised land-based exercises alone N = 20 N = 30 N = 40 N = 6000.100.100.110.10 δ/20.270.330.380.39 δ 0.520.640.730.753δ/20.750.880.940.93 N is the total sample size divided equally between interventions. Primary endpoint of a future Bayesian trial would be change from baseline at 6 months in linearised NSAA score, and the 'true treatment effect' refers to the underlying difference between average outcomes on each intervention. Future trial data are simulated according to the model \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \widehat{\theta} $$\end{document}θ^ ~ N(θ, 4σ2/N) and s2 ~ (σ2/N) χ N2 − 2 setting σ = 15 and δ = 9, where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \widehat{\theta} $$\end{document}θ^ is the difference between sample mean outcomes on each treatment arm (and θ is the true treatment difference) and s 2 is the pooled sample response variance BODY.DISCUSSION: This pilot trial has demonstrated that a standard full-scale randomised controlled trial would not be feasible, as originally planned, in the UK alone. Our data should help in planning future studies of both AT and LBT in DMD. The majority of UK centres we approached could not participate for a variety of reasons. Variability in pool availability and AT provision has been highlighted as a problem by a national charity [25]. NHS treatment costs (see the "Results" section) have long been known as a barrier to the timely completion of publicly funded research in the UK, and our study also suffered as a result of the lack of nationally implementable guidelines [26, 27]. In the participating centres, which included some of the largest in the UK, the majority of boys assessed did not meet our eligibility criteria (Fig. 1). In those who were ambulant and therefore eligible, many did not participate, mostly because of current or anticipated participation in other trials. In the time between planning our study and implementing it, several trials started, the existence of which we had not been aware. Although the protocols of some, such as the DMD Heart Protection Study (ISRCTN50395346), permitted co-enrollment, we were unable to obtain ethics committee approval for co-enrollment to our own study. All those randomised to the AT group completed the study, but three of the four in the control group did not, for the same reasons that prevented initial recruitment, as discussed above. Randomising as individuals meant that one family with more than one eligible boy had two boys in the AT group and another in the control group. Our study was not powered to detect clinically important differences when AT was added to LBT. The intended sample of 40 would have permitted power calculations for a future full-scale trial [20, 28]. There have not been any previous reported studies of AT in DMD. Due to problems associated with the 6MWD, the routinely collected NSAA [15, 29, 30] appears to be the most feasible outcome for any future full-scale trials. This could minimise data collection costs and loss of information due to the NSAA being routinely collected for DMD patients at clinic appointments. Rare disease groups can benefit from the use of the cohort multiple randomised controlled trial design (cmRCT) or Trials Within Cohorts (TWiCs) [31], which uses staged-informed consent to overcome drop-out and other problems associated with interventions in the absence of equipoise [32]. Pragmatic trials of physiotherapy interventions in DMD could be more feasible if the disease registries such as the NorthStar Project were able to adopt cmRCT/TWiCs functions in the future. When planning this study, we found that there were no agreed validated standardised protocols for either AT or LBT, either in terms of what to do or the dosage (frequency and duration). We therefore created our own manuals for both (reported separately). Conventionally, in the UK, AT is given as 6-week blocks but we chose a higher dosage to ensure that any benefit would not be missed through under-dosage. Independent review of the intervention optimisation showed that the dose varied and that the protocols were not always appropriately applied. In general, AT was well tolerated but in some, it caused pain or fatigue. For LBT, there was some evidence of an inverse relationship between dosage and compliance. In addition, the poor return of LBT data suggests poor compliance. One mother of a family with four affected boys told us she never performed home LBT as she did not have the time or resources. We have insufficient evidence to demonstrate whether AT provides value for money in terms of quality-adjusted life years gained. The direct (health system) costs (£1970 to £2734) are less than haemodialysis for children aged over 6 months (£10,296 to £46,352 for three sessions per week) or specialist paediatric services for cystic fibrosis (£2554 to £24,809 over 6 months) [33]. Based on experience accrued before and since the trial was designed, service users and therapists have proposed a number of ways that services could be made cheaper and more feasible.Health systems could increase the number of pool sessions offered by the health system by combining access to children with other neuromuscular conditions in the pool and requiring parents to be in the pool while a physiotherapist rotates between clients.Health systems should enable parents to safely deliver targeted AT techniques, through in-pool training and diagram sheets. Give them responsibility to access warm water pools between courses provided by the health system.Health systems and voluntary sector organisations could enable parents to link with each other, in order to share the documented high costs of warm-water pool hire and conduct AT exercises regularly at their convenience [25]. In some places, this 'hydrotherapy club' model has been enhanced by limited support from qualified and volunteer student physiotherapists, without being cost-prohibitive [34].For boys who are in special schools, access is already often better where technical instructors, physical therapy assistants and other staff members can deliver AT based on our manuals with limited oversight by health system physiotherapists. Both the qualitative research findings and the patient and public involvement underline how much families value AT in participation terms and dedicated parents are willing to negotiate complex systems in search of better access. We end this discussion section with a statement from our service user representatives, James and Victoria:The ideal is open-ended, weekly therapist-led AT, but the current service is sporadic four- to six-week blocks. James found he had to rebuild all his confidence when coming back to therapist-led AT after breaks in service. Sustaining water confidence and self-esteem is very important to self-management. If you have a big break then, each time you go back, the likelihood is you've physically deteriorated – so the break just highlights the physical deterioration. It's easier, psychologically, to experience gradual increasing difficulties over time, rather than perceive step changes in the decline of physical function. The problem then is, how do we do approach the ideal? BODY.CONCLUSIONS: Our study has shown that a full-scale RCT of AT, designed along frequentist lines, is not feasible in the UK alone. Any future studies should consider Bayesian approaches to achieve a statistically valid result with an achievable number of patients in a reasonable timeframe. Even then, the effect on motor function may not be very marked and a composite assessment also capturing individual patients well-being should be considered. We have demonstrated that AT has other benefits which are valued by people with DMD and physiotherapists. Two reviews and a conceptual framework [35, 36] offer guidance for selecting measures of participation which, we propose, should be considered as primary outcomes in future research. Our study highlights, once again [37], the lack of basic information on the effectiveness, selection/prioritisation, dosage, and practicability of valid therapy protocols for stretches/exercises in people with DMD. Our treatment manuals offer a basis for a service, but more work is needed to understand how it can best be made personalised [38–41], developmentally appropriate [42] and coordinated. To ensure new models of provision meet the needs of stakeholders, further service development of the work should be co-produced [39, 43, 44], involving DMD family members, specialist and community physiotherapists, representatives of schools and/or teaching assistants, community AT pool providers and third sector organisations. Participation is currently inequitable, being determined by income and location [25, 45]. The UN Convention on the Rights of Persons with Disabilities (Articles 19, 25 and 30) [46] and, in the UK, The Children and Families Act 2014 (special educational and health provision reasonably required) [47, 48] provide an ethical and legal basis to guide co-production of new services. Where the capacity of parents and teaching assistants allows [49], physiotherapists can build capacity and share responsibility for the work of rehabilitation in the child, family and community. This could involve providing training and printed information on exercises that can be performed safely and effectively with their boys, as well as improved signposting to services [50–55]. Any future studies should consider alternative strategies such as Bayesian analytical approaches to deliver a statistically valid result with an achievable number of patients in a reasonable timeframe. To improve participation by NHS trusts in future trials, we recommend a review of excess treatment cost provision, early engagement of NHS England, consideration of refunding travel costs to study participants and randomisation by family and not individual. BODY.ADDITIONAL FILES: Additional file 1:CONSORT. (DOCX 24 kb) Additional file 2:Hydrotherapy manual. (PDF 136 kb) Additional file 3:Land-based therapy manual. (PDG 256 kb)

TITLE: Tourniquet induced ischemia and changes in metabolism during TKA: a randomized study using microdialysis ABSTRACT.BACKGROUND: Tourniquet use in total knee arthroplasty (TKA) surgery is applied to minimize blood loss thereby creating better overview of the surgical field. This induces ischemia in the skeletal muscle resulting in reperfusion injury. Our aim was to investigate the in vivo metabolic changes in the skeletal muscle during TKA surgery using microdialysis (MD). ABSTRACT.METHODS: Seventy patients were randomly allocated to tourniquet group (n = 35) or non-tourniquet group (n = 35). Prior to surgery, catheters were inserted in the operated leg and non-operated leg. Interstitial dialysate was collected before and after surgery and at 20 min intervals during a 5 h reperfusion period. Main variables were ischemic metabolites: glucose, pyruvate, lactate and glycerol and L/P ratio. ABSTRACT.RESULTS: Significant difference in all metabolites was detected between the two groups, caused by tourniquet application. Tourniquet induced ischemia resulted in decreased levels of glucose and pyruvate to 54 and 60 % respectively, compared to baseline. Simultaneously, accumulation of lactate to 116 % and glycerol to 190 % was observed. L/P ratio was elevated indicating ischemia. In the non-tourniquet group the metabolite changes were less profound and normalized within 60 min. ABSTRACT.CONCLUSIONS: Microdialysis revealed that performing TKA with tourniquet is associated with increased ischemia. This affects all metabolites but the changes are normalized after 5 h BODY.BACKGROUND: In elective TKA the intraoperative use of pneumatic tourniquet is commonly used to minimize blood loss and enhance surgical overview. Despite knowing, that tourniquet induces ischemia and soft tissue damage surgeons still uses it, often not aware of the effects of the induced ischemia [1]. The kinetics of ischemic metabolites during periods of ischemia and reperfusion remains uncertain. The tourniquet pressure combined with ischemia has been investigated and inflicts a more profound damage to the skeletal muscle than ischemia alone [2]. The clinical aspects regarding tourniquet use has been vigorously investigated and cases of rhabdomyolysis have been described [1, 3–6]. The skeletal muscle in limbs is very sensitive to ischemic changes and a clinical assessment is not sufficient to evaluate the degree of ischemic tissue damage induced by the tourniquet [7, 8]. Thus, using an in vivo technique could provide a more accurate assessment of the metabolic events. Microdialysis (MD) is a minimally invasive technique that allows continuous in vivo monitoring of metabolism in extracellular space [9]. It was originally described by Ungersted and Pycock to monitor neurochemical changes [10]. Interstitial levels of metabolites as glucose, lactate, pyruvate, lactate/pyruvate (L/P) ratio were measured, because they serve as direct indicators of ischemia, whereas glycerol reflects cell damage [8, 11, 12]. During ischemia, lactate increases and pyruvate decreases, leading to an increased L/P ratio, where a ratio above 25 is considered abnormal [13]. Lactate/pyruvate ratio was calculated since it is a precise marker of ischemia and increase during ischemia [14]. The microdialysis catheter consists of a double-lumen linear tube that at the tip has a semipermeable membrane, the tube mimics the functions of a capillary blood vessel. The catheter is connected to a pump that, with a constant flow, pumps the fluid so it can pass the membrane. Diffusion along the concentration gradient occurs in the interstitial space and equilibrium takes place between the fluid and molecules. The molecules are collected in small vials, which reflects the composition of the interstitial space fluid and can then be analyzed immediately afterwards. The metabolites of interests have traditionally been pyruvate, glucose, lactate and glycerol. To our knowledge MD technique has not been applied in a randomized controlled setup to investigate tourniquet induced ischemia during TKA surgery, even though this could contribute to the understanding of the phenomenon. The aim of this study was to investigate the in vivo degree of ischemia in skeletal muscle with the use of MD during surgery and cuff inflation and during a period of reperfusion to estimate ischemia and cell damage. In particular the limb distally to the cuff was of particular interest and not directly beneath the cuff, where apparent ischemia takes place. BODY.METHODS: This prospective randomized clinical trial was conducted at Aalborg University Hospital, Aalborg, Denmark. A total of 70 primary TKA were performed between January 2011 and January 2012. Approval from the local Ethics Committee (approval no. N-20090045) and registration at ClinicalTrials.gov (NCT01309035) were obtained. All patients gave written consent and were enrolled in this study in accordance with the Consolidated Standards of Reporting Trials (CONSORT) and The Helsinki Declaration (Fig. 1).Fig. 1Flow chart of included participants This study was part of a larger randomized controlled trial where two other main aims were investigated: tourniquets effect on implant fixation and clinical outcomes. The ischemic conditions of tourniquet use are presented in this publication. BODY.METHODS.PATIENTS: Patients aged 50–85 scheduled for primary unilateral TKA were included and were comparable regarding demographics (Table 1). Exclusion criteria included rheumatoid arthritis, peripheral vascular disease, diabetes, prior knee surgery and use of anticoagulation medicine.Table 1Demographics and perioperative dataTourniquet (n = 31)Non tourniquet (n = 31)MeanSDMeanSDAge (yrs)68,38.468.27.8Sex m/f16/1517/14BMI25.12.025.22.5Operation time (min)69.55.371.34.5Cuff pressure mmHg)250Ischemia duration (min)74.43.7 Patients were block randomized using sealed envelopes and were allocated into two groups: 34 patients had surgery using a tourniquet (Tq group) and 33 patients had surgery without the use of a tourniquet (non-Tq group). The envelopes were opened when the surgeon was present in the operating theatre before surgery. Patients were unaware of the group to which they had been allocated. BODY.METHODS.MICRODIALYSIS: In this study we used CMA 60 (CMA Microdialysis AB, Sweden) catheters (length 30 mm, outer diameter 0.6 mm and molecular cut off 20 kDa) in skeletal muscle of the lower extremity. In both groups 2–3 ml lidocaine was injected subcutaneously in the gastrocnemius muscles (vastus medialis) before catheters were inserted parallel to the muscle fibers at an angle of 35°. The correct position of the catheter was verified by ultrasonography. In the non operated leg a catheter was inserted at same level, serving as a reference. Catheters were connected to a syringe filled with 4 ml perfusion fluid T1 (CMA Microdialysis AB, Sweden) that was placed in CMA 106 MD pumps, that were constant perfused at a rate 0.3 μl/min. Afterwards a period of 40 min of flushing and stabilization was allowed. The ISCUS MD analyzer (CMA Microdialysis AB, Sweden) with Reagent Set A was used to analyze all the collected MD samples and this was done immediately after sampling. Before surgery, MD catheters were inserted and the average of the first consecutive samples before performing surgery, were used to establish a baseline and defined as 100 % for metabolites (Table 2). Baseline was measured, after an initial 40 min flushing period followed by a stabilization period of 20 min. In the reference leg, baseline reached stable values within that period of time, and remained unchanged for the whole period of 300 min.Table 2Average interstitial baseline concentration at a constant flow rate of 0.3 μl/minGlucose (mmol/L)5.0 ± 2.0Pyruvate (μmol/L)64.9 ± 10.4Lactate (mmol/L)1.8 ± 0.4Glycerol (μmol/L)84.5 +/12.6L/P ratio28.3 ± 3 Immediately after surgery and tourniquet release if that was used, the first sample was collected which defined the time zero (t = 0). The dialysates were regularly collected every 20 min during a 5 h postoperative period, representing time of reperfusion. Samples from each patient were obtained and the changes in the operated leg was compared to the baseline and the patient's own reference leg. In addition, the non-tq group served as a reference group. Differences between the two groups were compared to evaluate tourniquet effects. Longer periods of ischemia leave the cells depleted of energy and the ability to regenerate metabolites decreases. The skeletal muscle must change from oxidative phosphorylation to anaerobic glycolysis to create energy and maintain homeostasis. Thus, a rise is seen in lactate production and decrease in glucose and pyruvate [15]. Glycerol is mainly derived from the degradation of phospholipids in cell membrane and increases due to cell damage. The ratio between the concentration of a metabolite in a dialysate and interstitial space is expressed as "relative recovery". Recovery of a given dialysate is affected by numerous factors such as molecular weight, surface area of membrane (length and diameter), perfusion flow rate, diffusion rate [9, 16]. Previous studies have described that relative recovery is inversely related to perfusion flow rate, i.e at a slow rate of nearly zero (0.33 μl/min) the relative recovery will be approaching 100 % [9]. The metabolite recovery in present study was defined as 100 %, as relative recovery was not investigated. In the literature most of the experiments are performed with incomplete recovery [8, 12, 15]. BODY.METHODS.SURGICAL TECHNIQUE: All procedures were standardized with regard to spinal anesthesia, operative technique and postoperative pain treatment and rehabilitation regimen. Both groups had an appropriately sized thigh tourniquet applied, but it was only inflated in Tq group. In the non-Tq group, it was placed on the thigh but not inflated, thereby serving as a safety device if uncontrollable bleeding should occur. In Tq group limb exsanguination was done by elevation for 2 min, the cuff was inflated to 250 mmHg. The cuff was not removed until the wound was closed and dressed. All knee implants were the NexGen® CR-Flex Fixed Bearing Knee (Zimmer, Warsaw, Indiana, USA) with use of Biomet Refobacin® Bone Cement R (Biomet, Warsaw, Indiana, USA). In all cases, the patella was resurfaced. Surgical procedures were all performed by the same surgeon. A two-stage cementation procedure was performed. The tibia and patella were implanted first, and then another package of cement was used to fixate the femoral component. This procedure, although more time consuming, was done to secure a careful cementation. Immediately after wound closure, dressings were applied, and the cuff was deflated in the Tq group and removed. BODY.METHODS.STATISTICAL ANALYSIS: Data for each metabolite over time in each group were analyzed by using analysis of variance (ANOVA), Student's t-test for comparison of the Tq group with the non-Tq group, and Wilcoxon rank sum test if assumption for t-test was not fulfilled. Data is presented as mean and standard deviation for normal distributed data. The metabolic changes during surgery and reperfusion are expressed in percentages of baseline values. The level of significance was set at 95 % confidence limit and P-value less than 0.05 was considered significant. Statistical analysis was performed by using STATA 11.0 BODY.RESULTS: Seventy patients were enrolled in the trial: 62 patients (33 male and 29 female) completed the study (Fig 1). Preoperative demographics were similar between groups and showed no significant differences regarding age, weight or gender. The duration of ischemia was 74.4 ± 3.7 min in the tourniquet group. BODY.RESULTS.COMPARISON BETWEEN TQ GROUP AND NON-TQ GROUP: Comparing the Tq group with non-Tq group differences were registered in all of the metabolites from beginning of reperfusion time and until a period of 180 min. After that there were no difference and the metabolites were restored back to initial levels. This is expressed in Fig. 2 where the mean differences between the two groups are shown.Fig. 2Tourniquet use induces significant ischemia and differences between the Tq group and the non-Tq group are detected in all of the metabolites from the beginning of reperfusion and until 180 min BODY.RESULTS.TOURNIQUET GROUP: All metabolite concentrations were affected following tourniquet induced ischemia (Fig. 3).Fig. 3Tq group: absolute values in percentile change from baseline. The ischemic changes are normalized after 300 min Glucose decreased by 54 % (2.3 ± 0.7 mmol/L; p < 0.001) and this reduction was detectable during time of reperfusion, but restored back to baseline 300 min postoperatively. Pyruvate concentration was initially reduced to 60 % (25.9 ± 5.6 μmol/L; p < 0.001), while it was dramatically elevated during first period of 30–60 min of reperfusion to 123 % (145.6+/10.9 μmol/L; p < 0.001). At 180 min it was restored back to baseline and no difference was detected (p = 0.118). Concentration of lactate increased significantly during reperfusion of 30–60 min up to 116 % (3.9 ± 0.8 mmol/L; p < 0.001). After 120 min of reperfusion it slowly returned to baseline (p = 0.129). After 300 min no significant difference was registered (p = 0.952) when comparing to baseline (Fig. 3). Concentration of glycerol also increased dramatically at beginning of reperfusion to 190 % (244.7 ± 12.5 μmol/L; p < 0.001) and stayed significant increased during 180 min of reperfusion (p < 0.001). At 300 min there was no significant difference (p = 0.634). L/P ratio increased significantly 79 % (107 ± 33.3; p < 0.001) after period of ischemia, but after 90 min of reperfusion initial level was restored. Significant differences in all metabolites were noted until 180 min. between operated leg and non operated leg (Fig. 4).Fig. 4Mean difference between operated leg and reference leg in the Tq group. Significant differences in metabolites are detected until 180 min All values returned to baseline values within 300 min in both legs and no difference was registered. BODY.RESULTS.NON-TOURNIQUET GROUP (NON ISCHEMIC REFERENCE GROUP): The metabolites were less affected an returned faster back to initial levels (Fig. 5).Fig. 5In the non-Tq group metabolite changes were smaller and restored within 60 min The first sample after surgery showed a glucose concentration that only decreased 11.5 % (4.6 ± 0.7 mmol/L; p < 0.001) during surgery and during 90 min of reperfusion normal levels were reached (p = 0.220). Pyruvate concentration was reduced to 13.5 % (53.8 ± 9.5 μmol/L) of the initial value and during a short reperfusion period of 30 min, it was back to baseline. Concentration of lactate increased during early reperfusion and at 30 min it reached a maximum of 30 % (2.6 ± 0.5 mmol/L). After 60 min. it was unaltered and no statistical significance was registered. Glycerol concentration was increased to maximum of 48 % (114.5 ± 15.4. μmol/L) 60 min postoperatively. During a longer postoperative period it slowly returned to normal (Fig. 5). L/P ratio also changed significantly reaching maximum at 30 min reperfusion, 45 % (42.2 ± 13.3; p < 0.001) but after this time of it was quickly restored. Difference between operated and non operated leg did not show greater ischemic conditions, rather cell damage as a response to surgery was registered, expressed as increase in glycerol (Fig. 6)Fig. 6Mean difference in the non-Tq group between operated leg and reference leg. Glycerol is affected due to cell damage as response to surgery. Ischemic metabolites are not affected BODY.DISCUSSION: Microdialysis is recognized as a useful tool when assessing metabolic changes in skeletal tissue in clinical settings [8, 15]. The present study establishes microdialysis as an effective way to monitor interstitial levels of metabolites during tourniquet induced ischemia. The study was conducted to investigate metabolic changes distally in a limb, since the ischemia underneath the tourniquet has been described previously in literature [15]. The period of interest was when the limb was exposed to ischemia during surgery and the following time of reperfusion. The main findings showed that tourniquet use causes ischemia and cell damage as measured by metabolites. Significant ischemia can be detected in the affected limb until 3 h because of tourniquet use. All Marker levels were normalized after 5 h. To our knowledge, this study is the first to assess the ischemic changes during TKA surgery caused by tourniquet use in a randomized setup. The difference between the two groups illustrate that tourniquet application induces changes in level of metabolic markers, which are manifested up to 180 min of reperfusion. The systemic response to surgery observed in the markers was adjusted for, by using the non-tourniquet group as reference and we concluded that the differences in marker levels are locally affected by the tourniquet induced ischemia. In the tourniquet group there were significant changes of all the metabolites in the operated leg, when comparing baseline to measurements during reperfusion. The differences were present in all metabolites and were statistically significant comparing from baseline during reperfusion, before slowly returning to normal levels. The non-operated leg, that served as indicator for systemic response, was compared to the operated leg and significant difference were also present at the individual measurement points. The difference between the operated leg and non operated leg represents the local skeletal muscle response in the operated leg. In the non-tourniquet group, levels of metabolites had changed in the operated leg when comparing baseline to postoperative measurements, but they returned to baseline values faster. Changes were similar to tourniquet group, but not as large or prolonged. When comparing the operated leg and reference leg there was no significant difference, indicating that a local response is not occurring and that the changes are due to an overall systemic response to surgery (Fig. 5). Thigh pain and swelling has been investigated in other studies, finding tourniquet application being the reason for increased pain and swelling due to ischemia and direct compression [4, 17]. Little is known about basal metabolite concentrations in interstial levels of resting skeletal muscle. Comparing to previous studies our baseline levels are in agreement with data reported earlier [8, 12, 15]. In these studies baseline levels ranged from 3.3–5 mmol/L for glucose, between 1.9 –2.4 mmol/l for lactate, between 40–96 μmol/L for glycerol, while pyruvate was only measured in one study at levels of around 40 μmol/L [15]. Östman et al. [15] used microdialysis to measure tourniquet induced ischemia in patients undergoing arthroscopic surgery. Here measurements were carried out over a time course of 120 min after tourniquet release. All the metabolites were affected and within 120 min the changes were restored back to normal values. The results are in consistence with this present study, where the same metabolite kinetics was observed. Glycerol is a component of the cell plasma membrane and released into the interstitial space when cells are damaged during surgery. Glycerol can be used as a marker of cell destruction. In addition, high levels of glycerol may also be due to the hormonal regulation of lipolysis and hypoglycemia during tourniquet use which facilitates a catecholamine response that initiate a lipolysis reaction in skeletal muscle [17]. This can partially be a reason for the high rise in glycerol concentration in the tourniquet group, combined with the mechanical compression of the tourniquet. Increased L/P ratio is a precise marker of ischemia and we observed a difference from baseline immediately after surgery when using tourniquet. After a period of 60 min reperfusion L/P ratio levels were back to normal. In the non-Tq group no difference was observed. Muscles are believed to be relatively resistant to ischemia, but even shorter or sudden periods of ischemia may result in an overload of calcium in the muscle and could in weaker patients induce secondary complications such as compartment syndrome and respiratory distress syndrome [8, 13]. This should be taken into consideration if tourniquets are applied. Microdialysis is a minimally invasive technique that has limited risks for patients and even with small concentration volumes, monitoring of the ischemic changes is allowed. It is applicable despite reduced blood flow during ischemia and a continuous sampling of interstitial fluids can be performed. Microdialysis contains a major limitation when estimating data because only an approximation can be stated. Recovery depends upon many factors that affect the equilibrium. To achieve the highest recovery we used the largest membrane recommend to skeletal tissue and the lowest perfusion rate allowed. Most of the clinical studies available use relative recovery and by using very low flow the recovery will be reaching nearly 100 %. BODY.CONCLUSIONS: This study shows that using tourniquet in TKA surgery is associated with increased ischemia during first the postoperative hours but the changes are normalized after 5 h.

TITLE: The Effect of Health Belief Model-Based Education on Knowledge and Prostate Cancer Screening Behaviors: A Randomized Controlled Trial ABSTRACT.BACKGROUND:: Prostate cancer has been reported as the second leading cause of cancer death among men in 2013. Prevention and early detection of cancer are considered as critical factors in controlling the disease and increasing the survival of patients. Therefore, we aimed to investigate the effect of Health Belief Model (HBM)-based education on knowledge and prostate cancer screening behaviors in a randomized controlled trial. ABSTRACT.METHODS:: This study was a non-blinded randomized controlled trial. We enrolled 210 men aged 50-70. Balanced block randomization method was used to randomize the final participants who had inclusion criteria into intervention (n=93) and control (n=87) groups. The participants of the intervention group attended training workshops based on HBM. Data were collected using three questionnaires, i.e. demographic questionnaire, Prostate Cancer Screening-Health Belief Model Scale (PCS-HBMS), and the Knowledge about Prostate Cancer Screening questionnaire, all given before and immediately one month after the intervention. ABSTRACT.RESULTS:: The mean scores of the perceived susceptibility, severity, barriers and benefits increased significantly after the intervention (P>0.05) in the intervention group. In the control group, such a difference was reported only for perceived susceptibility (P>0.05). The rate of participation in prostate cancer screening in the intervention group increased from 7.5% to 24% and 43.3% one month and three months after the intervention, respectively. ABSTRACT.CONCLUSION:: Our findings showed that the health education programs designed based on HBM could positively affect prostate cancer preventive behaviors of individuals by improving their knowledge level and leaving positive effects on perceived susceptibility and severity as well as considering the perceived barriers, benefits and health motivations. Trial Registration Number: IRCT2013090911691N3 BODY.I: Studies have reported that men are more prone to diseases and have a higher mortality rate than women.1 Previous investigations have shown that men adopt more inappropriate lifestyle choices, are less concerned about their health, ignore the warning signs of the disease, and also have late referral to medical centers compared with women.1 According to the official census published by American Cancer Society, prostate cancer was reported as the second leading cause of cancer death among American men after lung cancer and its incidence ranked the first among all cancers in 2013.2 However, in Iran, the rate of deaths from prostate cancer is found relatively higher than other types of cancers. Remarkably, with 1309 deaths in 2013, its mortality rate was estimated as 3.85 per 100.000 men in the same year. According to statistical surveys, this was higher than that of esophageal and laryngeal cancer but lower than that of gastric, lung and bronchial cancer.3 Based on the published statistics, its age-standardized incidence rate in Iran during 2003 to 2008 was reported as 4.69, 7.16, 14.04, 16.65, and 16.02 per 100,000 men, respectively, indicating an increasing trend of the disease in Iran during the mentioned years.4 About 97% of all prostate cancers occur in men aged 50 and older.2 Prostate cancer is fully and definitely treatable if diagnosed and detected early before the metastasis of the disease. Since such a cancer is often asymptomatic, it is diagnosed after its progress to the later stage that is incurable. At this stage, it has no definite treatment, so the mortality rate increases.5 In 2013, the American Cancer Society recommended that men aged older than 50 should be aware about screening for early prostate cancer detection and those who are at risk of developing the disease should receive information about such screening at earlier ages. Ethnicity, a family history of the disease, age and obesity are known as the risk factors of this cancer.2 Despite being asymptomatic, prostate cancer can be detected early using various diagnostic methods. Digital Rectal Examination (DRE) and Prostatic-Specific Antigen (PSA) are routine testing techniques for early prostate cancer diagnosis. Transrectal Ultrasound (TRUS- guided prostate biopsy is also another most commonly used method of diagnosing the disease.6 Furthermore, prevention and early detection of cancer are considered as critical factors in controlling the disease and increasing the survival of patients. Therefore, the importance of public health education should be emphasized in developing countries where people have inadequate information about screening methods.7 Various studies have shown that men with higher levels of knowledge show higher tendency towards such screening .3 When counseling and education is done based on a specific protocol, it could lead to a change in people's behavior.8 Health Belief Model (HBM) has been widely used to measure the health beliefs and behaviors about cancer screening. HBM is a cognitive model that tries to identify patterns of healthy behavior. The perceived susceptibility, severity, benefits, and barriers are four main components of the HBM. Behavior was explained by the HBM as ensuing from the combination of attitudes associated with four concepts. Perceived susceptibility refers to beliefs about the probability of obtaining a disease or condition. Perceived Severity: Feelings concerning the seriousness of acquiring a sickness or of leaving it untreated embody evaluations of each medical and clinical consequence (for example, death, disability, and pain) and potential social consequences (such as effects of the work, domestic life, and social relations). Perceived Benefits focus on the effectiveness of healthy behavior in reducing the threat of the condition.9,10 Perceived Barriers is the potential negative aspects of a particular health behavior, a kind of unconscious, cost-benefit analysis occurring when the individuals know the perceived barriers are more costly than the perceived benefits; then, they take action to do screening. For example, these barriers can be expensive, time consuming, unpleasant, painful or upsetting. These barriers may lead a person away from performing the healthy action. In addition to the four original concepts, health motivation has also been used as part of the HBM in predicting health related behavior. Health motivation refers to a generalized state of intent that results in behaviors to maintain or improve health. This concept was first introduced for inclusion in the HBM by Becker. The concept of health motivation used in combination with the original four HBM concepts has evidence of significant predictive ability.10 Therefore, in this study we used HBM focusing on prevention as a reference framework. Currently, there is a lack of consideration towards men's health, especially the middle-aged and elderly ones. Due to the increasing number of cases with prostate cancer reported by clinical specialists, which are caused by the late referral of the patients, we aimed to investigate the effect of HBM-based education with the purpose of increasing knowledge and the health belief about prostate cancer and prostate cancer screening behaviors. BODY.M: This study was approved by the Ethics Committee of Shiraz University of Medical Sciences (Ethics Committee Approval Number: CT-92-6721). In this non-blinded randomized controlled trial, 210 men aged 50-70 were enrolled during April to October 2013. We selected our participants from the population of men who were retired from Shiraz Department of Education, using a simple random sampling method. The researcher referred to the list of the males retired from Shiraz Education Department, using table of random numbers. Their positive and broader insights towards research projects could facilitate easy accessibility to them for further follow-ups and evaluation of the results. Shiraz General Department of Retirement Affairs was chosen as research setting due to the large number of referrals for welfare and administrative affairs. The sample size was calculated as 105 in each group based on the data of similar studies and using Power SSC statistical software (power: 80%, α: 0.05, mean difference: 1.6, loss rate=20% and SD: 3.2). A simple random sampling method was used to select 210 participants. Quadri- Balanced block randomization method was used to randomize the participants into intervention and control groups. In this study, we had two groups of control and intervention. Therefore, we used two variables, A and B, for them, respectively. By taking two variables A and B in quaternary blocks, six modes of movement were possible. According to the sample size (210), 53 blocks were needed. Then, the blocks were randomly written on paper and the researcher referred to the list of men and placed them in the blocks. Afterwards, 30 men were excluded due to their withdrawal from participation in the study, so the number of final participants was 93 and 87 in the intervention and control groups, respectively (figure 1). Figure 1CONSORT Flow diagram of participants Inclusion criteria were willingness to participate in the study, giving written informed consent, no history of prostate cancer and prostatic hyperplasia with obvious clinical symptoms, age of 50 to 70 years, and lack of severe vision and hearing impairment. However, exclusion criteria were absence in training sessions and participation in similar training courses. After explaining the aims of the study, written informed consent was obtained from all the participants and their anonymity and confidentiality were guaranteed. Data were collected by the researcher and a trained research assistant through face to face interview by using three different questionnaires including demographic questionnaire, Prostate Cancer Screening- Health Belief Model Scale (PCS-HBM) and the Knowledge Prostate Cancer Screening questionnaire. The demographic questionnaire was developed by the researcher; it included 13 questions about demographic characteristics of the participants. The questionnaire provided information about age, marital status, educational level and monthly income, history of prostate cancer and prostatic hyperplasia with obvious clinical symptoms, history of undergoing prostate cancer screening using DRE and PSA testing, a family history of the mentioned cancer, knowledge about the disease as well as the methods of acquiring knowledge about it for researcher. The Prostate Cancer Screening Knowledge questionnaire was developed by Weinrich et al. (2004) and contained 12 questions. Each question had three options "true", "false" and "I don't know". The correct responses were scored 1 and the wrong ones and those answered "I don't know" were scored 0. Scores ranged from 0 to 12 with higher scores reflecting a higher level of knowledge. Scores lower than 7, 7-9 and 10-12 were considered as low, intermediate, and good, respectively.11 Prostate Cancer Screening-Health Belief Model Scale (PCS-HBM)which was designed by Capik and Gozum (2011) included 41 items with a 5 point Likert scale anchored at 1=completely disagree and 5=completely agree. The scale consisted of 41 questions and 5 sub-scales including perceived susceptibility (5 items), perceived severity (5 items), health motivations (10 items), perceived barriers (15 items), and perceived benefits (7 items). An increase in the scores for the sub-scales of susceptibility, severity, motivation and benefit and a decrease in the score for the sub-scale of barriers reflected the positive effect of the intervention.12 PCS-HBM and Prostate Cancer Screening Knowledge questionnaire were translated in Persian using back translation technique, which includes the use of a panel of experts and interpreters to translate the items from the source language to the target language and then they were back-translated to the source language. Then, some changes were made to adapt this instrument to Iranian culture. After performing a pilot study on 30 men retired from Shiraz Department of Education, the reliability coefficient for PCS-HBM and Prostate Cancer Screening Knowledge questionnaires was calculated, using Cronbach alpha and Kuder Richardson 20technique. After analyzing the data, Kuder Richardson 20 coefficient was calculated as 0.98 for the Knowledge Prostate Cancer Screening Questionnaire and Cronbach's alpha was calculated as 0.83 for PCS-HBM questionnaire. To assess the prostate cancer screening behaviors of men in the intervention group, they were given referral forms for free consultation with a urologist and prostate cancer screening. Subsequently, the participation rate of men, who had not been screened within the last year, was examined one and three months after the intervention. Afterwards, the participants of the intervention group attended training workshops consisting of two four-hour sessions for two days, in groups of 15 participants. The educational program was designed based on pre-test results and structures of health belief model. Educational intervention was performed in the intervention group through lecture, group discussion with questions and answers, and brain storming. The learning process was facilitated by teaching aids, such as videos, photos and booklets. In the first session, first lecture method was employed due to little information in most of the subjects to make them familiar with prostate cancer, its anatomy, physiology, functions of prostate gland, pathology, and effective risk factors. Then, we used perceived susceptibility structure, talked about the incidence and prevalence rate of prostate cancer in Iran and the world, signs and symptom of prostate cancer, and the current treatment modalities of prostate cancer. Then, with regard to adults' education theory which considered free discussion as a necessary part of education, the subjects held group discussion. Then, by considering the perceived severity, those whose parents, relatives or a close friend had died as a result of prostate cancer were invited to talk about the severity of the complications of prostate cancer as someone who had experienced it. Next, group members freely discussed about their experiences about complications of prostate cancer. Finally, the complications of the lack of health, especially low levels of primary and secondary prevention, were discussed by the participants. In the second session, first lecture method was employed due to low level of information in most of the subjects to make them familiar with methods of prostate cancer screening. The subjects had group discussion on benefits and advantages of prostate cancer screening in prevention of prostate cancer, treatability of prostate cancer in the early stage, and cost efficacy of prostate cancer prevention. In order to help the subjects to brain storm in education, all the inhibiting obstacles in unimportant subjects' complications of diagnosis of prostate cancer and positive predictive value(PPV) and negative predictive value (NPV) of PSA test were indicated and related strategies were mentioned. Then, the clients discussed about the ear of prostate cancer screening. CDs and slides were shown. Finally, the referral center for prostate cancer screening was introduced to the clients. All the participants filled out the questionnaires at baseline and after one month. The men participating in the control group received no planned educational program, but the intervention sessions were offered to this group after the study was completed. The collected data were analyzed using SPSS software, version 18. Statistical qualitative tests, Analysis of Covariance (ANCOVA), Chi-square, independent and paired t-test were used as appropriate. The significance level was set at <0/05. BODY.R: The age range of the participants was 50-70 years and their mean±SD age was 58.1±4.8 and 56.8±5.3 in the intervention and control groups, respectively. Independent t-test showed no significant difference between the two groups with respect to their age P=0.08. 95% of the participants were married. There was a significant difference between the groups in terms of educational level and monthly income; those in the intervention group had a higher educational level and income compared of the participants in the control group (P>0.05). Regarding the randomized allocation of the participants, the results of ANCOVA showed that the significant difference found between the groups in terms of income and educational level had no compounding effect on the study. The rate of the participants who had no family history of prostate cancer and no experience of undergoing DRE and PSA testing for prostate cancer screening was reported 87.2%, 95.6% and 85.6%, respectively. 86.1% of the men had no knowledge about such screening; however, the other respondents knew about it and reported television (48%), magazines and newspapers (20%), a family member with the same disease (12%), radio (8%), physicians (8%), and friends (4%) as their source of knowledge. According to table 1 which compares the knowledge level between the intervention and control groups before and after the intervention, 95.7% of the men in the intervention group were at low and intermediate levels before the intervention, while their levels improved to intermediate and good after the intervention. Nevertheless, we observed no significant changes in the control group in this regard (table 1). Table 1 Comparison of knowledge level between the intervention and control groups before and after the intervention Knowledge Level Number (%) Groups Number (%) Time Before intervention 1 month after intervention Low Intervention 45 (48.4%) 6 (6.5%) Control 26 (29.9%) 23 (26.4%) Intermediate Intervention 44 (47.3%) 55 (59.1%) Control 47 (54%) 49 (56.3%) Good Intervention 2 (2.2%) 32 (34.4%) Control 6 (6.9%) 8 (9.2%) Paired t-test showed a statistically significant difference in the mean score of HBM components in the intervention group after being compared with that before the intervention (P>0.05). In the control group, such difference was reported only for perceived susceptibility (P>0.05), while there was no statistically significant difference in the mean scores of perceived severity, barriers, benefits, and motivation (P>0.05). Independent t-test revealed a statistically significant difference between the intervention and control groups with respect to the mean scores of the perceived susceptibility, severity, barriers and benefits after the intervention (P>0.05) compared with before it (P>0.05). The results of the data analysis showed a statistically significant difference between the intervention and control groups regarding the mean scores of knowledge and motivation before the intervention (P>0.05). To reach a more accurate result (to control the significance effect of the mean scores of knowledge and HBM components in the intervention and control groups before the intervention), the mean difference scores were compared after the intervention. According to the result of independent t-test, a statistically significant difference was observed between the groups with respect to the mean scores of all HBM components after the intervention (P<0.001) (table 2). Table 2 Comparison of the mean score (±SD) of HBM components and knowledge between the intervention and control groups Sub-Scales Intervention (n=93) Mean±SD Control (n=87) Mean±SD P value * Before Intervention Perceived Susceptibility 14.66±4 13.52±3.99 0.059 Perceived Severity 3.21±3.85 12.44±2.89 0.133 Health Motivation 40.84±6.21 38.45±6.47 0.012 Perceived Barriers 45.37±7.06 45.57±7.91 0.859 Perceived Benefits 29.98±3.2 29.48±3.99 0.348 Knowledge 7.37±1.7 8.33±1.43 <0.001 After Intervention P value ** Perceived Susceptibility -2.84±3.2 0.52±2.38 <0.001 Perceived Severity -4.32±2.95 -0.16±1.72 <0.001 Health Motivation -5.37±5.35 0.47±5.35 <0.001 Perceived Barriers 18.08±8.37 -0.49±3.41 <0.001 Perceived Benefits -3.5±3.23 -0.49±3.41 <0.001 Knowledge -2.54±1.34 -0.39±1.39 <0.001 * Independent t-test; ** Paired t-test Paired t-test indicated a significant difference in the mean score of knowledge in the intervention group after the intervention compared with before it (P<0.001), while no significant differences (despite a slight change) were observed in the control group (P=0.808). The participation rate of men in screening before the intervention, one month and three months after the intervention is shown in table 3. Table 3 The men participating in the intervention group in the screening Time Before intervention One months after intervention Three months after intervention Number (%) 7 (8%) 21 (24%) 36 (41.3%) BODY.D: The primary objective of this study was to increase participation in the screening and for this aim education based on the health belief model was implemented; then, we investigated the levels of knowledge, scores of the health belief components about prostate cancer, and the rate of participating retired men in prostate cancer screening. 86.1% of the retired men in this study had no knowledge about prostate cancer screening. Similarly, in a study which was conducted in North Florida, 0.83% African American men had some knowledge about prostate cancer screening and 17% did not have any knowledge about it.13 Another study also indicated that 58% of male New Yorkers were aware of prostate cancer screening in 2000.14 In our study and another study in Iran, the history of prostate cancer screening was 8.6% and 14.4%, respectively.15 This rate in the studies by Kris et al. (2007) was 67%; Allen et al.'s study (2010) reported 44%; and in Sheridan (2012) it was reported 59%.16-18 Comparison of the results of these studies with those of our study indicates a low level of awareness about prostate cancer screening and low participation rate in prostate cancer screening among Iranian men. Our findings were consistent with other studies indicating the significant increase of individuals' knowledge level about prostate cancer after the intervention.17,19-22 A study which was done in Turkey with this tool did not find a significant difference in the level of knowledge in men after an educational intervention by the web.23 Therefore, it can be concluded that for 50 to 70 year old men, face to face training and the group training could be more effective. In another study, it was confirmed that print arm is more effective than web arm and usual care to improve knowledge and reduce decisional conflicts about prostate cancer screening.24 All the aforementioned studies confirmed the importance of education and its effects on promoting the level of the individuals' knowledge. We also observed a significant increase in the mean score of perceived susceptibility in the intervention group following the educational intervention and such result was similar to other studies on prostate cancer screening, diabetes mellitus and breast self-examination.23,25-27 Moreover, most of our participants believed that they might be at risk of prostate cancer. Carmel had a critical review on 46 HBM-related investigations and concluded that "perceived susceptibility" could be the most powerful factor in predicting the behaviors.28 As to the perceived susceptibility, the belief that the disease can occur without any symptoms leads to initiation of screening behaviors.29 In our study, the mean score of such a component increased in the control group. Bakhtariaghdam et al. also reached a similar result and suggested it could be due to the fact that taking the pre-test had made the respondents sensitive to the subject.30 However, Ghaffari et al. believed that it resulted from the curiosity of the participants in the control group to evaluate and complete the questionnaire at the pre-test stage.31 Similarly, we can conclude that such increase lies in the curiosity of the participants to find out more about the disease and increase their knowledge about it during the interval between pre-test and post-test phases which makes them sensitive to the subjects discussed in the questionnaire. Reminding our participants of serious complications and the chronic nature of prostate cancer and considering loss of health and the problems caused by such disease as well as high costs of treatment have been important factors which led to improvement of their level of perceived severity. Several investigations showed that evaluation of clinical outcomes by the individuals could also affect this component.29 Moreover, we found a significant difference between the two groups after the intervention in terms of perceived severity. This finding was in agreement with other studies on the effect of HBM-based education on osteoporosis preventive behaviors and breast self-examination.29,32 Furthermore, independent t-test showed a statistically significant difference between the intervention and control groups with respect to the mean score of perceived benefits after the intervention. Other researchers found similar results in examining the effect of HBM-based educational program on urinary tract infection and Acquired Immune Deficiency Syndrome preventive behaviors.33,34 We believe that medical and health care staff should constantly consult with men about the risk of prostate cancer progression and benefits of screening. Men should also talk with the staff about their fears and obstacles which prevent them from participating in screening programs as it can increase their responsibility for their own health. There are two factors which can facilitate the men's participation in prostate cancer screening: 1-The belief that DRE and PSA tests help diagnose the disease before the appearance of symptoms.2-The belief that early diagnosis and treatment can improve the prognosis of the disease.35 In the incidence of preventive behaviors, perceived barriers are directly associated with early diagnosis and participation in prostate cancer screening,23 while education can remove such barriers and make men take action for early detection of the disease.36 According to both retrospective and prospective studies, "perceived barriers" is found to be the most powerful dimension of HBM in the expression and prediction of health protective behaviors.29 We observed a significant difference between the groups regarding the mean score of "perceived barriers". Likewise, other researchers found a significant decrease in the dimension of "perceived barriers" after HBM-based educational intervention in their studies on prostate cancer screening and nutritional behaviors associated with gastric cancer.23,37 Moreover, we tried to decrease the barriers significantly by increasing the participants' knowledge through education and providing free screening and consulting with a urologist. According to the results, the mean scores of health motivation appeared as significantly different between the groups. Our finding was similar to that of Capık and Gözüm who found an increase in the motivation mean score; however, such increase was not statistically significant.23 Insignificant increase of motivation could be attributed to the participants' low levels of knowledge and lack of sufficient information about prostate cancer and screening. Therefore, the significant increase of motivation in our study could be due to the proper knowledge level in the intervention group after training sessions and the efficiency of our educational intervention compared with internet and web-based education for men aged over 50. Capık and Gözüm reported that the rate of participation in the screening increased after the educational intervention.23 Furthermore, another study indicated that 48% of the participants who had not been screened within the last year were referred for screening again.19 Similarly, Weinrich et al. observed that 71.8% of those in the intervention group participated in free screening due to educational intervention.38 We found out that the participation rate in such screening increased from 7.5% to 24% and 43.3% one month and three months after the intervention, respectively. Finally, we observed that 36 men, who had not been screened within the last year, participated in prostate cancer screening. One limitation of the present study was the post-test one month after the intervention. Therefore, assessing information in several time intervals after the interventions is recommended in order to examine the long-term effects of interventions on prostate cancer screening behaviors and participation in decision-making regarding the subject. It is also recommended that the follow-up periods of screening should be increased to one year. Further investigations are also required to find out the most important potential barriers to prostate cancer screening in Iran. Another limitation of this study was selecting the samples from among a particular group of people such as teachers. It appears that the level of education and knowledge is so much higher than the general population. It is recommended that in future studies samples should be chosen from various groups of people such as rural ones to obtain more generalizable results. BODY.C: Our findings showed that the health education programs designed based on HBM could positively affect the prostate cancer preventive behaviors of our retired participants by improving their knowledge level and HBM components. Hence, we could confirm the efficacy of HBM in adopting the prostate cancer screening behaviors by the participants. Since this type of cancer is treatable in early stages, more attention should be paid to the educational design and planning based on educational theories and models so that we could increase the required knowledge about prostate cancer for early diagnosis and treatment of the disease.

TITLE: Efficacy of serology driven “test and treat strategy” for eradication of ABSTRACT: The treatment of choice of H. pylori infections is a 7-day triple-therapy with a proton pump inhibitor (PPI) plus amoxicillin and either clarithromycin or metronidazole, depending on local antibiotic resistance rates. The data on efficacy of eradication therapy in a group of rheumatology patients on long-term NSAID therapy are reported here. This study was part of a nationwide, multicenter RCT that took place in 2000–2002 in the Netherlands. Patients who tested positive for H. pylori IgG antibodies were included and randomly assigned to either eradication PPI-triple therapy or placebo. After completion, follow-up at 3 months was done by endoscopy and biopsies were sent for culture and histology. In the eradication group 13% (20/152, 95% CI 9–20%) and in the placebo group 79% (123/155, 95% CI 72–85%) of the patients were H. pylori positive by histology or culture. H. pylori was successfully eradicated in 91% of the patients who were fully compliant to therapy, compared to 50% of those who were not (difference of 41%; 95% CI 18–63%). Resistance percentages found in isolates of the placebo group were: 4% to clarithromycin, 19% to metronidazole, 1% to amoxicillin and 2% to tetracycline. BODY.INTRODUCTION: H. pylori eradication is strongly recommended in all patients with atrophic gastritis and peptic ulcer disease, but may also benefit subgroups of patients with dyspepsia, and patients who start with NSAID therapy [1–6]. H. pylori eradication therapy is an important component of guidelines concerning these patients [7, 8]. Currently, non-invasive management strategies and the widespread shortage in endoscopic capacity insure that many patients with H. pylori are managed without upper gastrointestinal endoscopy. The American College of Gastroenterology recommends that when an endoscopy is not performed, a serological test, which is the least expensive means of evaluating for evidence of H. pylori infection, should be done [9]. When endoscopy is indicated, biopsy specimens can be taken for microscopic demonstration of the organism, culture, histology or urease testing. Nowadays, in the Netherlands, biopsies are not routinely sent for culture and susceptibility testing of the infecting strain because of the high costs. Apart from patient compliance, resistance of Helicobacter pylori to antibiotics can decrease the success of H. pylori eradication therapy. Regimens of choice for eradication of H. pylori should be guided by local antibiotic resistance rates. In the Netherlands, the overall prevalence of resistance to clarithromycin and metronidazol was lower than in some surrounding countries possibly due to restrictive use of antimicrobials [10–12]. The advised treatment in the Netherlands consists of a proton pump inhibitor (PPI)-triple therapy for 7 days without prior susceptibility testing. An increase of resistance rates to antimicrobial agents is however expected because increasing number of patients treated and increasing consumption of antibiotics, in particular macrolides, was observed in recent years [13]. The aim of the present study was firstly, to determine the efficacy of 7-day PPI-triple therapy for H. pylori in a well-defined group of patients with a rheumatic disease and serologic evidence of H. pylori infection who were on long-term NSAID therapy and secondly, to get insight in the prevalence of antibiotic resistance of H. pylori in the studied population. BODY.METHODS: This study was part of a placebo-controlled randomized clinical trial of which the clinical results have been described elsewhere [14], wherein we described that H. pylori eradication has no beneficial effect on the incidence of gastroduodenal ulcers or occurrence of dyspepsia in patients on long-term NSAID treatment. Between May 2000 and June 2002, patients were recruited from eight rheumatology outpatient departments in six cities in the Netherlands. Patients with a rheumatic disease were eligible for inclusion if they were between 40 and 80 years of age, were positive for H. pylori on serological testing and were on long-term NSAID treatment. Forty-eight percent used a gastroprotective drug (7% H2 receptor antagonists [H2RA], 37% proton pump inhibitors [PPI], 7% misoprostol, 3% used a combination of these). Exclusion criteria were previous eradication therapy for H. pylori, known allergy for the study medication or presence of severe concomitant disease. Serologic testing for H. pylori IgG-antibodies was performed with a commercial enzyme-linked immunosorbent assay (Pyloriset® new EIA-G, Orion Diagnostica, Espoo, Finland) according to the manufacturer's instructions. A serum sample was considered positive for IgG antibodies to H. pylori if the test result was ≥250 International Units (IU). This assay has been assessed, in a population similar to the population in the presented trial, and has proven a sensitivity and specificity in the Netherlands of 98–100% and 79–85%, even in patients on acid suppressive therapy [15–17]. The study protocol was approved by research and medical ethics committees of all participating centers and all patients gave written informed consent. After stratification by concurrent use of gastroprotective agents (proton pump inhibitors, H2 receptor antagonists or misoprostol, but not prokinetics, or antacids), patients were randomly assigned to receive either H. pylori eradication therapy with omeprazole 20 mg, amoxicillin 1000 mg, and clarithromycin 500 mg (OAC) twice daily for 7 days or placebo. Patients with an allergy for amoxicillin were treated with omeprazole 20 mg, metronidazole 500 mg and clarithromycin 250 mg (OMC) or placebo therapy twice daily for one week in a distinct stratum. All study personnel and participants were blinded to treatment assignment for the duration of the study. The study protocol was approved by research and medical ethics committees of all participating centres and all patients gave written informed consent. At the 2-week follow-up visit, unused study medication was returned and remaining tablets were counted in order to check compliance. Patients were considered to be noncompliant if ≤6 days (85%) of study medication were used. Three months after baseline, and additionally if clinically indicated, patients underwent endoscopy of the upper gastrointestinal tract. A total of eight biopsies were taken during each endoscopy. Four samples, two from the antrum and two from the corpus were used for histology. All biopsies were stained with haematoxylin-eosin. The slides were scored independently by an experienced gastrointestinal pathologist and the investigator (HdL), blinded to treatment assignment and clinical data, according to the updated Sydney classification [18]. In case of discrepant results, the specimen was discussed until agreement was reached. The remaining four biopsies were sent to a microbiological laboratory for culture and storage at –70°C. A patient was considered H. pylori-negative when histology as well as culture was negative. All isolated strains were assessed for susceptibility to clarithromycin, metronidazole, tetracycline and amoxicillin at the central laboratory. Both biopsy specimens of corpus and antrum were streaked on Columbia agar (CA) (Becton Dickinson, Cockeysville, MD, USA) with 10% lysed horse blood (Bio Trading, Mijdrecht, the Netherlands), referred to as Columbia agar plates, and on CA with H. pylori selective supplement (Oxoid, Basingstoke, UK). Plates were incubated for 72 h at 37°C in a micro-aerophilic atmosphere (5% O2, 10% CO2, 85% N2). Identification was carried out by Gram's stain morphology, catalase, oxidase, and urea hydrolysis measurements. Inocula were prepared from an H. pylori culture grown on CA plates. MICs of metronidazole, clarithromycin, tetracycline and amoxicillin were determined by E-test (AB Biodisk, Solna, Sweden) on CA plates essentially as described by Glupczynski et al. [19]. CA plates were inoculated with a bacterial suspension with a turbidity of a 3 McFarland standard (2 × 108 CFU/mL ). CLSI (tentative) breakpoints 2009 for susceptibility (S) and resistance (R) were applied (metronidazole MIC ≤ 8 mg/l (S) and ≥16 mg/l (R), amoxicillin MIC ≤ 0.5 mg/l (S) and ≥ 2 mg/l (R); tetracycline MIC ≤ 2 mg/l (S) and ≥ 8 mg/l (R), and clarithromycin MIC ≤ 0.25 mg/l (S) and ≥ 1 mg/l (R)) [20]. Measurements with a Gaussian distribution were expressed at baseline as mean and SD, and measures with a non-Gaussian distribution were expressed as the median and interquartile range (IQR; expressed as the net result of 75th percentile–25th percentile). An additional analysis compared outcomes (presence of H. pylori after H. pylori eradication therapy or placebo) between stratum (patients on gastroprotective drugs (n = 165) and not on gastroprotective drugs (n = 182) by computing the homogeneity of the common odds ratio. SPSS software (version 17.0.0) was used to perform these analyses. Differences in the proportions of patients with susceptible and resistant H. pylori strains and for compliant and non-compliant patients were analyzed with 95% confidence interval using the Confidence Interval Analysis (CIA) software for Windows (version 2.2.0). The level of significance was set at p < 0.05, two sided. BODY.RESULTS: A total of 347 patients consented to be randomly assigned to eradication therapy (172 patients) or placebo (175 patients). Anti-H. pylori IgG antibodies were present in all patients (median titre 1689 [IQR 700-3732]). The treatment groups were similar in terms of demographic, rheumatic disease, NSAID and other drug use. Our eligibility criteria resulted in a study group with mainly inflammatory rheumatic diseases (rheumatoid arthritis 61%, spondylarthropathy 8%, psoriatic arthritis 7%, osteoarthritis 9%, other 15%). The most commonly used NSAIDs were diclofenac (29%), naproxen (18%), and ibuprofen (13%), most at full therapeutic doses (median relative daily dose 1 [IQR 0.5–1]). The mean age was 60 years (SD 10), 61% was female. Twenty-two patients had a known allergy for amoxicillin and received metronidazole instead (10 patients) or placebo (12 patients). Of these 347 patients, data on culture and histology of 304 patients were available (Table 1). In two cases only culture data were available and in one case only histology result was available; all three cases met the criteria for H. pylori-positivity and were found in the placebo group, but for clarity purposes were left out of Table 1. A total of 32 patients (with no significant differences between eradication and placebo groups) refused the 3-month endoscopy, withdrew informed consent, or could not undergo endoscopy because of adverse events. Seven patients used anticoagulant therapy ruling out biopsy sampling according the protocol, and in one patient no biopsy specimens could be obtained because of discomfort requiring early completion of the procedure. Table 1Results of culture and histology on H. pyloriCultureEradication group (n = 152)Placebo group (n = 152)HistologyTotal patients, n = 304H. pylori positiveH. pylori negativeH. pylori positiveH. pylori negativeH. pylori positive10 (6%)1 (1%)89 (57%)9 (6%)H. pylori negative9 (6%)132 (87%)22 (15%)32 (21%) At follow-up after 3 months, 79% (120 /152; 95% CI 72–85%) of the patients in the placebo group were H. pylori-positive by histology or culture of biopsy specimen. In the eradication group, this number was 13% (20/152; 95% CI 9–20%) (Table 1). Patients in the placebo group who were H. pylori negative at 3 months as assessed by culture and histology had significantly lower titers of H. pylori anti IgG antibodies at baseline than those who were H. pylori culture- and or histology-positive (mean difference −1582, 95% CI −2637 to −527, p = 0.004). There were no differences between strata according to the use of gastroprotective drugs for the presence of H. pylori by culture and or histology (p = 0.454). Compliance was 89% in patients in the eradication group and 98% in the placebo group with the assigned regimen (p < 0.001). In the eradication group, H. pylori could not be demonstrated in 91% of patients with full compliance (n = 136). In patients who did not take all 7 days of eradication therapy (n = 16), H. pylori was found in 50% (difference of 41%; 95% CI 18–63%). No differences were found in the placebo group. BODY.RESULTS.ANTIBIOTIC RESISTANCE RATES: A total of 105 clinical isolates of H. pylori were available for susceptibility testing (one isolate per patient; 95 isolates from the placebo group, ten from the eradication group) from the six participating laboratories in the Netherlands. The rates of resistance are summarized in Table 2. Table 2Antibiotic resistance of H. pylori isolatesAntibioticResistance ratesPlacebo group, n = 95Eradication group, n = 10Clarithromycin4%20%Metronidazole19%30%Tetracyclinea2%0Amoxicillina1%0All patients in the eradication group who were still H. pylori positive were assigned OAC (omeprazole 20 mg, amoxicillin 1000 mg, and clarithromycin 500 mg) and not OMC (omeprazole 20 mg, metronidazole 500 mg and clarithromycin 250 mg)a Intermediate susceptible In the placebo group (n = 95), resistance was found in 4% (4/95) to clarithromycin (MIC ≥1 mg/l), in 1% (1/95) and 2% (2/95) intermediate susceptibility to amoxicillin (MIC 1 mg/l) and tetracycline (MIC 4 mg/l), respectively, and in 19% (18/95) resistance to metronidazole. Amongst these 95 isolates two were resistant to metronidazole in combination with intermediate susceptible to tetracycline, and one strain was resistant to metronidazole and clarithromycin. The placebo group had an MIC90 for clarithromycin of 0.085 mg/l, for metronidazole >256 mg/l, for tetracycline 0.341 mg/l and for amoxicillin 0.16 mg/l. One H. pylori strain was resistant to clarithromycin and metronidazole, and intermediate susceptible to tetracycline and amoxicillin. No difference was found in H. pylori resistance rates between men and women (P = 0.217) or between patients who used gastroprotective agents and who those did not (p = 0.25). In the eradication group, two strains were resistant to clarithromycin and three to metronidazole. No strains were resistant to tetracycline or amoxicillin. BODY.CONCLUSION AND DISCUSSION: We report the results of a study on the efficacy of a test and treat strategy for H. pylori in rheumatology patients of the Netherlands who were positive for anti-H. pylori IgG-antibodies. The main findings in the studied patient population were: (1) a 7-day PPI-triple eradication therapy either with clarithromycin or metronidazole was efficacious with eradication rates of 87% (95% CI 80–91%) without prior testing for susceptibility of the infecting strain; (2) in 21% of the patients in the placebo group, the positive H. pylori serology test could not be confirmed by positive culture or histology; (3) compliance was an important factor for successful eradication of H. pylori; and (4) prevalence of antibiotic resistance in H. pylori was low. The main reason for not performing endoscopy at baseline was that this was not feasible in everyday rheumatology practice; therefore, serology was done to test for H. pylori. The reliability of serological kits for H. pylori infection has been widely confirmed [21], contributing to the reputation of serology as a simple, minimally invasive and inexpensive diagnostic and screening test. The best available serology test at the time of the study was the Pyloriset® an EIA-G from Orion Diagnostica, Espoo, Finland, with a specificity of 79–91% as assessed in previous studies in the Netherlands, including patients on acid suppressive therapy [22–24]. This specificity correlates well with our finding that in the placebo group, H. pylori could not be confirmed by culture or histology in 21% of the IgG positive patients. PPI usage (in this study 37% of the population) can result in false negative invasive and non-invasive diagnostic tests, such as culture, histology and 13-C urea breath, and should be stopped two weeks before testing [25]. This does not apply for serology. 13-C urea breath tests have better accuracy (>90%), but the serology test used in this study was less expensive and in all study centres easily available [26]. On the other hand, we must not overlook that conditions during transport of biopsies are critical for successful isolation of H. pylori. Possibly, the antibacterial effect of NSAIDs, as has been suggested in in vitro studies, might also partly explain a false positive rate of serology of 21% [27–29]. However, in a randomized clinical trial of 122 patients, aspirin in combination with a standard 7-day course OAC eradication was not significantly different compared to the standard therapy [29]. Based on culture and histology findings we conclude that one fifth of our patients were treated superfluously, with possible risk of side-effects of the eradication medication. Fortunately, both regimens were generally well tolerated [14]. Resistance to antibiotics in H. pylori is of particular concern because it is one of the major determinants in the failure of eradication regimens. Resistance rates for metronidazole and clarithromycin found in this study were similar as previously observed in other studies in the Netherlands in the years 1997–98 [10] and 1997–2002 [11, 12, 30, 31]. To our knowledge, there are no recent data available on H. pylori antibiotic primary resistance rates in the Netherlands [32]. In addition, in this study, compliance played a crucial role in success of eradication of H. pylori, i.e., treatment failure was as high as 50% in the non compliant group of patients. Possibly, the high number of tablets that has to be consumed during H. pylori eradication therapy is a contributing factor for non-compliance in this group of elderly patients who were also on other medications. In conclusion, serology driven test and treat strategy eradication of H. pylori with a 7-day PPI-triple therapy is successful in the majority of patients. Success of eradication is, also in this group of rheumatology patients, to a great extent determined by compliance.

TITLE: Video game training and the reward system ABSTRACT: Video games contain elaborate reinforcement and reward schedules that have the potential to maximize motivation. Neuroimaging studies suggest that video games might have an influence on the reward system. However, it is not clear whether reward-related properties represent a precondition, which biases an individual toward playing video games, or if these changes are the result of playing video games. Therefore, we conducted a longitudinal study to explore reward-related functional predictors in relation to video gaming experience as well as functional changes in the brain in response to video game training. Fifty healthy participants were randomly assigned to a video game training (TG) or control group (CG). Before and after training/control period, functional magnetic resonance imaging (fMRI) was conducted using a non-video game related reward task. At pretest, both groups showed strongest activation in ventral striatum (VS) during reward anticipation. At posttest, the TG showed very similar VS activity compared to pretest. In the CG, the VS activity was significantly attenuated. This longitudinal study revealed that video game training may preserve reward responsiveness in the VS in a retest situation over time. We suggest that video games are able to keep striatal responses to reward flexible, a mechanism which might be of critical value for applications such as therapeutic cognitive training. BODY.INTRODUCTION: Over the last decades, the video gaming industry has grown into being one of the biggest multimedia industries in the world. Many people play video games on a day-to-day basis. For example in Germany 8 out of 10 people between 14 and 29 years of age reported to play video games, and 44% above age 29 still play video games. Taken together, based on survey data approximately more than 25 million people above 14 years of age (36%) play video games in Germany (Illek, 2013). It seems as if human beings have a genuinely high motivation to play video games. Most frequently video games are played for the simple purpose of "fun" and a short-term increase in subjective well-being (Przybylski et al., 2010). Indeed, playing video games can satisfy different basic psychological needs, probably also dependent on the specific video game and its genre. Especially fulfillment of psychological needs like competence (sense of self-efficacy and acquisition of new skills), autonomy (personal goal-directed behavior in novel fictive environments), and relatedness (social interactions and comparisons) were associated with video gaming (Przybylski et al., 2010). Specifically, satisfaction of psychological needs might be mainly related to the various feedback mechanisms provided to the player by the game. This elaborate reinforcement and reward schedule has the potential to maximize motivation (Green and Bavelier, 2012). Due to the high use, video games have come into the research focus of disciplines such as psychology and neuroscience. It has been shown that training with video games can lead to improvement in cognitive performance (Green and Bavelier, 2003, 2012; Basak et al., 2008), and in health-related behavior (Baranowski et al., 2008; Primack et al., 2012). Further, it has been shown that video games can be used in the training of surgeons (Boyle et al., 2011), that they are associated with higher psychological quality of life in elderly participants (Allaire et al., 2013; Keogh et al., 2013), and that they can facilitate weight reduction (Staiano et al., 2013). Although it is known that video games are designed to be maximally rewarding by game developers, and video gamers achieve psychological benefits from the gaming, the underlying processes that account for psychological benefits are not fully understood. Green and Bavelier (2012) concluded from their research that beyond the improvements in cognitive performance, the "true effect of action video game playing may be to enhance the ability to learn new tasks." In other words, the effects of video game training might not be limited to the trained game itself; it may foster learning across a variety of tasks or domains. In fact, video game players learned how to learn new tasks quickly and therefore outperform non-video game players at least in the domain of attentional control (Green and Bavelier, 2012). The underlying neurobiological processes associated with video gaming have been investigated with different imaging techniques and experimental designs. A raclopride positron emission tomography (PET) study by Koepp et al. (1998) showed that video gaming (more specifically, a tank simulation) is associated with endogenous dopamine release in the ventral striatum (VS). Furthermore, the level of dopamine binding potential has been related to performance in the game (Koepp et al., 1998). The VS is part of the dopaminergic pathways and is associated with reward processing and motivation (Knutson and Greer, 2008) as well as acquisition of learning in terms of prediction error signal (O'Doherty et al., 2004; Atallah et al., 2006; Erickson et al., 2010). Using magnetic resonance imaging (MRI) to measure gray matter volume, Erickson et al. (2010) showed that ventral and dorsal striatal volume could predict the early performance gains in a cognitively demanding video game (in particular, a two dimensional space shooter simulation). Additionally, Kühn et al. (2011) found that on the one hand frequent compared to infrequent video game playing was associated with higher structural gray matter volume and on the other hand was related to stronger functional activation during loss processing (Kühn et al., 2011). Further, striatal functional magnetic resonance imaging (fMRI) activity during actively playing or passively watching a video game (space shooter simulation, Erickson et al., 2010) or during completing a different non-video game related task (in particular an oddball task) predicted the subsequent training improvement (Vo et al., 2011). Taken together, these studies show that neural processes that are associated with video gaming are likely to be related to alterations of the neural processing in the VS, the core area of reward processing. Moreover, video gaming seems to be associated with structural and reward processing related functional changes in this area. However, it is not clear whether video game related structural and functional properties observed in earlier studies represent a precondition, which biases an individual toward playing video games or if these changes are the result of playing video games. In summary, video games are quite popular and frequently used. One reason for that might be that video gaming may fulfill general human needs (Przybylski et al., 2010). Satisfied needs increase psychological well-being, which in turn is probably experienced as rewarding. Neuroimaging studies support this view by showing that video gaming is associated with alterations in the striatal reward system. Reward processing on the other hand is an essential mechanism for any human stimulus-response learning process. Green and Bavelier (2012) described video game training as a training for learning how to learn (learning of stimulus-response patterns is crucial to complete a video game successfully). We believe that video game training targets the striatal reward system (amongst other areas) and may lead to changes in reward processing. Therefore, in this study, we focus on striatal reward processing before and after video game training. Here, we conducted a longitudinal study to be able to explore reward-related functional predictors in relation to performance and experience in the game as well as functional changes in the brain in response to video game training. We used a successful commercial video game, because commercial games are specifically designed to increase subjective well being (Ryan et al., 2006) and therefore game enjoyment and experienced reward during the game may be maximized. According to the prediction hypothesis, we expect that ventral striatal response in a reward task before video game training predicts performance as already shown in a previous study with a different task (Vo et al., 2011). Furthermore, we want to explore whether ventral striatal reward responsiveness is related to experienced fun, desire, or frustration in the training group during the training episode. To investigate the effect of video game training, we conducted a second MRI scan after video game training had taken place. Based on the findings by Kühn et al. (2011) showing altered reward processing in frequent compared to infrequent video game players, we expected altered striatal reward signal during reward anticipation in participants that had received training compared to controls. If there are functional changes in the striatal reward system, these should be related to the effect of video game training. If not, the observed changes in the study by Kühn et al. (2011) may rather relate to a precondition of the frequent video game players. BODY.MATERIALS AND METHODS.PARTICIPANTS: Fifty healthy young adults were recruited via newspaper and internet advertisements and randomly assigned to video game training group (TG) or control group (CG). Preferably, we recruited only participants that played little or no video games in the last 6 months. None of the participants reported to play video games more than 1 h per week in the last 6 months (on average 0.7 h per month, SD = 1.97) and never played the training game ["Super Mario 64 (DS)"] before. Furthermore, the participants were free of mental disorders (according to personal interview using Mini-International Neuropsychiatric Interview), right-handed, and suitable for the MRI scanning procedure. The study was approved by the local Ethics Committee of the Charité – Universitätsmedizin Berlin and written informed consent was obtained from all participants after participants were fully instructed on the procedures of the study. Data of anatomical gray matter maps of these participants have been previously published (Kühn et al., 2013). BODY.MATERIALS AND METHODS.TRAINING PROCEDURE: The TG (n = 25, mean age = 23.8 years, SD = 3.9 years, 18 females) was instructed to play "Super Mario 64 DS" on the "Nintendo Dual-Screen (DS) XXL" handheld console for at least 30 min per day over a period of 2 months. This extremely successful platformer game was chosen based on its high accessibility for video gaming naïve participants, as it offers a well-suited balance between reward delivery and difficulty and is popular among male and female participants. In the game, the player has to navigate through a complex 3D environment using buttons attached to the console used for movement, jumping, carrying, hitting, flying, stomping, reading, and character specific actions. Prior to the training, participants were instructed on general control and game mechanisms in a standardized way. During the training period, we offered different types of support (telephone, email, etc.) in case frustration or difficulties during game play arose. The no-contact CG (n = 25, mean age = 23.4 years, SD = 3.7 years, 18 females) had no task in particular but underwent the same scanning procedure as the TG. All participants completed an fMRI scan at the beginning of the study (pretest) and 2 months after training or after a passive delay phase (posttest). The video game training for the TG began immediately after the pretest measurement and ended before posttest measurement. BODY.MATERIALS AND METHODS.QUESTIONNAIRES: During training, the participants of the TG were asked to record the amount of daily gaming time. Furthermore participants rated experienced fun, frustration and desire to play during video gaming on a 7-point Likert scale once a week in a word processing document (see, supplementary material for more details) and sent the electronic data files via email to the experimenters. The accomplished game-related reward (stars collected) was objectively assessed by checking the video gaming console after training period. The maximum absolute amount of stars was 150. BODY.MATERIALS AND METHODS.SLOT MACHINE PARADIGM: To investigate reward anticipation, a slightly modified slot machine paradigm was used that evoked strong striatal response (Lorenz et al., 2014). Participants had to go through the same slot machine paradigm before and after video game training procedure had taken place. The slot machine was programmed using Presentation software (Version 14.9, Neurobehavioral Systems Inc., Albany, CA, USA) and consisted of three wheels displaying two different sets of fruits (alternating fruit X and Y). At the two time points of measurement, a slot machine with cherries (X) and lemons (Y) or melons (X) and bananas (Y) were displayed in a counterbalanced fashion and equally distributed for the TG and CG. The color of two horizontal bars (above and below the slot machine) indicated the commands to start and stop the machine. At the beginning of each trial, the wheels did not move and gray bars indicated the inactive state. When these bars turned blue (indicating the start of a trial), the participant was instructed to start the machine by pressing a button with the right hand. After a button press, the bars turned gray again (inactive state) and the three wheels started to rotate vertically with different accelerations (exponential increasing from left to right wheel, respectively). When the maximum rotation velocity of the wheels was reached (1.66 s after button press) the color of the bars turned green. This color change indicated that the participant could stop the machine by pressing the button again. After another button press, the three wheels successively stopped rotating from the left to the right side. The left wheel stopped after a variable delay of 0.48 and 0.61 s after the button press, while the middle and right wheel were still rotating. The second wheel stopped after an additional variable delay of 0.73 and 1.18 s. The right wheel stopped rotating after the middle wheel with a variable delay of 2.63 and 3.24 s. The stop of the third wheel terminated the trial and a feedback about the current win and the total amount of reward was displayed on the screen. For the next trial, the button changed from gray to blue again and the next trial started after a variable delay that ranged between 4.0 and 7.73 s and was characterized by an exponential decreasing function (see Figure 1). FIGURE 1Structure of the slot machine task. FMRI analysis focused on stop of 2nd wheel, when the first two wheels display the same fruit (XX_) or when the first two wheels displayed different fruits (XY_) while the 3nd wheel was still rotating. The experiment contained 60 trials in total. The slot machine was determined with a pseudo-randomized distribution of 20 win trials (XXX or YYY), 20 loss trials (XXY or YYX), and 20 early loss trials (XYX, YXY, XYY, or YXX). Participants started with an amount of 6.00 euro representing the wager of 0.10 euro per trial (60 trials ∗ 0.10 euro wager = 6.00 euro wager) and gained 0.50 euro per trial, when all fruits in a row were of the same identity (XXX or YYY); if not, participants did not win (XXY, YYX, XYX, YXY, XYY, YYX) and the wager was subtracted from the total amount of money. Participants had no influence on winning or losing and the participants won the fixed amount of 10.00 euro (0.50 euro gain ∗ 20 win trials = 10.00 euro gain) at the end of the task. The participants were instructed to play the slot machine 60 times and that the aim in each trial is to get three fruit of the same kind in a row. Further, participants practiced the slot machine task before entering the scanner for 3–5 trials. No information was given that the task was a game of chance or any skill was involved. BODY.MATERIALS AND METHODS.SCANNING PROCEDURE: Magnetic resonance imaging scans were conducted on a three Tesla Siemens TIM Trio Scanner (Siemens Healthcare, Erlangen, Germany), equipped with a 12 channel phased array head coil. Via a video projector, the slot machine paradigm was visually presented via a mirror system mounted on top of the head coil. Functional images were recorded using axial aligned T2∗-weighted gradient echo planar imaging (EPI) with the following parameters: 36 slices, interleaved ascending slice order, time to repeat (TR) = 2 s, time to echo (TE) = 30 ms, field of view (FoV) = 216 × 216, flip angle = 80°, voxel size: 3 mm × 3 mm × 3.6 mm. For anatomical reference, 3D anatomical whole brain images were obtained by a three-dimensional T1-weighted magnetization prepared gradient-echo sequence (MPRAGE; TR = 2500 ms; TE = 4.77 ms; inversion time = 1100 ms, acquisition matrix = 256 × 256 × 176, flip angle = 7°, voxel size: 1 mm × 1 mm × 1 mm). BODY.MATERIALS AND METHODS.DATA ANALYSIS.IMAGE PROCESSING: Magnetic resonance imaging data was analyzed using Statistical Parametric Mapping software package (SPM8, Wellcome Department of Imaging Neuroscience, London, UK). EPIs were corrected for acquisition time delay and head motion and then transformed into the stereotactic normalized standard space of Montreal Neuroimaging Institute using the unified segmentation algorithm as implemented in SPM8. Finally, EPIs were resampled (voxel size = 3 mm × 3 mm × 3 mm) and spatially smoothed with a 3D Gaussian kernel of 7 mm full width at half maximum. BODY.MATERIALS AND METHODS.DATA ANALYSIS.STATISTICAL ANALYSIS: A two-stage mixed-effects general linear model (GLM) was conducted. On single subject level, the model contained the data of both fMRI measurements, which was realized by fitting the data in different sessions. This GLM included separate regressors per session for gain anticipation (XX_ and YY_) and no gain anticipation (XY_ and YX_) as well as the following regressors of no interest: gain (XXX and YYY), loss (XXY and YYX), early loss (XYX, XYY, YXY, and YXX), button presses (after bar changed to blue as well as green), visual flow (rotation of the wheels), and the six rigid body movement parameters. Differential contrast images for gain anticipation against no gain anticipation (XX_ vs. XY_) were calculated for pre- and posttest and taken to group level analysis. On the second level, these differential T-contrast images were entered into a flexible factorial analysis of variance (ANOVA) with the factors group (TG vs. CG) and time (pre- vs. posttest). Whole brain effects were corrected for multiple comparisons using a Monte Carlo simulation based cluster size correction (AlphaSim, Song et al., 2011). One thousand Monte Carlo simulations revealed a corresponding alpha error probability of p < 0.05, when using a minimum cluster size 16 adjacent voxels with a statistical threshold of p < 0.001. According to a meta-analysis by Knutson and Greer (2008), activation differences during reward anticipation were expected in the VS. Based on this a priori hypothesis, we further reported post hoc analysis within this brain area using a region of interest (ROI) analysis. To this end, we used a literature-based ROI for the VS (Schubert et al., 2008). These ROIs were created by combining previous functional findings regarding reward processing (predominantly monetary incentive delay task articles) with anatomical limits to gray matter brain tissue. Detailed information about the calculation of the VS ROI is described in supplementary material. Furthermore, we conducted a control analysis with the extracted mean parameters from the primary auditory cortex, because this region should be independent from the experimental manipulation in the reward task. Therefore we used an anatomical ROI of the Heschl's gyri as described in the Anatomic Labeling (AAL) brain atlas (Tzourio-Mazoyer et al., 2002). BODY.RESULTS.PREDICTION-RELATED RESULTS (PRETEST).BRAIN RESPONSE DURING GAIN ANTICIPATION: At pretest, during the slot machine task in both groups, gain anticipation (against no gain anticipation) evoked activation in a fronto-striatal network including subcortical areas (bilateral VS, thalamus), prefrontal areas (supplementary motor area, precentral gyrus, and middle frontal gyrus, superior frontal gyrus), and insular cortex. Additionally, increased activation in the occipital, parietal and temporal lobes was observed. All brain regions showing significant differences are listed in supplementary Tables S1 (for TG) and S2 (for CG). Note that the strongest activation differences were observed in the VS in both groups (see Table 1; Figure 2). For the contrast TG > CG, a stronger activation in the right supplementary motor area [SMA, cluster size 20 voxel, T(48) = 4.93, MNI-coordinates [x y z] = 9, 23, 49] and for CG > TG a stronger activation in the right pallidum (cluster size 20 voxel, T(48) = 5.66, MNI-coordinates [x y z] = 27, 8, 7) were observed. Both regions are probably not associated to reward-related functions as shown in the meta-analysis by Liu et al. (2011) across 142 reward studies. Table 1 Group by time interaction (TG: Post > Pre) > (CG: Post > Pre) of the effect of gain anticipation against no gain anticipation in the whole brain analysis using Monte Carlo corrected significance threshold of p < 0.05. TG, training group; CG, control group; H, hemisphere; MNI, Montreal Neurological Institute; L, left; R, right. Brain structure H Cluster size (vox) Z (peak) MNI coord. (mm) x y z Supplementary motor area R 36 5.32 9 20 49 Insula lobe/inferior frontal gyrus (p. orbitalis) L 23 4.81 -24 23 -2 Precentral gyrus R 22 4.58 39 5 31 Ventral striatum R 22 4.27 15 11 -8 Insula lobe/inferior frontal gyrus (p. orbitalis) R 20 5.10 30 26 -8 FIGURE 2Predictors of experienced fun. The effect of gain anticipation (XX_) against no gain anticipation (XY_) is shown on a coronal slice (Y = 11) in the upper row for the control group (CG) and training group (TG). The group comparison (CG <> TG) is shown in the bottom left panel. Imaging results are threshold with p < 0.05, Monte Carlo corrected. Correlation between right ventral striatal activity (ROI extracted data) and experienced fun (average over weekly questionnaires) is shown in the bottom right panel. a.u., arbitrary units. BODY.RESULTS.PREDICTION-RELATED RESULTS (PRETEST).ASSOCIATION BETWEEN VENTRAL STRIATAL ACTIVITY AND ASSOCIATED VIDEO GAMING BEHAVIOR: To test the hypothesis of the predictive properties of striatal reward signal toward video games, the ventral striatal signal was individually extracted using the literature-based ROI and correlated with questionnaire items as well as game success, which was assessed by checking the video gaming console. Due to a lack of compliance of participants, weekly questionnaire data of four participants was missing. Weekly questions about experienced fun (M = 4.43, SD = 0.96), frustration (M = 3.8, SD = 1.03) and video gaming desire (M = 1.94, SD = 0.93) were averaged across the 2 months. Participants collected 87 stars (SD = 42.76) on average during the training period. When applying Bonferroni correction to the calculated correlations (equal to a significance threshold of p < 0.006), none of the correlations were significant. Neither video gaming desire [left VS: r(21) = 0.03, p = 0.886; right VS: r(21) = -0.12, p = 0.614] nor frustration [left VS: r(21) = -0.24, p = 0.293; right VS: r(21) = -0.325, p = 0.15] nor accomplished game-related reward [left VS: r(25) = -0.17, p = 0.423; right VS: r(25) = -0.09, p = 0.685] were correlated with reward-related striatal activity. Interestingly, when using uncorrected significance threshold experienced fun during video gaming was correlated positively with the activity during gain anticipation in the right VS [r(21) = 0.45, p = 0.039] and a trend was observed in the left VS [r(21) = 0.37, p = 0.103] as shown in Figure 2 (bottom right panel). However, when applying Bonferroni correction to this exploratory analysis, also the correlations between experienced fun and ventral striatal activity remained non-significant. We further conducted a control analysis to investigate, whether this finding is specific for the VS. We correlated the same behavioral variables with the extracted parameter estimates of the Heschl's gyri (primary auditory cortex). The analysis revealed no significant correlation (all p's > 0.466). BODY.RESULTS.EFFECT OF VIDEO GAME TRAINING (PRE- AND POSTTEST): Analysis of gain anticipation against no gain anticipation during the slot machine task at posttest revealed activation differences in the TG in the same fronto-striatal network as observed at pretest (for details see Table S3). In the CG, this effect was similar, but attenuated (see Figure 3; Table S4). The interaction effect of group by time revealed a significant difference in reward-related areas (right VS and bilateral insula/inferior frontal gyrus, pars orbitalis) and motor-related areas (right SMA and right precentral gyrus) indicating a preserved VS activity in the TG between the time points, but not in the CG. Post hoc ROI analysis using the literature-based VS ROI confirmed the interaction result [Interaction group by time: F(48,1) = 5.7, p = 0.021]. ROI-analysis in the control region (Heschl's gyri) was non-significant. Additional t-tests revealed a significant difference between the time points within the CG group [t(24) = 4.6, p < 0.001] as well as a significant difference between the groups at posttest [t(48) = 2.27, p = 0.028]. Results for the interaction group by time are summarized in Table 1 and are illustrated in Figure 3. FIGURE 3Results of video game training effect. For posttest the effect of gain anticipation (XX_) against no gain anticipation (XY_) is shown using a coronal cut (Y = 11) in the upper row for control group (CG) and training group (TG). Imaging results of the interaction group by time are shown in the middle and bottom left panel (axial cut at Z = -8). ROI analysis for this interaction is in the middle (literature-based ROI in green) and bottom (bar graph of the ROI analysis displayed with standard error of means) right panel. Imaging results are threshold with p < 0.05, Monte Carlo corrected. ROI, region of interest; a.u., arbitrary units. BODY.DISCUSSION: The aim of the present study was twofold: We aimed at investigating how striatal reward responsiveness predicts video game related behavior and experience as well as the impact of video game training on functional aspects of the reward system. Regarding the prediction, we found a positive association between striatal reward signal at pretest and experienced fun during subsequent video game training. Regarding the effect of video gaming, a significant group by time interaction was observed driven by a decrease of the striatal reward signal in the CG. BODY.DISCUSSION.STRIATAL REWARD RESPONSIVENESS AND ITS PREDICTIVE PROPERTIES FOR VIDEO GAMING EXPERIENCE: A relationship between striatal reward signal and game performance or experienced desire and frustration was not observed. However, we were able to demonstrate a positive association of the striatal reward signal with experienced fun during video game training. Thus, we believe that the magnitude of striatal activity during reward processing in a non-video gaming related reward task is predictive for experienced fun during game play. However, this finding has to be interpreted with caution, since the observed correlation did not remain significant after correction for multiple testing. A possible explanation for the correlation between striatal reward signal and experienced fun during video gaming might be that the measured striatal reward signal during slot machine gambling reflects the individuals' reward responsiveness which may be associated with dopaminergic neurotransmission in the striatum. In accordance, previous studies showed that VS activity during reward anticipation is related to dopamine release in this region (Schott et al., 2008; Buckholtz et al., 2010). It has further been shown that also video gaming was associated with dopamine release in the same area (Koepp et al., 1998). Thus, the VS seems to be crucially involved in neural reward processing as well as video gaming, which involves many motivational and rewarding factors. Specifically, we are convinced that the observed relationship between VS activity and experienced fun might be related to a general responsiveness of the reward-related striatal dopamine system to hedonic stimuli. The VS has been associated with motivational and pleasure-elicited reactions in a recent review by Kringelbach and Berridge (2009). Thus, the observed association between ventral striatal activity and fun that refers to hedonic and pleasure-related experience during gaming seems well founded. Future studies should further investigate the relationship between striatal reward responsiveness and experienced fun during video gaming again to explore this relationship more deeply. As mentioned above, striatal dopamine release (Koepp et al., 1998), volume (Erickson et al., 2010), and activity during gaming (Vo et al., 2011) were previously associated with video gaming performance. The results of the current study did not show an association between video gaming performance and VS activity. The achieved reward was operationalized by the number of accomplished missions/challenges in the game. Typical missions within the game are exemplified by defeating a boss, solving puzzles, finding secret places, racing an opponent, or gathering silver coins. These missions represent the progress in the game rather than the actual gaming performance. Thus, these variables may not be a sufficiently precise dependent variable of performance. We were, however, not able to collect more game-related variables, because "Super Mario 64 DS" is a commercial video game and a manipulation of this self-contained video game was impossible. We further investigated the relationship between striatal reward signal and the experienced desire to play during video game training. Desire in this context is probably related to the need and expectations of video gaming's potential satisfaction and reward. Desire is not clearly separable from wanting, because it usually arises together with wanting. Neurobiologically, wanting involves not only striatal, but also prefrontal areas that are related to goal-directed behavior (Cardinal et al., 2002; Berridge et al., 2010). Therefore, a neural correlate of desire might not be limited to the striatal reward area. Indeed, Kühn et al. (2013) showed that structural gray matter volume changes in the dorsolateral prefrontal cortex induced by video game training are positively associated with the subjective feeling of desire during video game training. Thus, in the current study the striatal reward responsiveness might not be related to desire, because desire might rather be associated with prefrontal goal-directed neural correlates. Future studies may investigate this in detail. We expected a negative correlation between striatal reward responsiveness and experienced frustration during video game training since the VS activity is decreased at the omission of reward relative to the receipt of reward (Abler et al., 2005). However, this relationship was not observed. Previous studies showed that the insula is selectively activated in the context of frustration (Abler et al., 2005; Yu et al., 2014). Thus, future studies might also investigate insular activity in the context of omitted reward. BODY.DISCUSSION.EFFECT OF VIDEO GAME TRAINING ON THE REWARD SYSTEM: Kühn et al. (2011) showed in a cross-sectional study that frequent video game players (>9 h per week) demonstrated greater striatal reward-related activity compared to infrequent video game players. However, the question remained, whether this finding was a predisposition toward or a result of video gaming. In our present longitudinal study, gain anticipation during slot machine task revealed VS activity, which was preserved in TG over the 2 months, but not in CG. We assume that the striatal reward signal might reflect the motivational engagement during the slot machine task, which was still high in the TG at the posttest. The participants of the TG might preserve the responsiveness in reward processing and motivational willingness to complete the slot machine task at the second time point in a similarly engaged state as during the first time. An explanation for that finding might be that the video game training has an influence on dopamine-related reward processing during gaming (Koepp et al., 1998). Our results support this view, as this effect might temporally not be limited to the gaming session, but rather might have an influence on general striatal reward responsiveness in rewarding situations not related to video games. Kringelbach and Berridge (2009) showed that activity in the VS might represent an amplifier function of reward, and thus, video games might preserve reward responsiveness during game play itself, and even in the context of other rewarding tasks through amplification of pleasure-related activity. Thus, the video game training might be considered as an intervention targeting the dopaminergic neurotransmitter system, which might be investigated in the future. There is evidence, that dopaminergic interventions in the context of pharmacological studies can have a therapeutic behavior changing character. A recent pharmacological study using a dopaminergic intervention on older healthy adults by Chowdhury et al. (2013) showed that age-related impaired striatal reward processing signal could be restored by dopamine targeted drugs. Future studies should investigate the potential therapeutic effects of video gaming training on cognitive demanding tasks involving dopaminergic striatal signal. It would be highly valuable to uncover the specific effect of video gaming in the fronto-striatal circuitry. Our findings suggested an effect on reward processing, which in turn is essential for shaping of goal-directed behavior and flexible adaption to volatile environments (Cools, 2008). Therefore, tasks involving reward-related decisions such as reversal learning should be investigated in future longitudinal studies in combination with video game training. Multiple pharmacological studies have shown that a dopaminergic manipulation may lead to an increase or decrease in reversal learning performance, which probably depends on task demand and individual baseline dopamine levels (Klanker et al., 2013). The observed effect of video game training on the reward system was also driven by a decrease in striatal activity in the CG during posttest, which may in part be explained by a motivational decline in the willingness to complete the slot machine task at the re-test. A study by Shao et al. (2013) demonstrated that even a single training session with a slot machine task before the actual scanning session led to decreases in striatal reward activity during win processing compared to a group that did not undergo a training session. A further study by Fliessbach et al. (2010) investigated the re-test reliability of three reward tasks and showed that the re-test reliability in VS during gain anticipation were rather poor, in contrast to motor-related reliabilities in primary motor cortex that were characterized as good. A possible explanation of these findings might be the nature of such reward tasks. The identical reward at both time points may not lead to the same reward signal at the second time of task performance, because the subjective reward feeling may be attenuated by a lack of novelty. Obviously, in the present study the re-test was completed by both groups, but the decrease of the striatal reward activity was only observed in the CG, not in the TG. This preservation result in the TG may in part be related to the video game training as discussed above. Nevertheless, the CG was a no-contact group and did not complete an active control condition and thus, the findings might also represent a purely placebo like effect in the TG. However, even if not the specific video game training itself was the main reason for the preserved striatal response, our study may be interpreted as evidence arguing that video games lead to a rather strong placebo-like effect in a therapeutic or training-based setting. If video games would represent a stronger placebo effect than placebo medication or other placebo-like tasks is an open question. Moreover, during the scanning session itself participants were in the same situation in the scanner and one can expect that both groups produce the same social desirability effects. Still, the preservation effect should be interpreted very carefully, because placebo effect might confound the result (Boot et al., 2011). Future studies focusing on the reward system should include an active control condition in the study design. Another possible limitation of the study might be that we did not control the video gaming behavior of the CG. We instructed the participants of the CG not to change their video gaming behavior in the waiting period and not to play Super Mario 64 (DS). However, video gaming behavior in the CG might have changed and could have affected the results. Future studies should include active control groups and assess video gaming behavior during the study period in detail. In this study we focused on the VS. Nonetheless, we observed a significant training-related effect also in the insular cortices, SMA, and precentral gyrus. A recent meta-analysis by Liu et al. (2011) including 142 reward studies showed that besides the "core area of reward" VS also insula, ventromedial prefrontal cortex, anterior cingulate cortex, dorsolateral prefrontal cortex, and inferior parietal lobule are part of the reward network during reward anticipation. The insula is involved in the subjective integration of affective information, for instance during error-based learning in the context of emotional arousal and awareness (Craig, 2009; Singer et al., 2009). The activation during reward anticipation in the slot machine task may reflect subjective arousal and motivational involvement in the task. We believe that this significant training effect in the insula might – similar to the effect in VS – represent a motivational engagement, which was preserved in the TG at the posttest. Future studies could test this e.g., by applying arousal rating scales and correlate these values with insular activity. According to the differences in SMA and precentral gyrus, we want to highlight that these areas might not be involved in reward anticipation as it is not part of the suggested network of the mentioned meta-analysis (Liu et al., 2011). Instead, the SMA is involved in learning of motor-related stimulus-response associations among other functions (Nachev et al., 2008). With regard to the current study, SMA activity may reflect an updating process of the stimulus (slot machine with three rotating wheels) – response (button press to stop the slot machine) – consequence (here update of stop of the second wheel: XX_ and XY_) – chain. Speculatively, participants of the training group understand the slot machine after training as a video game, in which they could improve their performance by e.g., pressing the button at the right time point. In other words, the participants of the TG might have thought that they could impact the outcome of the slot machine by adapting their response pattern. Please note that the participants were not aware that the slot machine had a deterministic nature. As the precentral gyrus is also part of the motor system, the interpretation of the functional meaning of the SMA finding may be also valid for the precentral gyrus. Future studies might confirm these interpretations of SMA and precentral activation differences by systematically varying response-consequence-associations. BODY.DISCUSSION.VIDEO GAMING, SUPER MARIO, MOTIVATION, SUBJECTIVE WELL-BEING, AND THE REWARD SYSTEM: From a psychological view, joyful video games provide highly effective reward schedules, perfectly adjusted difficulty levels and strong engagement (Green and Bavelier, 2012). These specific properties potentially contain the opportunity to satisfy basic psychological needs such as competence, autonomy and relatedness (Przybylski et al., 2010). A study by Ryan et al. (2006) showed that participants feeling volitionally motivated by a 20 min training session of Super Mario 64 had an increased well-being after playing. This increased well-being was further associated with increases in the feeling of competence (e.g., experienced self-efficacy) and autonomy (e.g., acting based on interest). Together with the current finding of the preservation of the reward signal in a non-trained task, we believe that video games harbor the potential of a powerful tool for specific (cognitive) training. Depending on the video gaming genre and individual properties of the game, video games demand very complex cognitive and motor interactions from players to be able to reach the goal of the game and thus a specific training effect. The rewarding nature of video games may lead to a constant high motivational level within the training session. BODY.CONCLUSION: The current study showed that striatal reward responsiveness predicts the subsequent experienced video gaming fun suggesting that individual differences in reward responsiveness might affect motivational engagement of video gaming, but this interpretation needs confirmation in future studies. Furthermore, this longitudinal study revealed that video game training may preserve reward responsiveness in the VS in a re-test. We believe that video games are able to keep striatal responses to reward flexible, a mechanism which might be extremely important to keep motivation high, and thus might be of critical value for many different applications, including cognitive training and therapeutic possibilities. Future research should therefore investigate whether video game training might have an effect on reward-based decision-making, which is an important ability in everyday life. BODY.CONFLICT OF INTEREST STATEMENT: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

TITLE: Does improving maternal knowledge of vaccines impact infant immunization rates? A community-based randomized-controlled trial in Karachi, Pakistan ABSTRACT.BACKGROUND: In Pakistan, only 59-73% of children 12-23 months of age are fully immunized. This randomized, controlled trial was conducted to assess the impact of a low-literacy immunization promotion educational intervention for mothers living in low-income communities of Karachi on infant immunization completion rates. ABSTRACT.METHODS: Three hundred and sixty-six mother-infant pairs, with infants aged ≤ 6 weeks, were enrolled and randomized into either the intervention or control arm between August - November 2008. The intervention, administered by trained community health workers, consisted of three targeted pictorial messages regarding vaccines. The control group received general health promotion messages based on Pakistan's Lady Health Worker program curriculum. Assessment of DPT/Hepatitis B vaccine completion (3 doses) was conducted 4-months after enrollment. A Poisson regression model was used to estimate effect of the intervention. The multivariable Poisson regression model included maternal education, paternal occupation, ownership of home, cooking fuel used at home, place of residence, the child's immunization status at enrollment, and mother's perception about the impact of immunization on child's health. ABSTRACT.RESULTS: Baseline characteristics among the two groups were similar. At 4 month assessment, among 179 mother-infant pairs in the intervention group, 129 (72.1%) had received all 3 doses of DPT/Hepatitis B vaccine, whereas in the control group 92/178 (51.7%) had received all 3 doses. Multivariable analysis revealed a significant improvement of 39% (adjusted RR = 1.39; 95% CI: 1.06-1.81) in DPT-3/Hepatitis B completion rates in the intervention group. ABSTRACT.CONCLUSION: A simple educational intervention designed for low-literate populations, improved DPT-3/Hepatitis B vaccine completion rates by 39%. These findings have important implications for improving routine immunization rates in Pakistan. BODY.BACKGROUND: Vaccinating infants against childhood communicable diseases is one of the most cost-effective public health interventions worldwide [1]. Pakistan's Expanded Program on Immunization (EPI) schedule involves administrating BCG/OPV at birth, three doses of DPT/OPV/Hepatitis B vaccines at 6, 10 and 14 weeks of age, and measles vaccine at 9 and 15 months of age. In Pakistan, children receive vaccinations through EPI at fixed primary health centers. Additional supplementary activities are undertaken through outreach efforts, such as National Immunization Days. Recently, Pakistan has added the Haemophilus influenzae type b vaccine to be administered with the three doses of DPT/Hepatitis B vaccines. Despite recent efforts, immunization coverage rates in Pakistan remain low, with 59-73% of children aged 12 - 23 months receiving all three doses of DPT/Hepatitis B vaccine [2,3]. Therefore, low-cost innovative interventions, which can be implemented within Pakistan's existing health care infrastructure, are needed. Many studies have looked at factors that affect immunization completion rates. Low parental, specifically maternal literacy and knowledge regarding vaccines and immunization schedule, poor socioeconomic status, and residence in rural areas are associated with low immunization coverage [4-12]. Health provider factors that have been associated with increased immunization drop-out rates include parental difficulty of access to healthcare services and inadequate supervision of healthcare staff at health facilities [4-6,8]. Retention of proof of immunization by the infants' families has been associated with improved immunization coverage and facilitates documentation of vaccination status [6,10,11]. Increasing health awareness, knowledge about diseases, and their prevention or management has successfully improved many different health outcomes in high-income countries, especially among less literate populations [13-16]. Educational interventions promoting vaccine use have also proven cost-effective in improving immunization coverage rates in these settings [17,18]. However, there is limited data available from low-income countries [12]. Usman et al [12] recently reported findings from a randomized controlled trial in urban Pakistan evaluating the effect of center-based education to mothers of infants presenting at primary healthcare centers for first dose of DPT vaccine. The 2-3 minutes education session, conducted by trained study staff, emphasized the importance of completing the immunization schedule, and improved immunization completion rates by 31% among the cohort of infants whose mothers presented to an immunization center for their first vaccine [12]. To our knowledge, no study has looked at the impact of home-based vaccine promotion education among a population of mothers of newborns at high risk for not seeking immunization services for their children. Our study aims to close this gap. The main objective of this study was to assess the effect of short, home-based information sessions on importance of vaccines on DPT-3/Hepatitis B immunization rates in low-income urban and peri-urban communities in Karachi, Pakistan known for very low demand and care-seeking for vaccination services. BODY.METHODS.STUDY SETTING: This was a multi-site community-based, randomized controlled educational intervention trial conducted at five low-income sites in Karachi. Among these, one community was urban, whereas the other four were peri-urban, located about 45 minutes travel outside of Karachi. The population in the study areas has low literacy, with only 24% of the population being literate. The total combined population of all five study sites is approximately 260,000, with high infant and maternal mortality rates. The major income generating activities include fishing and livestock rearing, or employment in local small industries (garment and leather). The Department of Pediatrics and Child Health, Aga Khan University has well-established household-based surveillance for pregnancy and neonatal outcomes in these areas. A demographic surveillance round of the entire area is completed every three months. All pregnant women identified during this surveillance are visited frequently around the time of birth so that new births can be captured. BODY.METHODS.ELIGIBILITY AND ENROLLMENT OF PARTICIPANTS: All mothers living in the study areas, and having a live child ≤ 6 weeks old, were eligible to be enrolled in the study. Twin births, infants > 6 weeks of age, or infants born to mothers living outside the study surveillance areas were excluded. The cutoff of 6 weeks was used to ensure that the intervention was implemented before the first dose of DPT/Hepatitis B came due. Mothers of possibly eligible infants were identified through computerized surveillance databases of pregnant women and newborns maintained at each site. Families were approached with the help of local women, trained as community health workers (CHWs). The CHWs also obtained verbal consent from the mother of each eligible infant. The study protocol was approved by the Ethical Review Committee of the Aga Khan University. Informed consent was obtained from each participant at enrollment, and no breaches of confidentiality occurred. Each mother-infant pair, who consented to participate in the study, was assigned a unique study identification number. Information on household demographics and socio-economic status was collected using a pre-tested structured questionnaire. Data collected on the infant included age, sex, place of birth, and health status. The baseline interview also recorded mother's knowledge and beliefs about vaccines. A follow-up questionnaire was used to assess the outcome. Subjects were enrolled from August 2008 to November 2008. Study participants were followed up for assessment of outcome from December 2008 to March 2009, with each individual mother-infant pair approached four months after the educational intervention session. BODY.METHODS.RANDOMIZATION: Randomization lists, stratified for each of the five enrollment sites were generated by a computer and provided to the CHWs Upon consent, mother-infant pairs were assigned either to intervention or control arms through block randomization (n = 4), according to the computer-generated list. As the intervention was educational, blinding of study staff and participants was not possible. Outcome assessment was done by an investigator (BH) at each participant's house, four months after initial enrollment. The investigator was blinded to the exposure status of participants. BODY.METHODS.INTERVENTION: To address the needs of low literacy populations, easy-to-understand pictorial cards, using very simple language, to convey three key messages as part of the educational intervention were designed. The first key message highlighted how vaccines save children's lives. The second message provided logistic information about the address and location of the local vaccination centers. The third key message emphasized the significance of retaining immunization cards, and the role they could play at the time of the child's school admissions. A copy of these pictorial messages was left with the mother. These messages took about 5 minutes to impart, and were given by the trained CHWs to each participant at their household. The control group verbally received the general health promotion messages adapted from the curricula developed by the Pakistan Ministry of Health for the Lady Health Worker Program. These messages included information on hand-washing, breast-feeding, clean water, benefits of using oral rehydration solutions during diarrhea, bringing the infant to nearby health center when there are symptoms of acute respiratory illnesses, importance of antenatal check-ups for mothers, and some general information on vaccines. These messages were also given by trained CHWs. The length of each educational session in the control group was approximately 10-15 minutes. BODY.METHODS.STUDY OUTCOME: The study outcome in each study group was the immunization status of DPT-3/Hepatitis B at 4 months after enrollment (4 to 5 months of infant's age). Immunization rates of DPT-3/Hepatitis B vaccines for intervention and control groups were assessed by an investigator, and were divided into two categories: 1) Infants receiving all three doses of DPT/Hepatitis B vaccines (assessed through vaccination cards) were considered "DPT-3/Hepatitis B fully immunized". 2) Infants missing any dose of DPT/Hepatitis B or who had lost their vaccination cards were termed"DPT-3/Hepatitis B non-immunized". A participant was considered to be "DPT-3/Hepatitis B fully immunized" only if the mother/caretaker of the child was able to produce an EPI-issued or another health facility-issued vaccination card. Verbal responses of mothers regarding vaccine receipt without documentation on a vaccination card were not considered satisfactory evidence of their infant being fully immunized. BODY.METHODS.SAMPLE SIZE: We assumed a DPT-3/Hepatitis B immunization rate of 55% in the control group, and hypothesized a difference of 15% in the immunization rates between the intervention and control group. With 80% power and α = 0.05, we estimated a sample size of 163 in each arm. Adjusting for possible lost to follow-up, we enrolled 183 mother-infants pairs in each study group. BODY.METHODS.STATISTICAL ANALYSIS: Statistical analysis was performed using SAS Version 9.2 (SAS Institute, Inc., Cary, NC). Baseline characteristics of study participants were compared using proportions. Unadjusted risk ratio (RR) and 95% confidence interval (CI) were estimated for the study outcome (DPT-3/Hepatitis B fully immunized) using bivariate Poisson regression [19,20]. A multivariable Poisson regression model was built to assess the association between the study outcome, the study group and all other variables which were considered to be significantly associated with the study outcome at the bivariate level (p ≤ 0.20). All variables, having bivariate association with the study outcome (p ≤ 0.20) were also tested for interaction. The final model was interpreted using adjusted RR and corresponding 95% CI. The number needed to treat (NNT) in order to increase the completion of DPT-3/Hepatitis B immunization by one child was also estimated [21,22]. BODY.RESULTS: A total of 1157 mother-infant pairs were identified from surveillance databases at the five community sites were approached, and assessed for eligibility. Among these, 479 (41.4%) did not meet inclusion criteria, whereas 312 (27%) declined participation in the study (Figure 1), resulting in 366 (183 in each study arm) children being available for randomization. Four infants were lost to follow-up from the intervention group, and five were lost to follow-up from the control group during the study period and were excluded from the analysis. Therefore, 179 enrolled infants were included in the analysis from the intervention group and 178 from the control group (Figure 1). The distribution of enrolled mother-infant pairs among the five study sites was weighted to represent population size in each area and was as follows: Community A = 103; Community B = 96; Community C = 71; Community D = 47; and Community E = 40. Figure 1Flow diagram of study participant screening, allocation and follow-up. The distribution of baseline characteristics of the participants in the intervention and control arms is summarized in Table 1. No significant differences were observed between the two groups, although the proportion of mothers who had received no formal education was higher in the control group compared to those in the intervention group (75% vs. 66%). History of receipt of BCG vaccine and OPV (first dose) at birth was similar in both groups (76.4% in the control group vs. 77.1% in the intervention group). After the 4-month follow-up period, 129 (72.1%) infants in the intervention group had completed primary immunization with three doses of DPT and Hepatitis B vaccines compared to 92 (51.7%) in the control group. Therefore, DPT-3/Hepatitis B immunization rates in the intervention group were improved by 39% (unadjusted RR = 1.39; 95% CI: 1.07 - 1.82) (Table 2). Vaccination cards were retained by 81% of the study participants in the intervention group, and by 69.1% of the participants in the control group. Table 1 Distribution of baseline characteristics of study participants Control Group Intervention Group ( n = 183) ( n = 183) p-value Age at enrollment (mean days) 22.4 25.7 0.01 Male child (%) 50.3 44.8 0.30 Child was immunized at enrollment 76.4 77.1 0.88 Age of mother (mean years) 25.1 25.9 0.17 Education of mother 0.07  No formal education (%) 74.9 66.1  Primary (%) 12.0 20.8  Middle and above (%) 13.1 13.1 Occupation of mother 0.56  Housewife (%) 98.9 99.5  Other professions (%) 1.1 0.5 Education of father 0.09  No formal education (%) 54.6 53.6  Primary (%) 20.8 13.7  Middle and above (%) 24.6 32.8 Occupation of father 0.54  Fishermen (%) 33.9 27.9  Laborer (%) 20.2 22.4  Businessman (%) 13.7 10.9  Private job (%) 24.0 30.1  Other profession (%) 8.2 8.7 Ownership of house 0.80  Own (%) 79.2 80.3  Rented (%) 20.8 19.7 Construction material of house 0.79  Wooden structure or other (%) 6.6 5.5  Tin (%) 21.3 25.1  Concrete cement (%) 62.8 61.7  Cement (%) 9.3 7.7 Cooking fuel used in house 0.10  Wood or other (%) 20.8 14.2  Natural gas (%) 79.2 85.8 Place of birth 0.52  Home or other (%) 59.0 62.3  Hospital (%) 41.0 37.7 Mother knows about the impact of immunization on child's health 0.40  Don't know (%) 18.6 15.3  It prevents serious illness (%) 81.4 84.7 Mother knows where the nearest local immunization center is 0.34  No (%) 8.7 7.7  Yes (%) 91.3 92.3 Table 2 Effect of home-based vaccine education to mothers on study outcome Total N = 357 Received 3 doses of DPT/Hepatitis B vaccine Unadjusted RR (95% CI) Adjusted RR a (95% CI) n % Home-based vaccine education 179 129 72.1 1.39 (1.07-1.82) 1.39 (1.06-1.81) General health promotion messages 178 92 51.7 1.00 1.00 a Multivariable model adjusted for child's immunization status at enrollment. In the multivariable model, the child's immunization status at enrollment was the only variable significantly associated with the study outcome (p < 0.05). Adjusting for this variable did not change the effect estimate (adjusted RR = 1.39; 95% CI: 1.06 - 1.81) (Table 2). Place of residence of the study participants (urban vs. semi-urban) was not associated with the study outcome (p = 0.08). The number needed to treat (NNT) in order to increase the completion of DPT-3/Hepatitis B immunization by 1 child was also calculated. An estimated 5 mothers need to be educated in order to have one more child complete his/her DPT-3/Hepatitis B vaccinations. We also assessed if our results were subject to misclassification bias due to the requirement of documentation of vaccine receipt through vaccination cards for the infant to be classified as fully immunized at outcome assessment, and those without vaccination card as not fully immunized. There were 10 cases (3 in the intervention group and 7 in the control group) that could have been misclassified as "DPT-3/Hepatitis B non-immunized" due to lack of a vaccination card, but whose mothers recalled receipt of all three doses. Including these 10 children in the "DPT-3/Hepatitis B fully immunized" group, 132 (73.7%) infants in the intervention group had completed primary immunization with three doses of DPT and Hepatitis B vaccine, compared to 99 (55.6%) in the control group. Therefore, DPT-3/Hepatitis B immunization rates in the intervention group were improved by 33% (unadjusted RR = 1.33; 95% CI: 1.02 - 1.72). In the multivariable model, the child's immunization status at enrollment was the only variable significantly associated with the study outcome (p < 0.05). After adjusting for it, DPT-3/Hepatitis B immunization rates in the intervention group were improved by 32% (adjusted RR = 1.32; 95% CI: 1.02 - 1.71). BODY.DISCUSSION: This study demonstrates that providing vaccine-related targeted education to mothers at home is an effective and practical strategy to improve childhood immunization rates in low literacy settings such as ours. In this randomized controlled trial, a significant improvement in infant DPT-3/Hepatitis B vaccine immunization rates was observed in the group of mothers who received home-based education on the importance of vaccines, compared to those who received standard health promotion messages only. In low-income countries, efforts to improve infant vaccination completion rates have focused primarily on supply and/or provider factors, with little focus on creating demand for infant immunization services. The major thrust of the supply-side interventions to improve vaccination rates has been through mass immunization campaigns. These campaigns have been successful in improving vaccine coverage rates [23]. However, there are certain drawbacks of mass campaigns such as those done for polio vaccine in Pakistan and other developing countries [24-26]. They lead to a misconception on the parents' part that the child will be delivered all vaccines at home [25], and can result in a decline in the number of visits to the immunization centers, paradoxically driving down routine immunization coverage rates[26]. Furthermore, these mass immunization campaigns have resulted in declining performance of routine EPI activities in Pakistan[27]. Educational interventions have been successful in raising awareness regarding vaccine and increasing demand. Jacobson et al [17] were successful in increasing pneumococcal vaccine coverage rates among the elderly, by using low-literacy pamphlets encouraging study participants to "ask your doctor about the pneumonia shot". Kimura et al [18] were able to increase influenza vaccine coverage among workers in long-term care facilities with the help of an educational campaign and provision of free vaccines. In Pakistan, Usman et al [12] reported an increase of 31% in DPT3 completion among infants of mothers who received primary healthcare center-based education on their first immunization visit. The success of educational interventions in modifying health-seeking behavior may also be attributed to the focused nature of the interventions. This is certainly true in our study. The intervention group received a 5-minute educational session, focusing on the importance of immunization for a child's health. The control group, on the other hand, received a 10-15 minute verbal session on general child health promotion. Therefore, the group receiving the focused message may have been more likely to understand and retain its content, and modify their behavior, compared to the control group who may have had "information overload". We observed that maternal knowledge/perception regarding importance of vaccines was significantly associated with higher DPT-3/Hepatitis B immunization rates (RR = 2.11; 95% CI: 1.33 - 3.34). Our results are consistent with findings of other studies [4,9-11]. Surprisingly, infants living in rented houses (considered a crude proxy for lower socioeconomic status) were more likely to have received all 3 doses of DPT-3/Hepatitis B vaccines as compared to infants living in houses owned by their families (RR = 1.26; 95% CI: 0.93 - 1.71). However, this association is not significant. Another explanation could be that many of the households in these populations are considered illegal squatters but claim ownership of the land on which they've built their house. Therefore, paradoxically, households in rental accommodation could actually be socio-economically better-off. It is worth noting that even with the educational intervention specific to vaccines, only 72% of infants were fully immunized in the intervention group. This is because our study included very low-income, low-literacy populations of Sindh province where baseline immunization rates are 48% [2], much lower than the national reported figure of 73% [3]. The national average is a composite figure, including more prosperous and literate parts of the Pakistani population and the large province of Punjab which has a more functional EPI system and estimated vaccine coverage of 65% [2]. National surveys may overestimate or over-report vaccine coverage rates [28]. Different immunization centers use different vaccination cards, which differ in the design and method of recording proof of vaccination. This makes it difficult for data collectors to accurately determine an infant's immunization status. Furthermore, verbal reports, in lieu of vaccination cards, are often accepted as proof of immunization, but our experience using serological confirmation shows poor correlation between verbal recall and serological immunity [29]. Therefore, the true vaccine coverage rate for Pakistani children may be closer to the figure of 59% estimated by the recent Demographic and Health Survey of Pakistan [2]. Although ascertaining the reasons for low vaccine coverage was beyond the scope of this study, many barriers to improving immunization coverage remain in low-income communities and need to be systematically addressed. Our study also has a few limitations. First, the third key message provided education on retaining vaccination cards. Therefore, mothers in the intervention group were 17% more likely to save the card and provide proof of vaccination for outcome assessment. However, as shown above, using the stringent proof-of-vaccination via card to determine outcome did not bias our results significantly if infants with maternal recall of vaccine receipt were also included as fully immunized. Including these infants as fully immunized, DPT-3/Hepatitis B immunization rates in the intervention group were still 18 percentage points higher than the control group. A second limitation is the lack of blinding of CHWs and participants as the intervention was educational in nature. However, the investigator (BH) assessing the outcome four months after the intervention was administered was blinded to the exposure status of the study participants. Furthermore, chances of spillover effect, or contamination between the intervention and control arms were minimized by choosing mother-infant pairs from five different communities in Karachi, lowering the probability that households participating in our study with an eligible newborn would be located close to each other. We also observed a trend for mothers in the intervention group to be more educated compared to those in the control group (34% vs. 25%). However, this difference was not statistically significant and other measures of parental knowledge about vaccines did not favor the control group. Our study also had a high refusal rate (27%) which may have excluded participants less likely to accept vaccines from the trial. However, the most common stated reason for refusal was absence of the child's father when study staff visited the household for initial recruitment. Our educational intervention has the potential to be cost-effective. The cost of the intervention per CHW was estimated to be Pakistan Rs. 80 ($1). This includes the cost of laminated colored pictorial cards used by the CHWs to educate the mothers in the intervention group, as well as pamphlets of the pictorial messages left at each participant's house. We also estimated the cost of scaling-up this intervention nationally, through the Lady Health Worker Program. Given that there are 100,000 lady health workers working all over Pakistan, we estimate that the cost of the national scale-up will be approximately $200,000 for the national program ($100,000 for the cards and pamphlets, and $100,000 for training sessions). BODY.CONCLUSION: This study offers firm evidence that providing home-based focused education to mothers regarding the importance of vaccines, through pictorial messages using very simple language, is effective in improving infant immunization rates in low-income and low-literacy populations. Our trial intervention offers novel options to the current vaccine coverage enhancement efforts in low-income countries like Pakistan. Given the low "number needed to treat" to increase immunization completion by 20%, this intervention may prove to be quite cost-effective. The National Lady Health Worker Program is an ideal platform for building a more focused immunization promotion campaign, similar to what we designed, and drive up the demand for infant immunization services in Pakistan. However, the operational and logistical challenges involved in large-scale implementation of such an intervention need further evaluation. BODY.ABBREVIATIONS: EPI: Expanded Program on Immunization; BCG: Bacillus Calmette-Guérin; OPV: Oral Polio Vaccine; DPT: Diphtheria Pertussis Tetanus; CHW: Community health worker. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: AO provided the design and execution of the data analysis, as well as wrote the manuscript. BH contributed to manuscript writing and supervised field operations. ARS supervised manuscript preparation. AA supervised data analysis. AKMZ contributed to the design of the study, data analysis, and manuscript writing. All authors have read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/11/239/prepub

TITLE: Hepatitis B surface antigen levels: association with 5-year response to peginterferon alfa-2a in hepatitis B e-antigen-negative patients ABSTRACT.PURPOSE: To investigate the durability of response to peginterferon alfa-2a up to 5 years post-treatment and factors associated with response in hepatitis B e-antigen (HBeAg)-negative patients. ABSTRACT.METHODS: HBeAg-negative patients received peginterferon alfa-2a (180 μg/week) ± lamivudine (100 mg/day) for 48 weeks as part of a multicenter, randomized study. The planned 5-year efficacy analysis included patients (n = 230) enrolled in the long-term follow-up study. On-treatment hepatitis B surface antigen (HBsAg) decline kinetics were analyzed retrospectively in a subgroup of patients with HBsAg data available at baseline, weeks 12, 24, and 48 on-treatment, and 6 months post-treatment (n = 120). Receiver operating characteristic analyses identified the on-treatment HBsAg levels associated with response at 1 and 5 years post-treatment. ABSTRACT.RESULTS: HBV DNA ≤2,000 IU/mL and HBsAg clearance at 5 years post-treatment were achieved by 23 and 12% of patients, respectively. High rates of HBsAg clearance at 5 years post-treatment were achieved by patients with HBV DNA ≤2,000 IU/mL at 1 year post-treatment (28%). Rates of HBV DNA ≤2,000 IU/mL at 1 year post-treatment were 47.2 and 43.4% in patients with ≥10% decline from baseline at weeks 12 and 24, respectively, compared with 16.4% (p = 0.0003) and 13.2% (p < 0.0004) in patients with a <10% decline. Rates of HBsAg clearance at 5 years post-treatment were 22.6 and 22.4% in patients with ≥10% decline at weeks 12 and 24, respectively, compared with 7.5% (p = 0.0161) and 3.8% (p < 0.0001) in patients with <10% decline. ABSTRACT.CONCLUSIONS: Peginterferon alfa-2a results in increasing rates of HBsAg clearance during post-treatment follow-up in HBeAg-negative patients. On-treatment decline in HBsAg is significantly associated with long-term post-treatment response. BODY.INTRODUCTION: Chronic hepatitis B virus (HBV) infection is estimated to affect 350 million people worldwide and is associated with development of cirrhosis and hepatocellular carcinoma [1]. Serum hepatitis B surface antigen (HBsAg) is usually used as a qualitative marker for the diagnosis of HBV infection, and chronic hepatitis B (CHB) is defined as persistence of HBsAg in the circulation for more than 6 months. HBsAg clearance is considered a marker of complete and definitive remission of HBV activity [2], because it is associated with reduced incidence of hepatocellular carcinoma and improved rates of survival, and is the closest outcome to clinical cure of CHB [3–5]. Previous studies with conventional interferon alfa-2a in patients with hepatitis B e-antigen (HBeAg)-negative CHB demonstrated that HBsAg clearance could be achieved by this population. Approximately, one-third of patients who sustained a biochemical response for a median period of 7 years post-treatment achieved HBsAg clearance [6]. Subsequently, a finite course of peginterferon alfa-2a has been shown to result in HBsAg clearance, which increases in the years following the completion of the course of therapy. Indeed, rates of HBsAg clearance have been shown to reach 9% at 3 years post-treatment in HBeAg-negative patients [7]. Although HBsAg clearance is the ultimate goal in HBeAg-negative CHB [8], rates of response in the short-term are low and, therefore, it is not usually the primary efficacy endpoint in clinical studies. Alternative markers of response have been identified that are associated with subsequent increased rates of HBsAg clearance and long-term clinical benefits. In HBeAg-negative CHB, HBV DNA suppression ≤2,000 IU/mL is used to define sustained immune control with pegylated interferon in the shorter term (e.g., 1 year post-treatment) as this level of HBV DNA is associated with inactive disease and a low risk of hepatocellular carcinoma [2, 9, 10]. Patients who achieve sustained immune control following interferon-based therapy are likely to clear HBsAg during long-term treatment-free follow-up [11, 12]. The pivotal study of peginterferon alfa-2a in HBeAg-negative patients showed that approximately one-third of patients achieve sustained immune control [13]. Identification of responders either before or early during treatment would be of great benefit as it would not only ensure timely initiation of treatment in patients likely to respond but also allow modification of the treatment regimen in those patients unlikely to respond to the standard duration (48 weeks) of peginterferon alfa-2a monotherapy. Recent analyses have shown that serologic (e.g., HBsAg) and virologic (e.g., HBV DNA) markers either before or during treatment with pegylated interferon may help identify those patients most likely to respond post-treatment [14]. In a retrospective study, it was also shown that low pretreatment HBsAg levels and their decline during treatment with conventional interferon alfa predicted subsequent HBsAg clearance [15]. The current analysis describes the response rate at 5 years post-treatment in HBeAg-negative patients treated with peginterferon alfa-2a during the Phase 3 study—this is the final planned efficacy analysis from this pivotal trial. In addition, it investigates the potential of HBsAg quantification during treatment to predict long-term response. BODY.MATERIALS AND METHODS.STUDY DESIGN: HBeAg-negative patients received peginterferon alfa-2a (180 μg/week) ± lamivudine (100 mg/day) for 48 weeks as part of the large, multicenter, randomized Phase 3 study [13]. Efficacy was assessed as part of the long-term follow-up study [7], and results at 5 years post-treatment are reported. Additional retrospective analyses of data from the initial and long-term study were also conducted. HBsAg levels were analyzed retrospectively from stored samples collected at baseline, during therapy at weeks 12, 24, and 48 (end of treatment), and 6 months post-treatment (week 72). HBsAg levels were quantified using the Abbott Architect HBsAg assay (Abbott Laboratories, IL, USA; dynamic range 0.05–250.0 IU/mL) after 1:100 dilution. Samples with HBsAg >250.0 IU/mL at this dilution were retested at a final dilution of 1:1,000. Samples with HBsAg levels <0.05 IU/mL at 1:100 dilution were retested at the same dilution [16]. HBV DNA was measured using the AMPLICOR HBV test (Roche Molecular Diagnostics, Pleasanton, CA, USA; range 71–35,715 IU/mL); samples with HBV DNA >35,715 IU/mL were retested after 1:100 dilution according to the manufacturer's instructions. BODY.MATERIALS AND METHODS.EFFICACY ENDPOINTS: The original protocol stated that post-treatment efficacy should be determined at yearly time-points with the final assessment taking place 5 years post-treatment. The efficacy endpoints included in the current analysis were HBV DNA ≤2,000 IU/mL (≈10,000 copies/mL)—a marker of sustained immune control—and HBsAg clearance at 1 and 5 years post-treatment. Both parameters were assessed in patients enrolled in the long-term follow-up study (n = 230), and in patients included in the long-term follow-up study with HBsAg values available at baseline, weeks 12, 24, and 48 of treatment and 6 months post-treatment (n = 120). The HBsAg kinetic analysis was conducted in the subgroup of 120 patients with HBsAg levels available at all time-points. BODY.MATERIALS AND METHODS.ASSOCIATION OF ON-TREATMENT HBSAG AND HBV DNA LEVELS WITH RESPONSE POST-TREATMENT: As HBsAg decline from baseline has previously been shown to be similar in peginterferon alfa-2a and peginterferon alfa-2a + lamivudine-treated patients [17], data from both treatment groups were pooled. HBV DNA decline has been shown to be greater in patients treated with peginterferon alfa-2a + lamivudine compared with patients treated with peginterferon alfa-2a alone [17] and, therefore, wherever HBV DNA decline was assessed, the two treatment arms were analyzed individually. Only patients with HBsAg values available at baseline and at all other time-points (weeks 12, 24, 48, and 72) were included in this analysis. The association between HBsAg decline during treatment and response at 1 and 5 years post-treatment was investigated. Association between HBsAg decline and response at 6 months post-treatment was not investigated, due to the high rate of relapse between 6 months and 1 year post-treatment [7]. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Receiver operating characteristic analysis at weeks 12 and 24 of therapy was used to identify levels of HBsAg decline from baseline associated with high rates of response post-treatment. The target was to identify a cut-off value that would provide a negative predictive value (NPV) ≥95%. Logistic regression was used to analyze the chance of a response. In general, missing samples were treated as non-response. The only exception was when calculating the rate of HBsAg clearance, where last-observation-carried-forward (LOCF) methodology was used in patients with HBV DNA ≤71 IU/mL at the missing time-point. All statistical tests were considered exploratory and no adjustment for multiple testing was performed. Mantel–Haenszel Chi-square (MHχ2) test and Wald Chi-square (WCχ2) test were used as appropriate. The statistical analysis software used was SAS version 8.0 (SAS Institute, Cary, NC, USA). BODY.RESULTS: A total of 230 peginterferon alfa-2a ± lamivudine-treated patients were included in the long-term follow-up study. Of these, 120 (52%) had HBsAg values available at all time-points (i.e., baseline and weeks 12, 24, and 48 of therapy and 6 months post-treatment). Of the patients with HBsAg values available at all time-points, the majority (78%) was from the Asia–Pacific region. Baseline characteristics were similar between the two populations (Table 1).Table 1Baseline characteristics for peginterferon alfa-2a ± lamivudine-treated patients included in the long-term follow-up study (n = 230) and in those with HBsAg values at each time-point (n = 120)Long-term follow-up populationa (n = 230)Long-term follow-up population with HBsAg available at all time-pointsb (n = 120)Ethnicity (%) Caucasian/Asian/other27/72/132/67/2Gender (%) Male/female83/1775/25Genotypec (%) A/B/C/D7/28/42/2010/20/46/22Age (years) mean ± SD39.9 ± 11.041.3 ± 9.9HBsAg (log10 IU/mL) mean ± SD3.39 ± 0.613.40 ± 0.61HBV DNA (log10 IU/mL) mean ± SD6.46 ± 1.916.49 ± 1.85ALT (IU/L) mean ± SD87 ± 7592 ± 87Number of patients with HBsAg values available at all time-pointsNA120 China32 Hong Kong26 Thailand13 Italy11 Poland10 Spain9 Turkey7 Taiwan5 France4 New Zealand2 Greece1ALT alanine aminotransferase, NA not applicableaRef. [7]bPatients with HBsAg values at baseline and at weeks 12, 24, 48, and 72cSeven patients in the overall population and three patients in the current analysis population were not infected with one of the four main HBV genotypes BODY.RESULTS.EFFICACY OF PEGINTERFERON ALFA-2A AT 1 AND 5 YEARS POST-TREATMENT: In the long-term population (n = 230), 72 patients (31%) had HBV DNA ≤2,000 IU/mL at year 1 post-treatment and 11 patients (5%) had cleared HBsAg (Table 2). In the 72 patients with HBV DNA ≤2,000 IU/mL at year 1 post-treatment, 50% (36/72) had sustained suppression of HBV DNA ≤2,000 IU/mL at year 5 post-treatment. For comparison, in patients with available data at year 1 and year 5 post-treatment, 88% (36/41) sustained suppression of HBV DNA ≤2,000 IU/mL. The rate of HBsAg clearance at 5 years post-treatment was significantly higher in patients with HBV DNA ≤2,000 IU/mL at 1 year post-treatment (20/72; 28%) than in patients with HBV DNA >2,000 IU/mL at 1 year post-treatment (8/158, p < 0.0001). HBsAg clearance was achieved by 42.4% (14/33) of patients with HBV DNA <70 IU/mL at 1 year post-treatment.Table 2Response rates at 1 and 5 years post-treatment for peginterferon alfa-2a ± lamivudine-treated patients included in the long-term follow-up study (n = 230) and for patients included in the current analysis (n = 120)Long-term follow-up populationa (n = 230)Long-term follow-up population with HBsAg available at all time-pointsb (n = 120)Response at 1 year post-treatment, n (%) HBV DNA ≤2,000 IU/mL72 (31)36 (30) HBsAg clearance11 (5)6 (5)Response at 5 years post-treatment, n (%) HBV DNA ≤2,000 IU/mL54 (23)31 (26) HBsAg clearance28 (12)17 (14)aRef. [7]bPatients with HBsAg values at baseline and at weeks 12, 24, 48, and 72 In patients with HBsAg values available at all time-points (n = 120), 36 patients (30%) had HBV DNA ≤2,000 IU/mL at year 1 post-treatment and, of these, 14 (39%) achieved HBsAg clearance at 5 years post-treatment (compared with 3.6% (3/84) of patients with HBV DNA >2,000 IU/mL at 1 year post-treatment, p < 0.0001). Rate of HBsAg clearance in patients with HBV DNA <70 IU/mL at 1 year post-treatment was 61.5% (8/13). Rates of response at 1 and 5 years post-treatment in both populations are shown in Table 2. BODY.RESULTS.BASELINE HBSAG AS A PREDICTOR OF RESPONSE: Receiver operating characteristic analysis identified a baseline HBsAg level of 5,000 IU/mL that was associated with post-treatment response. This provided a positive predictive value (PPV) of 34% for HBV DNA ≤2,000 IU/mL at 1 year post-treatment and a PPV of 30% for HBV DNA ≤2,000 IU/mL at 5 years post-treatment. The NPVs generated were 78 and 84%, respectively. BODY.RESULTS.ASSOCIATION BETWEEN HBSAG DECLINE AND POST-TREATMENT RESPONSE: The patients were divided into three mutually exclusive response categories: (1) patients with HBsAg clearance, (2) patients without HBsAg clearance but with HBV DNA ≤2,000 IU/mL, and (3) patients without HBsAg clearance and with HBV DNA >2,000 IU/mL post-treatment. The three groups were determined based on response at year 1 (Fig. 1a) and at year 5 (Fig. 1b). HBsAg decline during treatment (48 weeks) and the initial follow-up period (24 weeks) was significantly more pronounced in patients with HBsAg clearance at either 1 or 5 years post-treatment when compared with patients not achieving HBsAg clearance or HBV DNA suppression. In patients with HBV DNA <2,000 IU/mL at 1 year post-treatment, the pattern of HBsAg decline was similar to that in patients with HBsAg clearance at 5 years post-treatment, although the overall decline was the greatest in patients with HBsAg clearance at 5 years post-treatment (Fig. 1c).Fig. 1On-treatment HBsAg decline according to response. a 1 year post-treatment. b 5 years post-treatment. c 1 year (HBV DNA ≤2,000 IU/mL) and 5 years (HBsAg clearance) post-treatment BODY.RESULTS.HBSAG AND HBV DNA DECLINE IN RESPONDERS, RELAPSERS, AND NON-RESPONDERS: The decline in HBsAg level in peginterferon alfa-2a ± lamivudine-treated patients with HBV DNA ≤2,000 IU/mL at the end of treatment and 5 years post-treatment (virologic responders, n = 31) was compared with that in patients with a response at end of treatment that was not sustained until year 5 (relapsers, n = 76) and in patients with HBV DNA >2,000 IU/mL at the end of treatment and 5 years post-treatment (non-responders; n = 13) (Fig. 2a). HBsAg clearance was more pronounced in responders than in relapsers and non-responders. Of the virologic responders, 17 (55%) achieved HBsAg clearance at 5 years post-treatment; however, none of the relapsing or non-responding patients achieved this endpoint.Fig. 2a On-treatment HBsAg decline in responders, relapsers, and non-responders. b On-treatment HBV DNA decline in responders, relapsers, and non-responders treated with peginterferon alfa-2a. c On-treatment HBV DNA decline in responders, relapsers, and non-responders treated with peginterferon alfa-2a + lamivudine. Responder: HBV DNA ≤2,000 IU/mL at end of treatment and year 5; relapser: HBV DNA <2,000 IU/mL at end of treatment; HBV DNA >2,000 IU/mL at year 5; non-responder: HBV DNA >2,000 IU/mL at end of treatment and year 5 In peginterferon alfa-2a-treated patients, on-treatment HBV DNA decline was similar in responders (n = 15) and relapsers (n = 31). The decline was significantly higher in responders and relapsers compared with non-responders (Fig. 2b). There was only one non-responder in the peginterferon alfa-2a + lamivudine-treated group, thus, making comparisons between groups difficult (Fig. 2c). On-treatment HBV DNA levels did not distinguish between responders and relapsers in either peginterferon alfa-2a treatment group. BODY.RESULTS.ASSOCIATION BETWEEN ON-TREATMENT HBSAG LEVEL AND RESPONSE AT 1 AND 5 YEARS POST-TREATMENT: Receiver operating characteristic analysis identified a ≥10% log10 HBsAg decline from baseline that was significantly associated with post-treatment response. The association between on-treatment HBsAg log10 decline and HBV DNA ≤2,000 IU/mL at 1 or 5 years post-treatment was investigated, using 10% log10 decline from baseline as a cut-off at weeks 12 and 24. More patients achieved ≥10% HBsAg log10 decline from baseline at week 24 (56%) than at week 12 (44%). Patients with a ≥10% log10 HBsAg decline from baseline achieved significantly higher rates of HBV DNA ≤2,000 IU/mL at both year 1 and year 5 post-treatment than patients with a <10% log10 decline from baseline (Fig. 3a). Applying the identified cut-off level at weeks 12 and 24 provided PPVs of 47 and 43%, respectively, and NPVs of 84 and 87%, respectively, at year 1. At year 5, PPVs were 42 and 36%, respectively, and NPVs were 87% at both weeks 12 and 24.Fig. 3Response rates at 1 and 5 years post-treatment according to HBsAg decline at week 12 or week 24 of treatment. a Response defined as HBV DNA ≤2,000 IU/mL. b Response defined as HBsAg clearance Rates of HBsAg clearance were also significantly higher in patients with a ≥10% log10 decline in HBsAg from baseline at week 12 (p = 0.0484 for response at 1 year post-treatment and p = 0.0184 for response at 5 years post-treatment) and week 24 (p = 0.026 for response at 1 year post-treatment and p = 0.0038 for response at 5 years post-treatment) than in patients with a <10% log10 decline (Fig. 3b). The PPV and NPV for HBsAg clearance at 5 years post-treatment, based on ≥10% log10 decline in HBsAg from baseline at week 12, were 23 and 93%, respectively. At week 24, the PPV and NPV were 22 and 96%, respectively. High rates of HBsAg clearance at 5 years post-treatment were achieved by patients with a ≥10% decline in HBsAg from baseline at week 12 and HBV DNA <2,000 IU/mL at 1 year post-treatment (40.0%, 10/25). Similarly, 44.8% (13/29) of patients with a ≥10% decline in HBsAg from baseline at week 24 and HBV DNA <2,000 IU/mL at 1 year post-treatment achieved HBsAg clearance at 5 years post-treatment. BODY.DISCUSSION: The pivotal trial of peginterferon alfa-2a in HBeAg-negative CHB has provided a wealth of information about the long-term post-treatment effects of a finite course of treatment. A previous analysis of this study demonstrated that rates of HBsAg clearance increased after completion of treatment, with 9% of patients treated with peginterferon alfa-2a ± lamivudine achieving HBsAg clearance 3 years after treatment [7]. In the protocol-defined analysis described, rates of HBsAg clearance were shown to further increase during longer-term follow-up, with 12% of patients achieving HBsAg clearance at 5 years post-treatment. HBsAg clearance is the ultimate treatment goal of patients with HBeAg-negative CHB as it is associated with improved outcomes [3–5]. Although HBsAg clearance has been observed during treatment of HBeAg-positive CHB with some of the newer nucleos(t)ide analogs [18, 19], the rates of HBsAg clearance in HBeAg-negative patients during nucleos(t)ide analog therapy are negligible [19]. In contrast, the current study clearly demonstrates that rates of HBsAg clearance after peginterferon alfa-2a therapy are substantial and durable. Patients achieving HBV DNA suppression at 1 year post-treatment achieved high rates of HBsAg clearance at 5 years post-treatment. As 28% of patients with sustained immune control (HBV DNA ≤2,000 IU/mL at 1 year post-treatment) achieved HBsAg clearance at 5 years post-treatment, this endpoint appears to be a valuable early indicator of long-term response. The analyses conducted in patients enrolled in this international, multicenter, randomized trial have considerably increased the knowledge of the potential value of peginterferon alfa-2a therapy in patients with HBeAg-negative CHB. As a result of the heterogeneous population enrolled, this study—the largest and the most extensive of peginterferon alfa-2a in HBeAg-negative CHB to date—closely reflects the global clinical situation. The long-term benefits achieved following a finite course of peginterferon alfa-2a in such a population are clearly encouraging. Early identification of patients who could benefit from this treatment approach would be valuable, as it would allow clinicians to motivate patients likely to achieve a long-term response to complete therapy while also identifying those patients for whom an alternative treatment regimen may be necessary. Recently, two groups showed that HBsAg levels vary considerably during the natural history of CHB and reflect the disease phase [20, 21], and previous results demonstrated that on-treatment HBsAg kinetics and HBsAg levels at the end of treatment are associated with sustained response to peginterferon alfa-2a [14, 17]. The observation that lower levels of HBsAg are associated with greater immune control has resulted in considerable interest in HBsAg kinetics during peginterferon alfa-2a therapy. A difference in HBsAg decline between responders and non-responders, as occurred in this study, was described initially by Brunetto et al. [17]. In the current analysis, differences in HBsAg decline patterns were also observed in patients achieving a long-term response to treatment compared with those relapsing after experiencing an on-treatment response. This confirms data from Moucari et al. [14] that showed the association between HBsAg decline and short-term post-treatment response. As variations in HBsAg kinetics between responders, non-responders, and relapsers to peginterferon alfa-2a therapy have been established in both short-term and long-term follow-up, research has focused on whether clinicians can use the knowledge of HBsAg kinetics to make disease-management decisions. In the current study, receiver operating characteristic analysis identified a decline in HBsAg levels at weeks 12 and 24, which was associated with high rates of post-treatment response. Patients with a ≥10% log10 IU/mL decline in HBsAg from baseline achieved significantly higher rates of HBV DNA ≤2,000 IU/mL and HBsAg clearance up to 5 years post-treatment than patients not achieving this level of decline. It is worth noting that 40–45% of patients with this level of on-treatment HBsAg decline and sustained immune control at 1 year post-treatment achieved HBsAg clearance at 5 years post-treatment. Although PPVs for long-term sustained immune control were slightly lower at week 24 than at week 12, the high rates of HBsAg clearance achieved by patients with a ≥10% decline during treatment and sustained immune control 1 year post-treatment suggest that on-treatment quantification at either time-point provides clinically important information. The target was to achieve NPV ≥95%, as this would identify patients unlikely to achieve a sustained response and allow physicians to consider stopping treatment. However, as the NPVs generated by this analysis did not reach the target level, using this as a stopping rule would mean that 13–16% of potential responders would have their treatment stopped prematurely. Some smaller studies have identified on-treatment HBsAg cut-off levels that generate higher NPVs. For example, Moucari et al. [14] showed that an HBsAg decline of 0.5 log10 IU/mL (68% decline from baseline) at week 12 generated an NPV of 90% and an HBsAg decline of 1.0 log10 IU/mL (90% decline from baseline) at week 24 gave an NPV of 97%. However, these levels of decline did not produce similar results in the current analysis (data not shown). The lack of consistent findings may be explained by differences in the study design, response parameters, and study populations between the two studies. For example, Marcellin et al. [7] defined post-treatment response as undetectable HBV DNA at 6 months post-treatment, rather than the HBV DNA <2,000 IU/mL and HBsAg clearance at 1 year and 5 years post-treatment employed in the current analysis. Given that relapse is known to occur between 6 months and 1 year post-treatment [7], 1 year may be a more appropriate time-point for assessment of sustained response. In addition, the differences in cut-off levels identified in these two populations may be explained, at least in part, by HBV genotype, which is known to affect post-treatment response [14, 17, 22, 23]. In the current analysis, the patients were infected predominantly with HBV genotype C (46%) and D (22%) with very few infected with HBV genotype A (10%); while in the Moucari et al. [14] analysis, 27% of patients were infected with HBV genotype A. The influence of HBV genotype on HBsAg kinetics in samples from patients who were also included in the current analysis was described initially by Brunetto et al. [17] who showed that patients infected with all the major genotypes achieve some level of HBsAg decline, but this was most pronounced in patients infected with genotypes A and B. Genotype is, therefore, likely to be a major influencing factor on decline of HBsAg levels in HBeAg-negative subjects. Further analyses are needed to elucidate the influence of genotype on HBsAg kinetics and to determine how knowledge of infecting genotype combined with HBsAg quantification can be used to predict response to peginterferon alfa-2a. While most investigations have examined on-treatment prediction of response, there is also potential value in identifying at baseline those patients likely to respond to peginterferon alfa-2a. In the current analysis, patients with HBsAg ≤5,000 IU/mL at baseline achieved the highest rates of response post-treatment, but the PPVs (approximately 30%) and NPVs (approximately 80%) calculated were lower than those relating to on-treatment HBsAg quantification. Consequently, on-treatment HBsAg quantification appears to be a more appropriate predictor than quantification of HBsAg at baseline. Unlike on-treatment HBsAg quantification, measurement of HBV DNA levels during treatment does not differentiate between treatment responders and relapsers [14]. This observation was based on response at 6 months post-treatment but was also seen in the current analysis, where a significant difference in HBV DNA decline between patients with a response at 5 years post-treatment and relapsers could not be demonstrated. In addition, there were differences in HBV DNA decline between peginterferon alfa-2a-treated patients and patients receiving combination therapy with lamivudine. Wherever there was a difference in HBV DNA decline between responders/relapsers and non-responders in monotherapy-treated patients, this was not present in the combination therapy group. Consequently, HBV DNA quantification may not be as valuable for identifying long-term responders to therapy as HBsAg quantification. A recent analysis of another study of peginterferon alfa-2a in HBeAg-negative patients investigated the potential of combining HBsAg and HBV DNA response during treatment to improve NPVs [24]. The NPV of 100% was reported in patients who did not achieve an HBsAg decline or an HBV DNA decline >2 log10 copies/mL at week 12 of treatment. The importance of infecting genotype on HBsAg kinetics was discussed earlier, and as most patients in this analysis were infected with HBV genotype D, further analysis is required to determine whether this rule can be used in patients infected with other genotypes. It is interesting to speculate how HBsAg quantification could be used in clinical practice to help individualize peginterferon alfa-2a therapy. Preliminary data from a study of extended peginterferon alfa-2a therapy in HBeAg-negative patients have shown that extension therapy improves sustained response rates as a result of a reduction in relapse [25]. An important future consideration will be whether patients likely to benefit from extended therapy can be identified early during the initial phase of treatment. Brunetto et al. [26] have studied in-depth HBsAg kinetics in the HBeAg-negative patients included in the Phase 3 peginterferon alfa-2a study and showed that response rate is linked to decline pattern. Although patients with a continuous HBsAg decline from baseline (≥10% decline from baseline to week 24 and ≥10% from weeks 24 to 48) achieved the highest rates of response, patients with a late decline (≥10% decline from baseline after week 24) also achieved high rates of response when compared with patients with a <10% HBsAg decline during the entire 48-week treatment period. It is possible that patients with a late HBsAg decline will benefit from an extended period of peginterferon alfa-2a therapy; however, this needs to be studied in prospective clinical trials, which also consider in more detail the role of infecting genotype. The current analysis has limitations. Only a proportion of patients included in the initial or follow-up studies had HBsAg levels determined during treatment and 6 months post-treatment. In addition, only patients with HBsAg data available at all on-treatment and post-treatment time-points were included and, consequently, there is the potential for selection bias. However, baseline characteristics and response rates in the 230 patients in the long-term analysis were similar to those achieved by the 120 patients in the current analysis, and the statistical methods used were conservative as missing samples were taken as non-responders. Wherever LOCF methodology was used, a secondary parameter was included (HBV DNA <71 IU/mL) to reduce the chance of false-positive data. In conclusion, a finite course of peginterferon alfa-2a resulted in increasing rates of HBsAg clearance up to 5 years post-treatment in HBeAg-negative patients. Sustained immune control at 1 year post-treatment was an early indicator of subsequent HBsAg clearance. Analysis of data from this large, long-term study has shown that HBsAg quantification may be an appropriate on-treatment tool for monitoring response to peginterferon alfa-2a in HBeAg-negative patients, thereby confirming observations in small-scale studies. Further prospective studies are required before clear clinical guidance on use of HBsAg monitoring can be provided for physicians. In the future, increased understanding of the kinetics of HBsAg decline in HBeAg-negative patients treated with peginterferon alfa-2a may help physicians make individualized treatment decisions that should ultimately increase the rate of response in patients with CHB.

TITLE: Effect of a participatory organizational-level occupational health intervention on job satisfaction, exhaustion and sleep disturbances: results of a cluster randomized controlled trial ABSTRACT.BACKGROUND: We examined whether the implementation of a participatory organizational-level intervention aiming to improve the working environment with a focus on the core task at work, increased job satisfaction and reduced exhaustion and sleep disturbances among pre-school employees. ABSTRACT.METHODS: The study sample consisted of 41 intervention group pre-schools with 423 employees and 30 control group pre-schools with 241 employees. The intervention lasted 25 months and consisted of seminars, workshops, and workplace specific intervention activities that were developed by focusing on the core task at work. We analyzed within-group changes in the three outcome variables from baseline to follow-up with t-tests for paired samples, separately for intervention and control group. Between-group differences in changes in the three outcome variables were analyzed using a mixed model with a repeated statement to account for the clustering effect of workplaces. ABSTRACT.RESULTS: Within-group analyses showed that exhaustion decreased statistically significantly in both the intervention and the control group. There were no statistically significantly changes in job satisfaction and sleep disturbances. Between-group analyses showed that there was no statistically significant difference between the two groups for changes in any of the outcome variables, neither in the unadjusted or in the adjusted analyses. ABSTRACT.CONCLUSIONS: We found no evidence that participating in an organizational-level occupational health intervention aiming to improve the working environment with a focus on the core task at work has an effect on pre-school employees' job satisfaction, exhaustion and sleep disturbances. ABSTRACT.TRIAL REGISTRATION: ISRCTN16271504, November 15, 2016. BODY.BACKGROUND: The relation of the psychosocial work environment with employees' health and well-being is likely highly complex and characterized by many factors [1]. Adverse psychosocial working conditions that had been related to health endpoints include for example mismatches between high demands and low control [2–4], and high effort and low reward [5, 6], poor management style [7] and organisational injustice [8]. Psychosocial resources at work, such as high workplace social capital [9–11] may contribute to the protection of employees' health. Organizational-level occupational health interventions aim for reducing health-hazardous and enhancing health promoting working conditions [12]. It has been argued, that in particular participatory organizational interventions may have a positive impact on employees' health, partly because these types of interventions improve employees' job control [13]. The participatory approach refers to employees' involvement and participation and implies that employees participate in workplace problem analysis and take an active part in developing and implementing intervention activities tailored their own workplace [14, 15]. However, results from organizational interventions are inconsistent and study quality is often low [16, 17]. According to the Stress-As-Offense-to-Self (SOS) theory, the distinction between core tasks and illegitimate tasks at work are key for understanding employees' health and well-being [18]. Core tasks are activities that are essential for fulfilling the purpose of the organization and are closely linked to the professional identity of an employee. For a nurse, for example, it is a core task to take care of the medical needs of a patient. In the SOS theory, illegitimate work tasks are defined as the opposite of core work tasks and regarded as stressors, potentially affecting employees' health and well-being. They are conceptualized as either unnecessary, i.e. they should not be done at all or as unreasonable, i.e. they are outside one's occupation or occupational status and should be done by others. Previous research has shown that carrying out illegitimate tasks, as opposed to core tasks, is associated with counterproductive work behavior [19], higher level of cortisol [20], elevated stress level [21], decreased mental health [22], sleep disturbances [23], lowered self-esteem [18, 24] and feelings of resentment towards ones organization and burnout [18]. In this article, we evaluate the effect of a participatory organizational intervention that aimed to improve the working environment with a focus on the core task at work. In a previous article, we had shown that the intervention predicted a lower risk of sickness absence in the intervention group compared to the control group [25]. In this article, we test the effect of the intervention on three variables: job satisfaction, exhaustion and sleep disturbances. We hypothesized that the intervention will lead to increased job satisfaction and reduced exhaustion and sleep disturbances in intervention group participants compared to control group participants. The hypothesis is built on the underlying assumption that a psychosocial workplace intervention focusing on the core tasks at work will reduce exposure to adverse psychosocial working conditions, i.e. work stressors, and that reduced exposure to work stressors will result in more job satisfaction and less exhaustion and sleep disturbances. We choose job satisfaction as a general measure of employees' well-being at work, as suggested in previous studies, for example Bond and Bunce [26], Pryce et al [27], and DeJoy et al [28]. There is a strong relationship between job satisfaction and health, in particular for aspects of mental health [29]. Exhaustion and sleep disturbances are important health problems that are suspected to be at least partly related to the work environment [30, 31]. Further, exhaustion is a core symptom of the burnout syndrome that is common among human service workers and is a key topic in work environment research [32]. Previous research concluded that burnout increases the likelihood of sickness absence [33], and suggested that changes in the psychosocial work environment can reduce risk of burnout [34]. The relationship between psychosocial working conditions and exhaustion was also found in other studies [31, 35]. With regard to sleep disturbances, a recent review showed that psychosocial work factors impact sleep disturbances and called for work environment intervention studies tackling sleep disturbances [36]. BODY.METHODS: The aim of this intervention study was to study municipal pre-schools. The intervention was an initiative from the Municipality of Copenhagen, Denmark. The intervention was implemented in pre-schools in the Children and Youth Administration in Copenhagen by eight professional working environment consultants from a private company. The research evaluation was conducted by the University of Aalborg and the Danish National Research Centre for the Working Environment (NRCWE). BODY.METHODS.STUDY DESIGN AND PARTICIPANTS: This is an organizational-level occupational health intervention study that was cluster randomized and parallel with two arms. Questionnaire measurements were conducted at baseline and at 24 months of follow-up. Seventy eight workplaces formed the cluster randomized controlled trial. The Municipality of Copenhagen decided to conduct the intervention at 44 pre-schools, whereas 34 pre-schools served as the control group. A statistician randomized the workplaces accordingly. Of the 44 intervention workplaces, three were lost during follow-up: one workplace did not receive the intervention, because the workplace was preoccupied with other project activities; two workplaces discontinued the intervention, one was closed during follow-up and one left the study because the management had a negative appraisal of the intervention. Of the 34 control group workplaces, four were lost because they did not provide baseline or follow-up measurements. Thus, the analyses were based on 41 intervention and 30 control group workplaces Employees were eligible for the study if they were employed and present at the intervention and control group workplaces during the time of the baseline questionnaire measurements. Figure 1 shows that at baseline (September 2011), 944 employees at the intervention group pre-schools and 616 employees at the control group pre-schools received the questionnaire. Of these, 775 in the intervention (82.1%) and 470 (76.3%) in the control group responded. Of the 775 intervention group baseline responders, 423 employees responded to the follow-up questionnaire 24 months later, whereas 352 employees were lost to follow up. Due to missing information on some of the outcome measures, the final study sample in the intervention group was n = 409 for job satisfaction, n = 411 for exhaustion, and n = 409 for sleep disturbances. Of the 470 control group baseline responses, 241 employees responded to the follow-up questionnaire, whereas 229 were lost to follow up. The final study sample in the control group was n = 228 for job satisfaction, n = 234 for exhaustion, and n = 226 for sleep disturbances. According to Danish law, research studies that use solely questionnaire and register data do not need approval from the National Committee on Health Research Ethics (Den Nationale Videnskabetiske Komité).Fig. 1Flow chart towards the final study sample BODY.METHODS.THE INTERVENTION: The intervention was a participatory intervention focusing on the core task at work. Employees participated in the workplace problem analysis and solution finding process to ensure employees' involvement, commitment and control and at the same time to ensure that intervention activities were tailored to the specific needs of the workplace. In each intervention workplace, the pedagogical leader and two employee representatives, the shop steward and the health and safety representative, formed a steering group that managed the intervention. Each steering group received implementation support from a professional working environment consultant for the full intervention period. The intervention consisted of intervention activities common for all steering groups, i.e. seminars and workshops on how to develop and implement workplace specific intervention activities using a participatory approach, change management, workplace culture and evaluation tools. Based on the common intervention activities and consultants implementation support, the steering groups developed and implemented workplace specific intervention activities involving all employees. The intervention followed a structured and step-wise approach. From September 2010 to September 2011, the intervention project leader team planned and coordinated the intervention study. For five months from September 2011, workplace specific intervention activities were developed by the steering groups with the participation of all employees. Consultants explained to workplaces that this intervention's focus on the core task at work was equivalent to develop activities to improve the performance of central work tasks and procedures. The implementation lasted from February 2012 to June 2013. Finally, the workplaces conducted a self-evaluation between March and June 2013. BODY.METHODS.EFFECT MEASURES: We measured effects on changes in job satisfaction, exhaustion and sleep disturbances with self-administered questionnaires at baseline and at 24 months of follow-up. Both intervention and control group employees received and responded to the questionnaires during working hours. We measured job satisfaction with one item (Regarding your work in general. How satisfied are you with your job as a whole, everything taken into consideration?'), rated on a four-point scale (very satisfied, satisfied, dissatisfied, very dissatisfied) [37]. We measured exhaustion ('Within the past two weeks, how much of the time have you felt lacking in energy and strength?') and sleep disturbances ('Within the past two weeks, how much of the time have you had trouble sleeping at night?') with one item each, derived from the Major Depression Inventory [38]. Responses were rated on a six-point scale (all of the time, most of the time, slightly more than half of the time, slightly less than half of the time, some of the time, at no time). Higher scores indicate more job satisfaction, more exhaustion and more sleep disturbances. BODY.METHODS.STATISTICAL ANALYSIS: All analyses were conducted using SAS 9.3. First, to test baseline differences between intervention and control group in the study sample, we used Chi-square test, two sample t-test and Proc GLM. Next, we calculated the baseline and follow-up mean scores for each outcome variable separately for the intervention and the control group. Using paired t-tests, we analyzed changes from baseline to follow-up for each outcome variable, separately within the intervention and within the control group. Next, using the Genmod procedure in SAS, we analyzed differences in changes of the outcome variables between the intervention and the control group during follow-up in a mixed model with a repeated statement to account for the clustering effect of workplaces. We calculated unadjusted estimates and estimates adjusted for sex and age (continuous) (Model 1) and further adjusted for job group (pedagogical leader, nursery nurse, nursery nurse assistant, other job group), workplace type (integrated, day care, kindergarten) and workplace size (continuous) (Model 2). Finally, we conducted post-hoc analyses, in which we repeated the between group analyses while adjusting for the baseline scores of the outcome variables. BODY.RESULTS.CHARACTERISTICS OF PARTICIPANTS: Table 1 shows employee and workplace characteristics in the intervention and control group. Compared to control group participants, intervention group participants were younger (mean age: 42.9 vs. 44.9 years, p = 0.02) and were employed at workplaces of greater size (mean size 23.4 vs. 21.8 employees, p = 0.02). The groups did not differ with regard to sex, job group, and workplace type.Table 1Employee and workplace characteristics and baseline scores of outcome variables in the intervention and the control group in the study sampleIntervention groupControl groupChi2 (p)t (p)MeanSD% n MeanSD% n Employee characteristic 423241 Age42.910.444.99.8 2.43 (0.02) Women87.036890.02171.36 (0.24) Job group2.05 (0.56) - Pedagogical leaders6.6286.215 - Nursery nurses57.224252.7127 - Nursery nurse assistants28.412030.774 - Other job groups7.83310.425 Workplace characteristics 4130 Size23.48.421.89.6 -2.25 (0.02) Workplace type2.26 (0.32) - Integrated77.132679.7192 - Day care18.77918.344 - Kindergarden4.3182.15 Baseline scores of outcome variables Job satisfaction3.190.574093.020.70228 -3.11 (0.002) Exhaustion2.721.134113.011.26234 3.06 (0.002) Sleep disturbances2.041.284092.341.40226 2.68 (0.008) Statistically significant results are printed in bold Table 1 also shows the baseline scores of the three outcome variables. Intervention group participants had more favorable scores on all three variables, i.e. they reported more job satisfaction and less exhaustion and sleep disturbances than control group participants. These differences remained statistically significant, when we adjusted the analyses for employee and workplace characteristics (age, sex, job group, workplace size and workplace type, data not shown but is available on request from the first author). BODY.RESULTS.COMPARISON OF THE STUDY SAMPLE WITH EMPLOYEES LOST DURING FOLLOW-UP: When comparing the participants in the study sample with the participants that dropped out during follow-up, we found that the drop-out pattern was similar in the intervention and control group with regard to age and sex. In both groups, younger employees compared to older employees and men compared to women were more likely to drop out of the study. In the intervention group, mean age was 39.7 years for dropouts and 42.9 years for non-dropouts (p < 0.0001) compared to 41.1 years for dropouts and 44.9 years for non-drop-outs in the control group (p = 0.0002). Proportion of men was 17.9% among dropouts and 13.0% among non-dropouts in the intervention group (p = 0.06) and 16.2% among dropouts and 10.0% among non-dropouts in the control group (p = 0.05). In addition, in the intervention group, the mean workplace size was 26.0 employees among those who dropped out of the study compared to 23.4 employees among employees in the study sample (p < 0.0001). There was no such pattern in the control group (workplace size: 22.1 vs. 21.8 for those who dropped out and remained, respectively, p = 0.71). In both intervention and control group, participants who dropped out had a higher exhaustion score at baseline compared to those who remained in the study. This difference was statistically significant in the intervention group (2.93 vs. 2.72, p = 0.01) but not in the control group (3.16 vs. 3.01, p = 0.20). BODY.RESULTS.EFFECT OF THE INTERVENTION ON JOB SATISFACTION, EXHAUSTION AND SLEEP DISTURBANCES: Table 2 shows within group changes from baseline to follow-up in job satisfaction, exhaustion and sleep disturbances. Exhaustion decreased statistically significantly in both the intervention group (-0.16 points, p = 0.01) and the control group (-0.29 points, p < 0.001). There was no statistically significant change in job satisfaction and sleep disturbances, neither in the intervention group nor the control group.Table 2Within group changes in job satisfaction, exhaustion and sleep disturbances during 24 months of follow-upIntervention group (n = 423)Control group (n = 241) n Baseline Mean (SD)Follow-up Mean (SD)changet p n Baseline Mean (SD)Follow-up Mean (SD)changeT p Job satisfaction4093.19 (0.57)3.20 (0.54)+0.010.290.772283.02 (0.70)3.09 (0.62)+0.071.210.23Exhaustion4112.72 (1.13)2.56 (1.17)-0.16-2.50 0.01 2343.01 (1.26)2.73 (1.16)-0.29-3.48 <0.001 Sleep disturbances4092.04 (1.28)1.97 (1.22)-0.08-1.140.262262.34 (1.40)2.25 (1.36)-0.09-0.940.35Statistically significant results are printed in bold Table 3 shows the between-group changes for job satisfaction, exhaustion and sleep disturbances. There was no statistically significant difference between the intervention and control group for any of the three variables, neither in the crude nor in the adjusted analyses.Table 3Intervention effect on job satisfaction, exhaustion and sleep disturbances in the intervention group compared to the control group during 24 months of follow-up n UnadjustedModel 1Model 2Est95% CI p Est95% CI p Est95% CI p Job satisfaction637-0.06-0.21–0.100.47-0.04-0.20–0.110.59-0.06-0.21–0.090.40Exhaustion6450.13-0.12–0.370.310.12-0.12–0.370.310.15-0.08–0.380.20Sleep disturbances6350.01-0.28–0.310.930.01-0.29–0.310.940.03-0.24–0.310.82Interaction change x group analyses: Unadjusted; Model 1: Adjusted for sex and age (continuous); Model 2: further adjusted for job group (pedagogical leader, nursery nurse, nursery nurse assistant, other job group), workplace type (integrated, day care, kindergarden) and workplace size (continuous). Workplace identification number is included in a repeated statement BODY.RESULTS.POST-HOC ANALYSES: Because we had found that intervention and control group differed statistically significantly in the baseline scores of the three outcome variables (see Table 1), we conducted post-hoc analyses that repeated the between-group analyses in Table 3, while adjusting for the baseline values of the outcome variables. The estimates from this post-hoc analyses were similar to the estimates reported in Table 3 (data not shown but is available on request from the first author). BODY.DISCUSSION: The hypothesis that this intervention, which was a participatory organizational-level intervention aiming to improve the working environment with a focus on the core task at work, would improve job satisfaction and reduce exhaustion and sleep disturbances was not confirmed. There were no statistically significant differences between the intervention and control group during a 24 months follow-up. In a previous article of the same intervention study, we had shown that intervention group participants had a decreased risk of sickness absence during follow-up compared to control group participants [25]. Taken the previous and the current finding together, it seems that the intervention was efficacious with regard to sickness absence but not with regard to job satisfaction, exhaustion and sleep disturbances. However, one has to be cautious with drawing conclusions by comparing the two analyses, because the two samples were only partly overlapping. In the analysis on sickness absence, we used register data to assess the outcome variable and therefore we were able to analyze sickness absence for all employees at all workplaces, including employees who left the workplace during follow-up (who were excluded on the day they left their workplace) and employees who newly started at a workplace during follow-up (who were included on the day, they entered the workplace) [25]. In the current analysis on job satisfaction, exhaustion and sleep disturbances, register data was not available and therefore the analysis was restricted to employees who filled in the questionnaire at both baseline and follow-up. Moreover, sickness absence was assessed with monthly updates throughout the whole follow-up period, whereas job satisfaction, exhaustion and sleep disturbances were only assessed twice, at baseline and at the follow-up measure after 24 months. When an intervention study failed to show an impact of the intervention, two main explanations have to be considered: theory failure or implementation failure [39]. Theory failure refers to that the theory was wrong. In the case of this study, this would mean that the theoretical assumption was wrong that a participatory organizational-level intervention aiming to improve the working environment with a focus on the core tasks at work would result in less job stress, which subsequently would result in more job satisfaction and less exhaustion and sleep disturbances. Implementation failure refers to that the theory was correct, but that the intervention was not appropriately implemented and that therefore the impact of the intervention could not be demonstrated. We cannot decide whether theory or implementation failure or other mechanisms are the most likely explanations for the null findings. A previous qualitative process evaluation of the implementation of the intervention at four selected workplaces [40] showed that the four workplaces implemented specific intervention activities to solve organizational and professional conditions that were necessary to improve the performance of the core task. Thus, this qualitative process evaluation indicates that the intervention was appropriately implemented in at least some workplaces. In addition, the effect on risk of sickness absence [25] suggests that implementation failure is not likely. Both the intervention and the control group showed a statistically significant reduction in exhaustion. We cannot rule out that the reduction in the control group was partly an effect of the intervention, if we assume that intervention knowledge has been spread from intervention group pre-schools to control group pre-schools. Such a contamination was theoretically possible as there was contact and exchange, including meetings, between managers and employees' representatives of the intervention and control group pre-schools. However, if contamination actually had happened and if this contamination explains the reduction in exhaustion in the control group we do not know. In addition to theory or implementation failure, methodological issues also may be an explanation of the null findings. At baseline, there was a highly significant difference between the intervention and control group in all three outcome variables, with the intervention group showing more job satisfaction and less exhaustion and sleep disturbances. These differences could not be explained by different employee or workplace characteristics in intervention and control group. Because of these differences in baseline scores of the outcome variables, it was more difficult for the intervention group than for the control group to show improvements during follow-up. We do not have a clear explanation why the two groups differed at baseline. One possible explanation is that this was due to chance as this was a cluster- and not an individual-randomized trial with only 78 clusters. Another explanation could be the setting when the baseline questionnaire was filled in. Intervention and control group participants filled in the questionnaire after they had been informed about the result of the randomization and it is possible that this has resulted in a better mood in the intervention group compared to the control group, which may have caused reporting of more job satisfaction and less exhaustion and sleep disturbances. It seems that attrition rate was higher among younger employees than older employees and among those with high levels of exhaustion at baseline compared to those with low levels of exhaustion. This might have led to underestimation of the intervention effect, if the intervention was particularly efficacious for younger employees and those with high levels of exhaustion. Conversely, an intervention effect would have been overestimated if the intervention was particularly non-efficacious among younger employees and among those with high levels of exhaustion. Strengths of the study are the cluster-randomized design and the comprehensive, structured and step-wise intervention approach. Response rate at baseline was high in both intervention and control group. Limitations of this study, in addition to that intervention and control group participants filled in questionnaires after randomization, were the use of single items to measure outcome variables and the rather long follow-up period. By measuring each outcome variable with one question only, we only measured limited aspects of job satisfaction, exhaustion and sleep disturbances. It is possible that results would have been different, if we had measured these three variables more comprehensively. Finally, 24 months is a rather long follow-up period, which was mainly due to that the intervention itself was conducted over a longer period that lasted, at least in some pre-schools, from September 2011, when the first intervention activities were planned, until June 2013, when the last intervention activities had been implemented. It is possible that there were effects at some point during the follow-up period that did not remain after 24 months, but also possible that some effects only occurred at the end of the intervention. In hindsight, it would have been better, if we would have assessed the endpoints more frequently, for example at 6, 12, 18 and 24 months of follow-up. This would have allowed us to more closely monitor how trajectories in health and well-being changed in relation to the intervention. BODY.CONCLUSION: We found no evidence that participating in an organizational-level occupational health intervention aiming to improve the working environment focusing on the core task at work has an effect on pre-school employees' job satisfaction, exhaustion and sleep disturbances.

TITLE: Hickman catheter and implantable port devices for the delivery of chemotherapy: a phase II randomised controlled trial and economic evaluation ABSTRACT.BACKGROUND:: In the United Kingdom, totally implantable venous access systems (TIVAS) are not routinely used. Compared with Hickman catheters, these devices are more expensive and complex to insert. However, it is unclear whether the higher costs may be offset by perceived greater health benefits. This pilot trial aimed to generate relevant data to inform the design of a larger definitive randomised controlled trial. ABSTRACT.METHODS:: This was a phase II prospective, randomised, open trial from two UK oncology centres. The primary end point was overall complication rate. Secondary end points included individual complication rates, time to first complication and quality of life. Analysis was by intention to treat. An economic evaluation was also carried out. ABSTRACT.RESULTS:: A total of 100 patients were randomised in a 3 : 1 ratio to receive a Hickman or a TIVAS. Overall, 54% of patients in the Hickman arm suffered one or more complications compared with 38% in the TIVAS arm (one-sided P=0.068). In the Hickman arm, 28% of the devices were removed prematurely due to a complication compared with 4% in the TIVAS arm. Quality of life based on the device-specific questionnaire was greater in the TIVAS arm for 13 of the 16 questions. The economic evaluation showed that Hickman arm was associated with greater mean cost per patient £1803 (95% CI 462, 3215), but similar quality-adjusted life years −0.01 (95% CI −0.15, 0.15) than the TIVAS arm. However, there is much uncertainty associated with the results. ABSTRACT.CONCLUSIONS:: Compared with Hickman catheters, TIVAS may be the cost-effective option. A larger multicentre trial is needed to confirm these preliminary findings. BODY: When intravenous chemotherapy is needed it can either be given through a peripheral cannula (typically in a forearm vein) or through a central venous access device where the catheter tip is placed in a large central vein (typically the superior vena cava). Peripheral administration of chemotherapy frequently causes local vein irritation and thrombosis. This results in rapid exhaustion of the forearm veins, interruption to treatment, patient discomfort and a genuine fear of cannulation (Cheung et al, 2009). When the catheter tip lies centrally in a large vein, the damage is mitigated due to rapid blood flow and large vessel diameter. These advantages make central devices the obvious choice for longer drug regimes. There are three main types of central device: (i) tunnelled central catheter commonly referred to as a Hickman; (ii) peripherally inserted central catheter (PICC); and (iii) totally implanted venous access system (TIVAS) commonly referred to as a port (Bishop et al, 2007). A recent informal survey (personal communications) of nine large UK cancer units indicated Hickman (58%) to be the most common followed by PICC (33%), with TIVAS only used in 9%. The TIVASs are more expensive, more complex and invasive to insert, and many healthcare staffs are unfamiliar with their aftercare. However, there is some evidence that TIVAS may have the lower complication rate and lead to greater patient satisfaction with less interruption to treatment regimens (Kulkarni et al, 2014). The evidence is weak and the studies are heterogeneous, in terms of patient populations, methodological approach and definition of outcomes. Therefore, the magnitude of this reduced risk is still unclear. There is a need to evaluate the value of these devices to the UK NHS by looking at clinical and cost-effectiveness. It is unclear whether the higher purchasing costs of TIVAS may be offset by the perceived clinical benefits of lower complication rates and greater patient satisfaction. This phase II pilot trial aimed to inform the design of a larger definitive randomised controlled trial (RCT) by generating information about potential recruitment rates, incidence and distribution of outcome events, and the potential cost-effectiveness of the devices. BODY.MATERIALS AND METHODS.STUDY DESIGN AND PARTICIPANTS: This study was a phase II prospective, randomised, open trial conducted at two regional oncology centres in Scotland. All oncology patients with solid tumours, aged 18 years and over, who required a central venous access device for the delivery of chemotherapy, were eligible to participate in the study. Those who had evidence of any medical or psychiatric disorders that would be a contraindication to study participation and those with life expectancy of <3 months were excluded. This trial was reviewed and approved by the Multicentre Research Ethics Committee (11/AL/0083). BODY.MATERIALS AND METHODS.RANDOMISATION AND MASKING: All eligible patients were centrally randomised using minimisation, with respect to body mass index (BMI; <20, 20 to <30, 30 to <40, ⩾40), with a random element. A 3 : 1 (Hickman : TIVAS) randomisation ratio was used because of the limited availability and the cost of TIVAS. It was not feasible to mask participants and nurses to the allocated treatment. BODY.MATERIALS AND METHODS.PROCEDURES: All devices were placed at one site under local anaesthesia with the patient option of conscious sedation. Hickman catheters were either single or double lumen; TIVASs were single lumen devices. The majority of the devices were placed by senior interventional radiologists, with a small number of Hickman catheters placed by a nurse-led venous access team. All devices were placed using jugular veins for access with ultrasound guidance. The positioning of the Hickman catheters was confirmed by fluoroscopy or chest X-ray; fluoroscopy was routinely used to position the TIVAS. A standardised approach to catheter care was adopted, which included weekly heparin flush and dressing change for the Hickman catheters, and monthly heparin flush for TIVAS. Unlike the Hickman catheters, TIVASs were not in routine use at either of the two centres before the study. Therefore, chemotherapy nursing staff received training before the start and during the study to minimise the potential impact of the 'learning curve'. BODY.MATERIALS AND METHODS.OUTCOMES: The primary end point was overall complication rate. Complications included infection (blood stream infection, wound or exit site infection) and mechanical complications (line occlusion, migration, accidental withdrawal, flipping, central venous thrombosis, wound haematoma and skin breakdown or ulceration). Secondary end points included incidences of individual complications, time to first complication, health-related quality of life and resource use. Time to first complication was defined as the time from study registration until confirmed complication. Patients who did not experience a complication were censored at the date of device removal, date of last chemotherapy if the device had not been removed, the date of withdrawal if the patient withdrew from the study before experiencing complications or date of death. Health-related quality of life was assessed using a specifically designed 16-question device-specific questionnaire (Supplementary Appendix I) and the EuroQoL 5D (EQ-5D). The EQ-5D was recorded at baseline and monthly thereafter until device removal, death or end of follow-up. Resource use was recorded as consultations with healthcare professionals (inpatient stay, outpatient visits and general practitioner consultations). Patients were recruited between August 2011 and July 2013; the 12-month follow-up was completed in July 2014. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The sample size calculation was based on a randomised phase II screening approach to provide initial evidence of the effect of TIVAS in lowering the complication rate relative to Hickman catheters (Rubinstein et al, 2005). Only one UK study had previously compared Hickman and TIVAS-associated complications in patients undergoing chemotherapy (Ng et al, 2007), and reported a complication rate of ∼60% with Hickman catheters. The current phase II trial was designed to have 82% power to produce a statistically significant result at the 20% one-sided level of statistical significance if the true complication rate with TIVAS is 40%. This corresponds to an odds ratio (OR) of 2.25, which is at the low end of the estimates obtained from the wider literature (Carde et al, 1989; Kappers-Klunne et al, 1989; Mueller et al, 1992; Dillon et al, 2004; Johansson et al, 2004). The intention was to randomise 75 patients to Hickman catheters and 25 patients to TIVAS. All analyses were performed on the intention-to-treat principle. Logistic regression was used for the primary analysis to compare the proportion of patients on each arm experiencing one or more complication; the model included the stratification variable used in the randomisation (BMI). Time to first complication was analysed as a secondary end point using a Cox regression, also including BMI in the model. Quality of life analysis was based on the device-specific questionnaire. Overall, 16 questions were graded on a scale of 1 (not at all) to 4 (very much). The worst score reported during the study was established for each question and these were compared across study arms via Mann–Whitney U-tests. The P-values for the individual questions were adjusted for multiple comparisons using the false-discovery rate approach (calculated using the p.adjust function of the stats library in R (http://www.r-project.org). BODY.MATERIALS AND METHODS.PRE-TRIAL ECONOMIC MODELLING: A probabilistic decision analytical model was used to evaluate the potential cost-effectiveness of Hickman catheters and TIVAS from the perspective of the UK NHS over the trial period (12 months). A simple decision tree structure was adopted to identify patients who may and may not experience complications. Data relating to complication rates, resource use, costs and health utilities were based on the results of the current phase II trial. The cost of Hickman catheters and TIVAS were costed at £80 and £300, respectively. The costs associated with the devices were calculated by applying unit costs to healthcare resource use. Health utilities and quality-adjusted life years (QALYs) were calculated from the EQ-5D data. Multiple imputation was used to impute missing values of the EQ-5D five dimensions (Rubin and Schenker, 1986), and mean QALYs were estimated using the area-under-curve approach (Dolan, 1997). Where appropriate, cost-effectiveness was expressed as incremental cost per complication averted and incremental cost per QALY gained. Probabilistic (via a 1000 iteration Monte Carlo simulation) and univariate sensitivity analyses were undertaken to assess uncertainty. To examine whether conducting a larger RCT of Hickman lines versus TIVAS may be worthwhile, an expected value of perfect information (EVPI) analysis was carried out (Drummond et al, 2007). The analysis combined the probability and the cost of making the wrong decision, in terms of forgone health benefit and wasted resources based on uncertainty in the existing data. For the model, it was assumed that the life of technology is 5 years and the number of eligible patients per annum has been estimated at 425 000 per annum in the United Kingdom (HES data 2009–2010). A sample size calculation for a future trial was also undertaken based on the results of the economic evaluation using the net monetary benefit (NMB) approach (Supplementary Appendix II; Briggs, 2000). The estimates for both the cost and the effects were combined to determine the sample size for a cost-effectiveness outcome, using the traditional statistical methods for mean effectiveness, but based on the expected change in NMB (i.e., the change in monetarised effect minus the change in cost between the two alternatives; Briggs, 2000; Armitage et al, 2002). BODY.RESULTS: Seventy-four patients were randomised to Hickman catheters and 26 to TIVAS (Figure 1). One patient randomised to the TIVAS arm received a Hickman catheter due to administrative error. Three patients withdrew from the study before device insertion (two Hickman arm and one TIVAS arm). Devices were all successfully placed in 97 patients. The majority (Hickman 93% and TIVAS 84%) were inserted on the day of randomisation, and the remainder within 6 days. No immediate complications occurred during device placement. The two arms were well balanced for demographic and clinical baseline characteristics (Table 1). Colorectal, breast and pancreatic cancers made up the majority of the tumour types. BODY.RESULTS.COMPLICATIONS: Forty (54%) Hickman patients reported one or more complication compared with 10 (38%) TIVAS patients (Table 2). On the basis of logistic regression model, taking into account BMI stratification, Hickman catheters were associated with a statistically significant increased risk (the threshold for statistical significance was based on the pre-defined statistical plan of this phase II study) of one or more complications compared with TIVAS devices (OR 2.07; 80% CI 1.11, 3.88; exact one-sided P=0.068). There were 28 blood stream infections in total, 27 in 20 Hickman patients and in 1 TIVAS patient. Blood stream infection was the commonest complication in the Hickman arm, accounting for 45% of the complications. Fifteen patients, all in the Hickman arm required device removal due to blood stream infection. There were 30 line occlusions, 19 in 15 Hickman patients and 11 in 6 TIVAS patients. Line occlusion was the commonest complication in the TIVAS arm accounting for 55% of the complications. These were primarily resolved through simple catheter flushes and none required device removal in the TIVAS arm. In contrast, two patients in the Hickman arm required device removal due to occlusion. One patient in each arm had a confirmed central venous thrombosis; there were no reported pulmonary embolic events and no devices removed due to venous thrombosis. Overall, 21 devices were removed due to complications—20 from the Hickman arm and 1 from the TIVAS arm. In the Hickman arm, these were for infection (15), line occlusion (2), device malfunction (1), wound/exit site infection (1) and other (1); in the TIVAS arm, one single device was removed due to device malfunction. The median time to first complication for the Hickman arm was 30 weeks (80% CI 19, not estimable). The median time to first complication was not calculable for the TIVAS arm, as <50% of the patients experienced a complication. Chemotherapy was interrupted due to complications in 12 patients in the Hickman arm and two in the TIVAS arm. In the Hickman arm, the duration of chemotherapy interruption ranged from 4 to 41 days, and in the TIVAS arm both interruptions were for 1 day only. BODY.RESULTS.QUALITY OF LIFE: Overall, quality of life based on the device-specific questionnaire was better in TIVAS patients than Hickman patients. The adjusted one-sided P-values indicated that there were statistically significant differences at the 20% level in favour of TIVAS for all but three of the questions relating to 'getting in and out of a car', 'using public transport' and 'going out shopping' (Table 3). BODY.RESULTS.COST-EFFECTIVENESS: In consequence to the higher complications rate, patients in the Hickman arm incurred significantly greater healthcare resource use than the TIVAS arm (Supplementary Appendix III). The health utilities fluctuated over the 12-month period in both the arms. In base-case analysis, Hickman catheters were associated with substantially greater mean cost (£2515 vs £712), fewer complications averted (62 vs 46, based on a cohort of 100 patients) and lower mean QALYs than TIVAS over a 1-year period (Table 4). However, the observed difference in QALYs between the devices is extremely small (0.64 vs 0.65). Overall, the Hickman arm was associated with greater costs and lower health benefits, suggesting that TIVAS is the dominant strategy. Univariate sensitivity analysis was undertaken to examine the impact of complication rates by adopting the data from the wider literature. The probabilities of complications were estimated from pooling the results from the current phase II trial with two existing RCTs using on a random effects model (Carde et al, 1989; Kappers-Klunne et al, 1989). The estimated pooled OR for any complications was 3.05 (95% CI 1.08, 8.64), this was used in the analysis. The difference in cost between Hickman catheters and TIVAS increased, but the impact on the QALYs was remained extremely small (Table 4). The healthcare resource use among patients in the TIVAS arm was extremely low in the current phase II trial, this was also tested in the sensitivity analysis. The mean cost of patient with complications was assumed to be the same in both arms, this has little impact on the overall results. However, the model is most sensitive to the health utility estimates. When the QALY estimates for the Hickman arm was increased by 20%, and when all health utilities estimates were adjusted for censoring, TIVAS was no longer the dominant strategy. The results of the probabilistic sensitivity analysis following 1000 replications of the model are presented on the cost-effectiveness plane (Figure 2). The majority of the point estimates suggest that Hickman catheters were associated with greater costs than TIVAS, but there is substantial uncertainty in the difference in QALYs between the two devices. The value of information analysis suggested that, given current decision uncertainty, and at a willingness to pay threshold of £20 000, additional research is potentially worthwhile if future research costs less than £42 million. On the basis of the base case, a sample of 507 per arm will be sufficient to show a positive NMB in favour of TIVAS, given the likely improvement in QALYs, rate of complications and potential cost savings compared with Hickman. However, when taking into account the additional evidence from existing literature using the pooled OR for any complications, the estimated NMA becomes greater in favour of TIVAS, the resultant required sample per arm was lower (323 per arm). BODY.DISCUSSION: This pilot study found that Hickman catheters were associated with significantly greater risk of complications than ports (OR 2.07; 80% CI 1.11, 3.88). These findings are in line with the existing evidence (Kulkarni et al, 2014; Coady et al, 2015). The most commonly reported complication in the Hickman arm was blood stream infection. This is likely related to the external component of the device plus the need for more regular flushing (weekly). In contrast with a totally implanted device, only one case of infection was observed. In the TIVAS arm, the most commonly reported complication was line occlusion (defined as inability to aspirate blood). The decision analytical model showed that, despite the lower device costs, taking into account complications, Hickman catheters were associated with greater costs, fewer complications averted, but similar QALYs compared with TIVAS. The TIVAS is the dominant strategy and is the cost-effective option. However, the estimates were associated with substantial uncertainty, and the findings were highly sensitive to health utility estimates. The expected costs of uncertainty can be interpreted as the EVPI, based on the assumption that perfect information can eliminate the possibility of adopting the wrong decision. This also represents the maximum that the healthcare system should be willing to pay for additional evidence to inform this decision in the future through further research. At a cost-effectiveness threshold of £20 000 per QALY, based on the assumption that 425 000 patients may be eligible for venous catheters in the United Kingdom per year and a conservative expected lifetime of 1 year for the catheter, the EPVI for the effective population is approximately £42 million. This represents the maximum that the healthcare system should be willing to pay for additional evidence to inform this decision in the future. This pilot trial was designed to generate information about potential recruitment rates, incidence of distribution of outcome events and the potential cost-effectiveness, to inform the design of a larger definitive RCT by. In terms of recruitment, the recruitment rate was poor at the initial 12 months. However, this was resolved by introducing dedicated staff to act as 'trial champion'. The champion interacted with the patient pathway at all the important stages and successfully engaged with both healthcare staff and patients. In term of assessing complication rates, the definitions of complications were clear, but further refinements to the definitions of mechanical complications and line occlusions would ensure more accurate classification and coding. For instance, line occlusion was the most frequently observed complications among patients with TIVAS. Further investigations found that on several occasions when nursing staff was not able to aspirate blood return, this was resolved by the medical staff successfully re-sitting the needle into the TIVAS. It is likely that several of these were misclassified as apparent 'line occlusions'. Training is important with both these devices to minimise complications. At the start of this trial, a TIVAS user-training programme was instituted as these devices were not in regular use. Training and nurse confidence improved over the study period. This could be a potential confounder in future trials. There were also limitations to the economic evaluation. Healthcare resource use recorded in the TIVAS arm was surprisingly low, especially when compared with the Hickman arm. This may reflect potential performance bias; the two senior radiologists who were responsible for insertion of the TIVAS were often involved in resolving TIVAS complications. As a result, the costs associated with TIVAS may have been underestimated. On the other hand, the EuroQol 5D was used to estimate health utilities associated with using the two devices, and showed very small differences between the two arms. This may be explained by the results being dominated by the toxicity of the chemotherapy and disease status. The device-specific quality of life questionnaire in contrast appeared sensitive to differences between the two devices with 13 of the 16 questions showing statistically significant differences. The QALYs associated with TIVAS may have been underestimated. Due to the small sample size, correlation between the two questionnaires was not explored. The uncertainty associated with the QALY estimates was an important driver to the EVPI results. There is a clear need for more accurate estimates of QALYs, which supports the conclusion that further research to reduce overall uncertainty is worthwhile. This study suggests that the most expensive and least used device (TIVAS) may in fact be the most cost-effective. If confirmed with a larger trial, TIVAS could become the dominant strategy. This will require a programme of both training and education across the United Kingdom where currently TIVAS are only used in a highly selective manner and almost exclusively placed by medical staff. A much larger multicentre trial is needed that should also include PICC to establish clinical and cost-effectiveness. The NIHR (HTA) has recently funded a large RCT comparing Hickman lines, TIVAS and PICC (HTA 11/67/01). This trial (CAVA) of up to 2000 subjects, based on the sample size calculation that took into account the data from this phase II study and the wider literature, is currently underway. BODY.CONCLUSIONS: Cancer is a leading cause of death and many patients are treated with chemotherapy. Intravenous chemotherapy often necessitates a long-term venous access device. This pilot study provided preliminary evidence of a lower complication rate with TIVAS compared with Hickman catheters in patients receiving chemotherapy. This difference resulted in the Hickman arm being associated with greater costs and lower health benefits than the TIVAS arm and hence being less cost-effective. These preliminary findings need confirmation from a larger multicentre phase III trial that should also include PICCs, which are currently the most common device used for chemotherapy delivery in the United Kingdom.

TITLE: Tackling tuberculosis patients' internalized social stigma through patient centred care: An intervention study in rural Nicaragua ABSTRACT.BACKGROUND: We report a patient-centered intervention study in 9 municipalities of rural Nicaragua aiming at a reduction of internalized social stigma in new AFB positive tuberculosis (TB) patients diagnosed between March 2004 and July 2005. ABSTRACT.METHODS: Five out of 9 municipal teams were coached to tailor and introduce patient-centered package. New TB patients were assigned to the intervention group when diagnosed in municipalities implementing effectively at least TB clubs and home visits. We compared the changes in internalized stigma and TB treatment outcome in intervention and control groups. The internalized stigma was measured through score computed at 15 days and at 2 months of treatment. The treatment results were evaluated through classical TB program indicators. In all municipalities, we emphasized process monitoring to capture contextual factors that could influence package implementation, including stakeholders. ABSTRACT.RESULTS: TB clubs and home visits were effectively implemented in 2 municipalities after June 2004 and in 3 municipalities after January 2005. Therefore, 122 patients were included in the intervention group and 146 in the control group. After 15 days, internalized stigma scores were equivalent in both groups. After 2 months, difference between scores was statistically significant, revealing a decreased internalized stigma in the intervention group and not in the control group. ABSTRACT.CONCLUSION: This study provides initial evidences that it is possible to act on TB patients' internalized stigma, in contexts where at least patient centered home visits and TB clubs are successfully implemented. This is important as, indeed, TB care should also focus on the TB patient's wellbeing and not solely on TB epidemics control. BODY.BACKGROUND: Tuberculosis (TB) is usually presented as a global public health problem: there were an estimated 9 million new TB cases and 2 million TB deaths in 2004 [1]. TB can also be seen as an individual health problem. TB patients experience psychological, social suffering and their basic rights may be negated [2]. Amongst problems met by TB patients, social stigma is increasingly recognized, often in association with HIV[3]. Social stigma is "an undesirable or discrediting attribute that an individual possesses, thus reducing that individual's status in the eyes of society [4]." It is also "a social process to be understood in relation to the concept of power, domination and difference. It is a process worsening already existing inequalities and exclusions [5]." Two types are usually distinguished: (1) enacted stigma concerns discrimination due to social inferiority, highlighted through people 'running away' from TB patients, while (2) perceived or internalized stigma, used as an outcome in this research, is a sense of inferiority, resulting from fear of enacted stigma, shown by patients hiding their diagnosis from others, or feeling ashamed of having TB [6]. Both types of stigma have been assessed for TB: enacted alone [7], perceived stigma alone [8,9] or both together [10,12]. Some interventions have been reported for their potentiality to reduce tuberculosis social stigma. This has been assessed through qualitative methods, for example in the case of TB clubs [13,14] or involvement of former TB patients in advocacy and mobilization activities [15]. However, no published studies have attempted to compare prospectively changes between a group of TB patients benefiting from these interventions and a control group. We report hereby an intervention study comparing changes in internalized stigma and treatment outcome between a group of TB patients benefiting from a patient-centered interventions package including at least TB clubs and home visits, and a group control in rural Nicaragua. The basic assumption behind that package design was that, by increasing power-sharing between the health personnel and TB patients (i.e. giving more power to the patient in the health care provider – patient interaction), TB patients' internalized stigma would decrease. BODY.METHODS: In this section, we describe the setting, the population, the definition of control and intervention groups, and the data collection. Data collection was not limited to outcome measurement. We emphasized comprehensive documentation of process before and during interventions package implementation. Indeed, we wanted to capture contextual factors (i.e. those factors external to the intervention package itself, including stakeholder's characteristics) that could influence package implementation. BODY.METHODS.SETTING AND POPULATION: Nicaragua is the second poorest country in America. First-line government health services (FLGHS) consist of health posts and health centers. They are managed by a Ministry of Health (MOH) team at the municipal level, under the regional level MOH authorities. TB care is delivered through a MOH program, strongly organized at the FLGHS level. It ensures the availability and quality of free sputum examination in government health laboratories and free TB treatment provided exclusively in FLGHS. In each health center, a nurse is usually responsible for the TB patients care. This intervention research project involved different stakeholders: (1) it was conceived by the authors of this paper, (2) adapted for its operational design by national TB program coordinator, and MOH team at the municipal and regional level; (3) implemented by MOH teams at municipal level for the operational component and by the authors of this paper for the research component; and (4) it benefited from financial and other supports, for the research component, from Damian foundation project in Nicaragua and Global Fund administrating institution in Nicaragua (NICASALUD). This research project included 9 municipalities of rural Nicaragua, selected by the MOH national TB program coordinator because of their need to be strengthened for the local management of the care to TB patients. Strengthening local management of the TB program was initially mainly foreseen through operational analysis exercise, explained here bellow. It targeted new AFB positive TB patients diagnosed between March 2004 and July 2005. Table 1 presents the population, health services and TB diagnosis data for 2003. Table 1 general characteristics of the municipalities (2003 values) 2003 values Population (proportion living at less than 5 kms from a health facility) N° consultation/inhabitant and by year No° of new AFB+ TB cases (N° TB cases diagnosed per 100000 inhabitants) Number of abandons (proportion of patients starting their treatment) Siuna 76099 (90%) 0.85 41 (81) 9 (10.7%) Bluefield 49331 (23%) 0.8 18 (36) 1 (5%) Waslala 45014 (80%) 2.1 34 (89) 13 (14.3%) San Jose de Bocay 33635 (90%) 2.3 17 (50) 5 (28%) La Dalia 48553 (53,5%) 1.42 17 (35) 1 (6%) Nueva Guinea 117097 (80,3%) 0,42 14 (13) 0 (0%) El Rama 50 038 (57%) 0.6 22 (43) 2 (10%) Juigalpa 61234 (80.9%) 1.6 19 (31) 3 (16%) Wiwili 49443 (-) 1.68 25 (50) 1 (4%) BODY.METHODS.THE PROCESS BEFORE IMPLEMENTING THE INTERVENTION: The process before implementing the intervention covered 5 out of 9 municipalities: Siuna, Bluefield, Waslala, San Jose de Bocay and Nueva Guinea. It was planned in 3 phases: an operational analysis, a qualitative exploration with the design of an intervention package thereafter introduced in the municipalities. During the first phase, operational analysis was performed. This is a method for identifying problems in the care process for TB patients. It is done through the use of a simple step-by-step model, the operational model. It is based on the passive detection of AFB-positive cases and the main steps that a patient must go through from the first symptoms and up to TB recovery [16]. It makes a distinction between technical (i.e., biomedical) problems, operational (i.e., organization of health services) and social (i.e., behaviour of specific actors) problems. In Nicaragua, the expected benefit from the operational analysis was to improve the abilities of local health personnel to analyze TB patients' care and, thereby, improve the relevance of interventions on TB patients' care. The operational model was meant to be used as part of a "problem-solving" or "managerial" process. During the second phase, a qualitative exploration of the social stigma meanings and determinants was used as an input for the design of an intervention package in order to reduce internalized stigma. During the third phase, feedback of the exploration, training on self-esteem and tailoring the intervention package through negotiation with stakeholders were planned. BODY.METHODS.THE INTERVENTIONS PACKAGE TO ACT ON TB STIGMA: We initially designed a set of three types of interventions for internalized stigma reduction. Firstly, we wanted to strengthen TB patients through TB clubs taking the form of self-help groups. Secondly, we wanted health personnel to better know TB patients and their realities through performing patient centered home visits and case discussion centered on the problems experienced by TB patients. The objective of the patient centered home visit was to know better the social network of the patient, identify strength and weaknesses of the network, and plan activities to support the patient. Health professionals were expected to listen to the relatives and neighbours of the patients, identify their feelings towards the patient (their fear, disgust, support, etc), and plan with the social network activities to support the patient during his treatment. A specific guide was conceived to assist health care providers. Thirdly, we planned a revised DOT provision through the involvement of a supporter at the first consultation or through reviewing the patient pathway by decreasing unnecessary isolation and decentralizing care where possible. Of the three types of initially designed interventions, at least TB clubs and patient centered home visits were implemented in the intervention municipalities. All these interventions were conceived in their implementation with the full participation of MOH authorities. Furthermore, TB clubs had been included in the 2005 Global Fund grant for Nicaragua although it was initially piloted in the intervention municipios by this research project. BODY.METHODS.DEFINITION AND CHARACTERIZATION OF THE CONTROL AND INTERVENTION GROUPS: We allocated patients in two groups: intervention and control. TB patients were included in the intervention group if diagnosed in a municipality organizing at least the two most consistent interventions of the package: TB clubs and home visits. Others were allocated to control group. We compared both groups in relation to basic socio-economic indicators: age, gender, declared income, literacy, and distance from the TB treatment center. Additionally, we compared their self-esteem, using the Rosenberg scale [17], at the start of the treatment. Indeed, it is recognized that self-esteem is a strong determinant of internalized stigma. BODY.METHODS.IMPLEMENTATION PROCESS MONITORING: Researchers performed six monitoring visits between February 2004 and December 2005 in each of the five municipalities where interventions package was introduced. During each visit, they documented the progress in implementing interventions and possible municipal health team's initiatives. They performed care process observations and they systematically interviewed various local stakeholders (patients, nurses in charge of TB patients, members of the municipal health team). BODY.METHODS.OUTCOME MEASUREMENT: Outcomes of this study were TB treatment outcome and TB internalized social stigma. TB treatment outcome classical indicators were computed with data from the municipal TB register crosschecked with the TB patient cards. The internalized social stigma was measured through a scale that has been largely inspired by the scale developed by Boyd Ritsher in the context of mental illnesses [18,19]. It includes 10 statements (see Table 2), has been pre-tested [20] and has a relatively good internal consistency (Cronbach alpha = 0,7). It measures four sub-dimensions: alienation, perceived discrimination, stereotypes endorsement and social withdrawal. The scale was applied to the same TB patients at various stages of their treatment (after 15 days, after 2 months), in order to measure the change in their perceptions as a consequence of the care process and exposure to the intervention package. Informed consent was sought before applying the scale. Confidentiality was stressed throughout the research process. Table 2 Proportion of TB patients agreeing with the 10 statements of the stigma scale in control and intervention groups, after 15 days and 2 months of treatment Statement Time since starting TB treatment Proportion of patients that agrees fully with the statement Control n = 146 19 missing values Intervention n = 122 14 missing values 1. I don't have anybody to help me solving economical, familial or sentimental problems After 15 days 40.00% 35.50% After 2 months 46.20% 17.60% 2. People that are not "phtisics" with a lot of cough can't understand my problems After 15 days 49.60% 55.10% After 2 months 45.30% 38.00% 3. I feel ashamed to have tuberculosis After 15 days 50.40% 46.20% After 2 months 45.30% 17.60% 4. We, TB patients, are contagious After 15 days 55.70% 32.70% After 2 months 51.30% 20.40% 5. People do not consider me and don't listen to me because I have tuberculosis After 15 days 44.30% 35.80% After 2 months 42.70% 19.40% 6. People treat me with pity because I have tuberculosis After 15 days 52.20% 37.40% After 2 months 47.00% 16.70% 7. People don't get close to me because I am phtisic, with my lungs damaged and a lot of cough. After 15 days 43.50% 32.70% After 2 months 42.70% 15.70% 8. I am not looking for new relations because I am phtisic, with my lungs damaged and a lot of cough After 15 days 45.20% 29.90% After 2 months 37.60% 13.90% 9. I don't socialize with others because I am afraid to give my point of view and I want to avoid that my family or friends feel ashamed or have problems. After 15 days 44.30% 33.60% After 2 months 39.30% 17.60% 10. I stay away from the people that are not phthisic, with my lungs damaged, to avoid being rejected. After 15 days 51.30% 36.40% After 2 months 47.00% 22.20% Univariate analysis of TB treatment outcome and internalized TB stigma variable was done using the EPIINFO 2000 program. BODY.METHODS.ETHICAL REVIEW: This proposal didn't pass through a formal ethical review. However, it was selected through an independent peer review process to receive financial support from Damian Foundation. It was further on submitted to Nicaragua Ministry of Health authorities for approval. BODY.RESULTS: A total of 268 AFB positive TB patients were enrolled in the study over the 9 municipalities. We report the process that leads or not to the implementation of intervention, the assignment of TB patients in the control or intervention group, the TB treatment results and the internalized stigma changes. BODY.RESULTS.THE PROCESS BEFORE IMPLEMENTING INTERVENTIONS: The three phases of the process before implementing the intervention were coached by researchers in Siuna, Bluefield, Waslala, San Jose de Bocay and Nueva Guinea. Upon request of the nurse in charge of TB patients in La Dalia, this municipality was also coached. The municipalities of El Rama, Juigalpa and Wiwili were just visited for data collection. BODY.RESULTS.OPERATIONAL ANALYSIS: The operational analysis was performed before January 2004 in Bluefield, Siuna, Waslala, San Jose de Bocay and Nueva Guinea. The process and results of this exercise were unequal. In Siuna, decentralization from the health center to the health post was quickly successful. For example, some village health workers transferred sputum samples to the health post and patients from a particular area received their treatment directly from a cured TB patient. Some health posts also provided treatment to TB patients. In Bluefield, the decentralization from health center to health post was difficult at first due to a lack of personnel and resources. However, as of July 2004, some patients received their treatment at the health post or even within their families. In Waslala, the decentralization process was slow but some patients had their treatment supervised by their families after July 2004. BODY.RESULTS.CONTEXT EXPLORATION AND INTERVENTION DESIGN: External researchers (i.e. the authors of this paper) performed a qualitative exploration of the meanings and determinants of social stigma in Siuna, Bluefield, Waslala, San Jose de Bocay and Nueva Guinea at the end of 2003 [21]. This consisted in in-depth interviews and focus groups with key stakeholders involved in the TB patients care. The patient centered package design was inspired by this study results. BODY.RESULTS.INTRODUCTION OF THE INTERVENTION PACKAGE TO ACT ON TB STIGMA: Workshops were organized in each of the 5 municipality during the first semester 2004. Firstly, training on self-esteem was organized by a psychologist in each of the municipality. Secondly, a draft of the interventions package was discussed in each municipality. Each local health team chose amongst the initial package, the interventions to be implemented. In Bluefield, the whole municipal health team and the TB program regional coordinator participated to the workshop together with nurses from the health center and the health post. They decided to focus on TB patients' clubs and home visits. By the end of the first semester 2004, a meeting was organized by the director of the health center with the whole staff in order to discuss perception and behavior towards TB patients. The regional authorities also organized the broadcast of TB related messages on the local radio. In Siuna, members of the municipal health team, nurses from the health center and the health posts attended the workshop. Initially, the municipal team decided to implement only home visits but very quickly started also TB patients' clubs. In Waslala, the whole nursing staff from the municipality participated in the initial workshop but the municipal health director was partly present. The nurse responsible for the care to TB patients in La Dalia, although not supposed to be involved in the study, participated also. The Waslala municipal health team members committed themselves to start home visits and to improve the first consultation process. In July 2004, a follow-up visit was done by researchers and several problems were encountered. A newly appointed medical doctor was performing the first consultation for TB patients. He did not know about the proposed methodology. Concerning home visits, the nurse in charge of TB patients at the municipal level complained of the lack of resources. He performed only two of them, and did not follow the suggested methodology. In San Jose de Bocay, no authority was present at the initial workshop with the exception of the TB program regional coordinator. The workshop was unprepared and attended only by part of the staff. The decision was taken to decentralize treatment wherever possible. In a second workshop organized in April 2004, few people, different from the first workshop, were present. Many others were involved in the mass vaccination campaign. The follow-up of the February workshop showed little changes and no decentralization. In July 2004, a monitoring visit was done. The municipal director, the nurse and the doctor in charge of TB patients were newly appointed. As a consequence, little changes were observed. In Nueva Guinea, the workshop mainly discussed the involvement of village health workers in TB suspected cases' referrals. In April 2004, a second workshop was organized. There was confusion as community health workers rather than health staff members were invited to the workshop. It was however decided to organize TB cases discussion and to focus on the reorganization of the TB patient's pathway. During 2004 and 2005, only one case discussion was organized during a monitoring visit. It was very much centered on TB control and not much on the difficulties met by TB patients. The revision of the patient's pathway was not really done because of changes in health staff. In May 2005, TB clubs that had been planned were not organized due to the lack of funds. BODY.RESULTS.IMPLEMENTATION OF THE INTERVENTION PACKAGE TO ACT ON TB STIGMA: Three groups of municipalities were identified according to the period home visits and TB clubs were effectively implemented. In the first group, composed of Siuna and Bluefield, it was effectively implemented as of July 2004. In the second group, composed of Waslala, La Dalia and San Jose de Bocay, it was implemented as of January 2005. Finally, home visits and TB clubs were never effectively implemented in the third group composed of Nueva Guinea, Juigalpa, El Rama and Wiwili. In Bluefield, TB clubs in their new form that is aiming at a maximum of TB patients interactions started operating early 2004. The director and the doctor in charge of epidemiology at the health center were initially present. Home visits were also performed by two health staff members. The regional level proposed a special format for home visits providing information on the TB patients' psychosocial conditions in addition to the usual contact tracing data. In January 2005, the TB clubs were chaired by TB patients themselves. They also decided to appoint an executive board. In Siuna, home visits were done mainly close by the health center. Quickly after, TB clubs started first in the health center upon the initiative of the nurse in charge of TB patients and the doctor responsible of epidemiology. Later on, a TB club executive board constituted of TB patients was set up. In other areas, two cured TB patients took the initiative of starting a club with 5–6 patients. They actively supported the work of the ministry of health in those areas. Another cured TB patient talked on the radio and on TV about tuberculosis and the ways to cure it. Finally, a local NGO decided to support TB clubs meetings. In Waslala, TB club meetings started to be regularly organized in 2005 with around 20 people present each time. Home visits were done, using the guideline developed in Bluefield. Not all TB patients were systematically visited because some were living too far away. In La Dalia, the nurse in charge of TB patients also started TB patients' club meetings and home visits with the support of the responsible at the regional level. In San Jose de Bocay, a new staff was appointed at the end of 2004. As a consequence, home visits, and TB clubs started. Additionally, the staff of this municipality took the initiative of organizing special counseling for the patients' families, aiming at reducing isolation and increasing support. Finally the decentralization of the diagnosis and treatment at the health post level started for some patients. Some health posts started sputum smear slide preparations for transfer to the health center laboratory. BODY.RESULTS.CHARACTERISTICS OF TB PATIENTS INTERVENTION AND CONTROL GROUPS: Hundred and twenty two AFB positive TB patients were diagnosed in municipalities implementing TB clubs and home visits at that time. They were assigned to the intervention group. The other 146 TB patients included in the study were assigned to the control group (Table 3). Table 3 Number of new AFB+ cases diagnosed by period, by group of municipalities , and by control or intervention group Municipalities TB patients Dates of diagnosis Group 1 (TB Clubs and home visits started as from 01/07/2004) Group 2 (TB Clubs and home visits started as from 01/01/2005) Group 3 (No intervention) Didn't benefit from TB Clubs and home visits (control group) Benefited from TB Clubs and home visits (intervention group) From 01/03/2004 till 01/07/2004 19 23 19 61 (= 19+23+19) 0 From 01/07/2004 till 31/12/2004 40 29 24 53 (= 29+24) 40 From 31/12/2004 till 30/06/2005 35 47 32 32 82 (= 35+47) Total 94 99 75 146 122 There was no statistically significant difference between the control and intervention groups for socioeconomic variables (Table 4). The mean age in both groups was around 35 years, there were more males than females, and distance to the treatment center averaged 17 kms. Both groups can also be categorized as poor people, with low literacy status and relatively low self-esteem as measured with the Rosenberg scale. Table 4 Characteristics of intervention group compared with control group Control n = 146 19 missing values Intervention n = 122 14 missing values Stat. sign. (p < 0.05) Average age 36.2 years 34.4 years NS Ratio male/female 1.28 1.28 NS Average distance between the TB treatment center and patient home 17.7 Km 17.03 Km NS Proportion of illiterates 44.9% 40.9% NS Proportion without declared income 54.1% 50% NS Rosenberg self-esteem scale (between 10 and 40) 22.2 22.6 NS BODY.RESULTS.TUBERCULOSIS TREATMENT OUTCOMES: The results of the treatment do not show statistically significant difference between the intervention and control groups. Both groups can be considered as having a good outcome: 90% of the TB patients in the control group and 93% of the ones in the intervention group were either cured or completed their treatment; only 5/146 (3%) of the patients in the control group and 4/122 (2%) of the patients in the intervention group abandoned their treatment. BODY.RESULTS.INTERNALIZED STIGMA SCALE: Details of the scale are presented on Table 2. We report the proportion of TB patients completely agreeing with the 10 statements after 15 days and 2 months of treatment. In the intervention group, there is an important diminution in the proportion of TB patients agreeing with the 10 statements between 15 days and 2 months of treatment. In the control group, there is no or only slight diminution. We computed the stigma scale score, attributing 5 points when a TB patient completely agrees with a statement and 1 point when he completely disagrees with it. The total of the scale score amounts therefore between 10 and 50 points. We compared the mean rate for the control group with the mean rate for the intervention group. For both groups, we calculated two internalized stigma scores, one for 15 days of treatment and another one for 2 months of treatment. After 15 days, scores were equivalent in both groups. However, after 2 months, difference between scores was statistically significant, revealing a decreased internalized stigma in the intervention group and not in the control group (Table 5). Table 5 Scale mean score after 15 days and 2 months of treatment in control and intervention group Scale mean value Control n = 146 19 missing values Intervention n = 122 14 missing values Stat sign After 15 days 34,6 31,7 p = 0,08 After 2 months 33,1 27,4 p = 0,001 Difference between 2 months and 15 days 1,5 4,3 p = 0,03 BODY.DISCUSSION: Most of the published studies identified the ignorance about tuberculosis, false communities' beliefs and fears as the reasons for tuberculosis social stigma. Suggested solutions are then informing and educating the community [22-24]. We took in this study a different approach. The intervention package tested was centered on the relation between health care providers and the TB patients. The central ingredients of a successful intervention package were the home visits and TB clubs. This was the result of a whole preparation process (see Figure 1). The hypothesis was that by participating in the introduction and the implementation of these core interventions, health personnel would progressively change their attitude towards TB patients, and that TB patients would feel in a stronger position to interact with other stakeholders. Figure 1The process before implementing interventions. Our study results show that TB patients' internalized stigma is significantly reduced, when a package of interventions including TB clubs and patient centered home visits is successfully implemented. However, the treatment outcome indicators which were already far above WHO targets in both the intervention and control groups didn't change significantly. Reducing only the internalized stigma within a context of good case holding management is important. The need to look at the TB patient's wellbeing, in addition to the TB epidemics control, has indeed been acknowledged in recent international reflections and policies on TB control [25-28]. We should consider this study as an initial step and conclusion concerning outcome measure would need to be confirmed in bigger scale study. Indeed, outcome of this type of intervention should have been probably better analyzed by grouping TB patients by municipios. This would give stronger evidences on the relationship between a given context, stakeholders, intervention package and the outcome. However, given the small scale and the low detection rate of TB patients in the municipios of study, we had to analyze all municipios together. Additionally, this study has been performed in a specific context, i.e. municipios chosen by the national TB coordinator among the least performing in remote area. Conclusion of this study, and mainly external validity, would probably be stronger if similar process would be performed in multiple contexts. Finally, risk of information bias (i.e. patients being aware of the importance of a change between their initial and their second response in the filling of the stigma scale), was prevented in two ways. Firstly, patients were not aware of being part of the intervention or the control group. Secondly, the internalized stigma scale was "self applied" by TB patients with eventually the assistance of a patient's relative, outside of the health care facility and the influence of health care professionals. These results should not be analyzed mechanically, by trying to link a standardized package of intervention to a given outcome. Acting on TB patients' internalized stigma is a complex issue. Successful interventions depend from the context and the stakeholders and quantitative measures only capture part of the TB patients' feelings. Firstly, interventions effect cannot be separated from the context. It would be indeed naïve to believe that any type of TB clubs and home visit independently from the context and the stakeholders would improve internalized stigma in TB patients. Indeed in our study, the implementation of the patient centered package has been highly dependent on the context and more specifically on the stakeholders at the local level. Two central stakeholders were critical for a successful intervention package: the TB patients and the nurse in charge of the TB patients. In the first group of municipalities (Siuna and Bluefield), the TB patients played an active role in the TB club activities and other support activities for TB patients, thanks to some individualities' leadership. In the same municipalities, and also in La Dalia, nurses caring for TB patients where highly motivated, knew the community members very well, and were used to working with TB patients, as they have a long lasting experience in such position. In those cases, nurses were supported by authorities: the nurse of Siuna benefited from a dynamic municipal health management team stable during the time of the research; the nurse of Bluefield benefited from the proximity of an enthusiastic TB program responsible at the regional level. Finally, in both Bluefield and Siuna, the work of the nurse responsible of the TB patients, was supported by local NGO's. These findings are congruent with recent policies and experiences emphasizing the pivotal role of frontline health workers in TB care [29-31]. Secondly, quantitative measures only capture part of the TB patients' feelings. In our study, we used a 10 items scale to measure internalized stigma. The presentation of scales to measure TB social stigma in a context of low HIV prevalence has not yet been published in peer reviewed journals. We identified in the "grey" literature five studies that quantified the TB social stigma: Two studies used only one binomial variable [32,33]; three other studies specified sub-dimensions of TB stigma and two used specific scales [10,11]. The scale we used to measure changes in internalized stigma has been applied to TB patients only in our study. Using it in other studies might strengthen its validity. However, as explained in the methodology, it was carefully designed and may be considered as valid for the Nicaraguan context. Further to its local benefits, this project has been able to influence national policies about the care of TB patients in government health services. The final result of this has been the production of a "manual de trabajo sobre estigma de la tuberculosis a nivel municipal en Nicaragua." Part of it has already been used in expanding PATB clubs and home visits in all areas of Nicaragua. It has been discussed and validated by a new commission, the "Commission Nacional the Apoyo a Personas Afectadas por Tuberculosis" (CONAPAT) created during, and partly as a consequence of this research project. It involves the main national stakeholders in TB control, i.e. the National TB control program of the Nicaraguan ministry of health, the administrator of the Global Fund (the NGO Nicasalud), the Damian Foundation, a Belgian NGO and the Centro de Investigaciones y Estudios de la Salud (CIES), a public health school. By its role of coaching, the research team of the CIES has been in a position to support the local implementation of the intervention package, but also to collect information about difficulties and the success in the local processes. This was particularly useful for discussions in the CONAPAT. Indeed, as a consequence of the intertwined process of information collection during coaching activities and discussion of it during CONAPAT meetings, scaling up of tools that were developed in this project was made possible. For example, early 2006, more than 50 municipios created PATB clubs with tools developed by this research. BODY.CONCLUSION: Increased attention is given to the TB social stigma and its consequences on TB patients suffering. This study provides initial evidences that it is possible to act on TB patients' internalized stigma, in contexts where at least patient centered home visits and TB clubs are successfully implemented. Outcomes are not dependant of a standardized set of intervention but also of the context, and mainly the capacity of frontline nurse and TB patient to establish a fruitful dialogue. Our findings are relevant particularly, in context where TB control is already effective. Through this paper, we hope to have contributed to promoting an expanding field of research which is highly needed in order to develop valid instruments to measure the stigma, to understand the optimal mix of contextual factors, stakeholders' interaction and interventions' package. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: JM conceived and designed the study; supervised the data collection; analyzed and interpreted the data and wrote the paper. AS conceived and designed the study; supervised the data collection; analyzed and interpreted the data and participated to the writing of the paper. GM conceived and designed the study; supervised the data collection; analyzed and interpreted the data and participated to the writing of the paper. PM conceived and designed the study; supervised the data collection; analyzed and interpreted the data and participated to the writing of the paper. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here:

TITLE: Habitual intake of flavonoid subclasses and risk of colorectal cancer in 2 large prospective cohorts ABSTRACT: Background: Flavonoids inhibit the growth of colon cancer cells in vitro. In a secondary analysis of a randomized controlled trial, the Polyp Prevention Trial, a higher intake of one subclass, flavonols, was statistically significantly associated with a reduced risk of recurrent advanced adenoma. Most previous prospective studies on colorectal cancer evaluated only a limited number of flavonoid subclasses and intake ranges, yielding inconsistent results. Objective: In this study, we examined whether higher habitual dietary intakes of flavonoid subclasses (flavonols, flavones, flavanones, flavan-3-ols, and anthocyanins) were associated with a lower risk of colorectal cancer. Design: Using data from validated food-frequency questionnaires administered every 4 y and an updated flavonoid food composition database, we calculated flavonoid intakes for 42,478 male participants from the Health Professionals Follow-Up Study and for 76,364 female participants from the Nurses' Health Study. Results: During up to 26 y of follow-up, 2519 colorectal cancer cases (1061 in men, 1458 in women) were documented. Intakes of flavonoid subclasses were not associated with risk of colorectal cancer in either cohort. Pooled multivariable adjusted RRs (95% CIs) comparing the highest with the lowest quintiles were 1.04 (0.91, 1.18) for flavonols, 1.01 (0.89, 1.15) for flavones, 0.96 (0.84, 1.10) for flavanones, 1.07 (0.95, 1.21) for flavan-3-ols, and 0.98 (0.81, 1.19) for anthocyanins (all P values for heterogeneity by sex >0.19). In subsite analyses, flavonoid intake was also not associated with colon or rectal cancer risk. Conclusion: Our findings do not support the hypothesis that a higher habitual intake of any flavonoid subclass decreases the risk of colorectal cancer. BODY: See corresponding editorial on page 3. BODY.INTRODUCTION: Flavonoids are a group of bioactive polyphenolic compounds naturally occurring in plant-based foods such as fruits, vegetables, grains and herbs, and drinks such as tea, wine, and juices. Based on their structure, flavonoids can be subclassified as flavonols, flavones, flavanones, catechins (flavan-3-ols or flavanols), isoflavones, anthocyanins, and oligomeric or polymeric flavonoids. Experimental studies have shown cancer-protective effects of flavonoids through several biological mechanisms, including antioxidative and anti-inflammatory properties, induction of apoptosis, and inhibition of proliferation and angiogenesis (1). Specifically, flavonoids have been shown to inhibit growth of human colon cancer cell lines in vitro and restrict colorectal carcinogenesis in animal models (2, 3). A proportion of dietary flavonoids is absorbed in the small intestine and reaches plasma concentrations that could have biological effects. In addition, substantial amounts reach the colon, where they are further metabolized into metabolites that may also mediate some biological activity (4). Current observational studies investigating the association between flavonoid intake and risk of colorectal cancer (CRC)12 have been inconsistent. Although several case-control studies suggest an inverse association between high flavonoid intake, particularly flavonols and anthocyanins, and CRC (5–9), prospective cohort studies have generally failed to detect an association (10–14). However, an inverse association between catechin intake and risk of rectal cancer has been suggested by a cohort study in women (15). In addition, in a secondary analysis of a US-based randomized controlled trial, the Polyp Prevention Trial, higher intake of flavonols was statistically significantly associated with a 76% reduced risk (OR: 0.24; 95% CI: 0.11, 0.53) of advanced recurrent adenoma, a precursor for CRC (16). In the only study investigating biomarkers of flavonoid intake in relation to CRC incidence we are aware of, urinary catechins at baseline were inversely related to later risk of colon cancer (17). Dietary flavonols and flavones and CRC risk have been prospectively investigated in the Health Professionals Follow-Up Study (HPFS) and Nurses' Health Study (NHS) previously, and no association was observed (13). A drawback of most prospective studies, including the previous analysis of NHS and HPFS data, is that only a small number of flavonoid subclasses (e.g., flavonols and flavones) were evaluated and intake ranges were limited (10–13, 15). In the present study, we have updated our flavonoid food composition database based on an updated and expanded USDA database (18) to examine the association between habitual intake of various flavonoid subclasses (i.e., flavonols, flavones, flavanones, flavan-3-ols, and anthocyanins) and CRC risk in the NHS and HPFS, with longer follow-up and a larger number of incident cases compared with the previous analysis (13), thus enabling us to investigate long-term habitual flavonoid intake as well as flavonoid intake with different latencies to better understand whether timing of intake makes a difference. BODY.METHODS.STUDY POPULATION: Details of the HPFS and NHS prospective cohorts have been published elsewhere (19, 20). In brief, the HPFS was initiated in 1986 and included 51,529 US male health professionals (dentists, optometrists, osteopaths, podiatrists, pharmacists, veterinarians) who were 40–75 y old at baseline. The NHS began in 1976, when 121,701 US-registered female nurses aged 30–55 y provided information on their medical history and lifestyle. In both cohorts, questionnaires were administered biennially to update lifestyle factors and elicit new medical diagnoses. Follow-up rates have been >90% in each 2-y cycle, and the cumulative follow-up rate (i.e., the percentage of potentially collected person-years) was 94% in the HPFS and 93% in the NHS. Dietary intake data were collected every 4 y in both the NHS and HPFS via a mailed food-frequency questionnaire (FFQ). In the present study, we used 1984 as baseline for the NHS and 1986 for the HPFS, the respective years in which dietary intakes were assessed by using a validated 116- to 131-item FFQ. Among the participants who returned the baseline FFQs, we excluded those who had a history of cancer (except for nonmelanoma skin cancer) or ulcerative colitis, reported very high or very low energy intakes (<600 or >3500 kcal/d for women, <800 or >4200 kcal/d for men), left >70 food items blank on the FFQs, or had missing information on flavonoid intake. After these exclusions, 76,364 women and 42,478 men were included in the analysis. The study protocol was approved by the Institutional Review Board at Brigham and Women's Hospital and the Harvard School of Public Health. BODY.METHODS.ASSESSMENT OF FLAVONOID INTAKE: Validated FFQs (20, 21) were administered in 1984, 1986, and every 4 y thereafter in the NHS. In HPFS, similar FFQs were administered in 1986 and every 4 y thereafter. In the semiquantitative FFQs, food and beverage items are specified by commonly used units or portion sizes. In each FFQ, participants were asked to report how often, on average, they consumed a serving of each food item during the previous year with 9 possible response categories ranging from "never or less than once per month" to "6 or more times per day." Individual nutrient intakes were calculated from FFQ data by multiplying the nutrient content per serving of each food by the indicated frequency of consumption (servings of that food per day) and summing over all foods. A database for the calculation of habitual intake of different flavonoid subclasses was developed as has been described previously (18). For the present analysis, 5 main flavonoid subclasses (expressed as aglycones) commonly consumed in the US diet were considered: flavonols (quercetin, kaempferol, myricetin, isorhamnetin), flavones (apigenin, luteolin), flavanones (eriodictyol, hesperetin, naringenin), flavan-3-ol monomers (catechin, epicatechin, epigallocatechin, epicatechin-3-gallate, epigallocatechin-3-gallate), and anthocyanins (cyanidin, delphinidin, malvidin, pelargonidin, petunidin, peonidin). Total flavonoid intakes were derived by the sum of the components flavonols, flavones, flavanones, flavan-3-ols, anthocyanins, and polymeric/oligomeric flavonoids (including proanthocyanidins, theaflavins, and thearubigins). As has been described previously, the main food sources of flavonols are tea and onions, those of flavones and flavanones are oranges, those of flavan-3ols are tea, those of anthocyanins are blueberries, and those of oligomeric and polymeric flavonoids are tea, apples, and strawberries (18, 22). Nutrient intakes were energy adjusted by the residual method (23). To represent long-term dietary intake, we calculated cumulative average intakes for a given questionnaire cycle by averaging the intake for the current and preceding FFQs (24). Because FFQs administered before 1990 contained fewer questions on some flavonoid-rich fruits and vegetables (e.g., onions were not listed in questionnaires before 1990), in sensitivity analyses, we used 1990 as baseline for both the HPFS and NHS. Reproducibility and relative validity of the FFQs have been investigated previously (25, 26). For the major food sources of flavonoids, correlations between dietary records and FFQs in the HPFS and NHS were as follows: 0.77–0.93 for tea, 0.83–0.90 for red wine, 0.74–0.76 for oranges, and 0.70–0.80 for apples or pears (25, 26). In recent analyses in the HPFS and NHS, flavonoid subclasses have been associated with lower risk of several chronic diseases, including hypertension (18), ovarian cancer (27), and Parkinson disease (28), giving further qualitative support for the ability of the FFQ to reasonably estimate dietary intake of flavonoid subclasses. BODY.METHODS.ASSESSMENT OF COVARIABLES: Information on lifestyle and other health-related factors, such as body weight, cigarette smoking, physical activity, participation in gastrointestinal endoscopic examinations, use of aspirin and other nonsteroidal anti-inflammatory drugs, menopausal status, and postmenopausal hormone use (in women only), was collected and updated in the biennial follow-up questionnaires. Family history of CRC in a first-degree relative was assessed in the 1982, 1988, 1992, 1996, and 2000 questionnaires in the NHS and in the 1986, 1990, 1992, and 1996 questionnaires in the HPFS. BODY.METHODS.ASCERTAINMENT OF CRC CASES: In both the NHS and HPFS, participants who reported a diagnosis of CRC on one of the biennial follow-up questionnaires were asked for permission to obtain their medical records and pathologic reports. Given the colorectal continuum model (29), we included colon cancers and rectal cancers. A study physician, blinded to exposure information, reviewed the records to confirm CRC diagnosis and to extract information on anatomic location, stage, and histologic type of the cancer. The National Death Index was queried to discover deaths and to ascertain any diagnosis of CRC that contributed to death or was a secondary diagnosis. In the case participants who had died of CRC were not captured during our regular disease follow-up, we contacted next of kin to ask for permission to obtain and review medical records to confirm a diagnosis of CRC. Deaths of all cohort members were identified through the National Death Index, the state vital statistics record, and death certificates that were mailed by next of kin of deceased participants, resulting in >98% complete ascertainment in mortality surveillance (30, 31). CRC was defined based on the International Statistical Classification of Diseases, Injury and Causes of Death (Ninth Revision) as a combination of tumors of the colon (C18.0–C18.7), tumors that were overlapping or unspecified (C18.8–C18.9), and tumors of the rectum (C19–C20). All cases included in the present analysis were confirmed by medical records. Between return of the baseline questionnaires and 2010, a total of 1458 CRC cases (1151 colon, 307 rectum) were documented in the NHS and 1061 (831 colon, 230 rectum) in the HPFS. BODY.METHODS.STATISTICAL ANALYSIS: Each participant contributed person-time of follow-up from the date of return of the baseline questionnaire to the date of CRC diagnosis, death, or end of analysis follow-up (31 May 2010 for the NHS, 31 January 2010 for the HPFS), whichever came first. We used Cox proportional hazards regression models calculating HRs (as an estimate of RR) and 95% CIs to quantify the associations of habitual intake of different flavonoid subclasses with overall and subsite-specific risk of CRC within each cohort. To control as finely as possible for confounding by age and calendar time, we stratified Cox models jointly by age in months at the start of follow-up and calendar year of the current questionnaire cycle. We tested the proportional hazards assumption by using likelihood ratio tests comparing models with and without interaction terms between flavonoid subclass variables and age or follow-up time and did not observe any violation. In multivariable models, on the basis of our knowledge from previous investigations within the HPFS and NHS (32–34), we simultaneously adjusted for CRC risk factors that may confound the association between flavonoid subclasses and CRC risk. These factors included pack-years of smoking before age 30 y, history of CRC in a parent or sibling, history of lower bowel endoscopy, regular aspirin use, menopausal hormone use (in women only), BMI, physical activity, alcohol consumption, total calorie intake, vitamin D intake, calcium intake, and consumption of red meat and processed meat. To assess whether potential collinearity between dietary variables may have affected our results, we also constructed a "basic model" (35), which included all nondietary covariates, alcohol, and total energy intake and then added each dietary variable separately to the model. To represent long-term dietary and lifestyle patterns, we used cumulative averages for BMI, physical activity, regular aspirin/nonsteroidal anti-inflammatory drug use, and dietary intakes throughout follow-up to control for confounding in regression models. As a sensitivity analysis, we also adjusted for the diet quality scores from the Alternate Healthy Eating Index–2010 (36) and Dietary Approaches to Stop Hypertension (37). Our primary exposure of interest was cumulative average flavonoid intake between baseline and follow-up period representing long-term habitual flavonoid intake and minimizing the impact of random measurement errors in the dietary assessment by FFQs. In sensitivity analyses, we evaluated the possible modification by time in the association between intake of flavonoid subclasses and CRC incidence. For this purpose, Cox models based on dietary data collected at different time points were constructed: 1) flavonoid intake at the beginning of follow-up, 2) recent (simple updated) flavonoid intake reported on the most recent FFQ before each follow-up interval (latency 0–4 y), and 3) flavonoid intake reported at different latencies (i.e., 4–8 y, 8–12 y, 12–16 y) before diagnosis as described previously (38). For example, for latency of 4–8 y, flavonoid intake assessed on the 1986 FFQ was related to CRC cases diagnosed between 1990 and 1994, flavonoid intake assessed on the 1990 FFQ was related to CRC cases diagnosed between 1994 and 1998, flavonoid intake assessed on the 1998 FFQ was related to CRC cases diagnosed between 2002 and 2006, and so forth. Because the FFQs were administered every 4 y, the simple updated model 2 can be considered a 0- to 4-y latency analysis. Intakes of flavonoid subclasses were categorized into quintiles based on the distribution in the study population. Tests for linear trend over quintiles were performed by assigning the median value to each category, entering these values as a continuous variable to the model, and evaluating its statistical significance by using the Wald test. All analyses were conducted in the NHS and HPFS separately, and risk estimates were pooled by using random-effects models (39) if there was no indication of heterogeneity by sex. We tested whether the association between flavonoid subclass intake and risk of CRC was modified by age (<65 y vs. ≥65 y), smoking status (ever vs. never), alcohol intake (high vs. low, based on sex-specific median value), or physical activity (high vs. low, based on sex-specific median value) by adding cross-product terms to the multivariable models and employing Wald tests. All analyses were conducted by using SAS software (version 9.3; SAS Institute Inc.). A 2-sided P value of <0.05 was considered statistically significant. BODY.RESULTS: Participants' characteristics, averaged according to proportion of person-time in each quintile of total flavonoid intake, are shown in Table 1 (equivalent tables for intake of flavonoid subclasses are available as Supplemental Tables 1–5). In both men and women, physical activity was higher in participants with high flavonoid intake than in participants in the lower quintiles of flavonoid intake. Dietary intakes of red meat and processed meat were lower in the upper quintiles of flavonoid intake than in the lower quintiles. In the HPFS, during 419,133 y of follow-up, 1061 cases of CRC occurred (2.53 cases per 1000 person-years). In the NHS, 1458 CRC cases were registered during 927,994 person-years of follow-up (1.57 cases per 1000 person-years). TABLE 1 Age-standardized characteristics of person-years according to quintiles of total flavonoid intake in the NHS and HPFS 1 HPFS NHS Q1 Q3 Q5 Q1 Q3 Q5 Age at baseline, y 51.4 ± 9.3 2 52.4 ± 9.4 52.9 ± 9.3 49.3 ± 7.1 50.4 ± 7.1 50.1 ± 7.2 BMI, kg/m 2 26.3 ± 3.8 25.8 ± 3.5 25.7 ± 3.5 26.6 ± 5.5 26.1 ± 5.1 26.0 ± 5.0 Physical activity, METs/wk 28.3 ± 35.8 35.5 ± 40.5 34.8 ± 40.2 13.6 ± 19.3 18.2 ± 22.6 18.0 ± 23.1 Current/past smokers, % 10.1 4.1 4.1 22.1 10.9 11.2 Pack-years of smoking before age 30 y 11.7 ± 6.7 10.7 ± 6.5 10.8 ± 6.6 7.5 ± 5.3 6.8 ± 5.4 6.9 ± 5.5 Family history of CRC, % 11.0 11.8 11.7 12.9 13.3 13.4 History of endoscopy, % 19.0 21.0 21.2 18.7 20.1 19.9 Regular aspirin or NSAID use, % 39.8 40.7 40.6 41.6 41.0 42.0 Dietary intake  Alcohol, g/d 12.1 ± 16.8 11.0 ± 14.2 9.9 ± 13.9 6.5 ± 12.0 5.5 ± 9.3 4.8 ± 9.2  Calcium, mg/d 952 ± 470 973 ± 446 968 ± 475 1097 ± 595 1197 ± 586 1184 ± 610  Vitamin D, IU/d 423 ± 304 451 ± 301 461 ± 326 395 ± 303 431 ± 301 433 ± 313  Red meat, g/d 74.1 ± 55.3 59.2 ± 45.6 53.0 ± 46.0 61.9 ± 44.0 53.3 ± 38.5 50.8 ± 38.9  Processed meat, g/d 13.4 ± 16.6 10.1 ± 12.4 8.4 ± 11.8 10.1 ± 12.3 7.8 ± 9.6 7.2 ± 9.5 Total flavonoids, mg/d 116 ± 36 267 ± 33 769 ± 355 107 ± 62 251 ± 62 808 ± 390 Flavonols, mg/d 10.5 ± 6.5 16.3 ± 8.2 32.0 ± 14.3 9.1 ± 5.8 14.6 ± 7.2 31.9 ± 13.6 Flavones, mg/d 1.5 ± 1.3 2.9 ± 1.6 3.4 ± 2.4 1.3 ± 1.1 2.4 ± 1.5 2.5 ± 2.0 Flavanones, mg/d 25.9 ± 23.3 57.9 ± 38.3 68.9 ± 59.7 21.4 ± 21.2 48.7 ± 35.8 49.8 ± 45.5 Flavan-3-ols, mg/d 11.9 ± 7.2 26.5 ± 10.8 128 ± 88 9.3 ± 5.1 24.1 ± 10.3 149.5 ± 95.1 Anthocyanins, mg/d 5.5 ± 5.4 14.5 ± 11.1 25.5 ± 36.1 5.5 ± 5.4 14.6 ± 11.3 22.5 ± 31.6 Polymers, mg/d 60.4 ± 26.3 149 ± 39 517 ± 279 54.1 ± 23.6 141.2 ± 35.5 565.8 ± 301.6 1 All variables (except age) are age standardized. Physical activity is described by the product sum of the MET of each specific recreational activity and hours spent on the activity per week. Regular aspirin or NSAID use is defined as ≥2 standard (325-mg) tablets of aspirin or ≥2 tablets of NSAIDs/wk. CRC, colorectal cancer; HPFS, Health Professionals Follow-Up Study; MET, metabolic equivalent; NHS, Nurses’ Health Study; NSAID, nonsteroidal anti-inflammatory drug; Q, quintile. 2 Mean ± SD (all such values). In relation to the different flavonoid subclasses, flavonol, flavone, flavanone, or flavan-3-ol intake was not associated with risk of CRC in pooled analyses or cohort-specific analyses (Table 2). Anthocyanin intake was statistically significantly associated with lower risk of CRC in age-adjusted models in men (RR: 0.78; 95% CI: 0.64, 0.94; P-trend = 0.03), which was attenuated after multivariable adjustment (RR: 0.88; 95% CI: 0.72, 1.07; P-trend = 0.35). We explored the reason for this attenuation by adding 1) nondietary potential confounders one by one to the age-adjusted model and 2) dietary potential confounders one by one to the basic model (for more detail on the basic model, see Statistical analysis section). Largest attenuations were observed after individual adjustment for physical activity, smoking before age 30 y, and red and processed meat intake (data not shown). However, no association between anthocyanin intake and risk of CRC was observed in women, and there was no substantial heterogeneity by sex (P-heterogeneity = 0.18). TABLE 2 RRs (95% CIs) of colorectal cancer and subsites according to quintiles of flavonoid intake 1 RR (95% CI) Q1 Q2 Q3 Q4 Q5 P -trend P -het Flavonols  Colorectal   Men (1061 cases)    Age adjusted 1 (reference) 0.82 (0.67, 1.01) 0.88 (0.72, 1.07) 1.06 (0.88, 1.28) 0.93 (0.76, 1.12) 0.72    Multivariable 1 (reference) 0.83 (0.68, 1.02) 0.92 (0.75, 1.13) 1.12 (0.92, 1.36) 1.00 (0.82, 1.22) 0.25   Women (1458 cases)    Age adjusted 1 (reference) 0.96 (0.81, 1.13) 0.87 (0.74,1.03) 1.01 (0.86,1.19) 1.00 (0.85, 1.18) 0.57    Multivariable 1 (reference) 0.99 (0.84,1.17) 0.92 (0.78, 1.09) 1.07 (0.91,1.27) 1.06 (0.90, 1.26) 0.23   Pooled (2519 cases), multivariable 1(reference) 0.92 (0.78, 1.09) 0.92 (0.81, 1.05) 1.10 (0.97, 1.24) 1.04 (0.91, 1.18) 0.10 0.92  Colon   Men (831 cases), multivariable 1 (reference) 0.79 (0.62, 1.00) 0.94 (0.75, 1.18) 1.14 (0.92, 1.42) 1.02 (0.82, 1.28) 0.16   Women (1151 cases), multivariable 1 (reference) 0.95 (0.79, 1.15) 0.86 (0.71, 1.05) 1.02 (0.85, 1.23) 1.01 (0.83, 1.22) 0.54   Pooled (1982 cases), multivariable 1 (reference) 0.88 (0.73, 1.06) 0.90 (0.77, 1.04) 1.07 (0.93, 1.23) 1.01 (0.86, 1.17) 0.16 0.50  Rectum   Men (230 cases), multivariable 1 (reference) 0.96 (0.63, 1.47) 0.84 (0.54, 1.30) 1.05 (0.69, 1.61) 0.92 (0.59, 1.42) 0.86   Women (307 cases), multivariable 1 (reference) 1.13 (0.78, 1.66) 1.18 (0.81, 1.72) 1.31 (0.90, 1.89) 1.30 (0.90, 1.90) 0.15   Pooled (537 cases), multivariable 1(reference) 1.05 (0.80, 1.40) 1.01 (0.73, 1.41) 1.19 (0.90, 1.57) 1.11 (0.79, 1.57) 0.37 0.29 Flavones  Colorectal   Men (1061 cases)    Age adjusted 1 (reference) 0.96 (0.79, 1.17) 0.95 (0.78, 1.16) 0.91 (0.75, 1.11) 0.90 (0.74, 1.09) 0.26    Multivariable 1 (reference) 0.99 (0.81, 1.21) 1.03 (0.84, 1.25) 1.02 (0.83, 1.24) 1.04 (0.85, 1.27) 0.67   Women (1458 cases)    Age adjusted 1 (reference) 0.94 (0.80, 1.12) 0.94 (0.79, 1.11) 0.90 (0.76, 1.06) 0.90 (0.76, 1.06) 0.17    Multivariable 1 (reference) 0.99 (0.83, 1.17) 1.01 (0.85, 1.19) 0.98 (0.83, 1.17) 0.99 (0.83, 1.18) 0.91   Pooled (2519 cases), multivariable 1 (reference) 0.99 (0.87, 1.13) 1.02 (0.89, 1.16) 1.00 (0.88, 1.14) 1.01 (0.89, 1.15) 0.81 0.71  Colon   Men (831 cases), multivariable 1 (reference) 1.00 (0.80, 1.25) 1.04 (0.83, 1.30) 0.99 (0.79, 1.25) 1.12 (0.89, 1.40) 0.33   Women (1151 cases), multivariable 1 (reference) 0.97 (0.80, 1.18) 1.01 (0.84, 1.23) 1.00 (0.83, 1.22) 1.00 (0.83, 1.22) 0.66   Pooled (1982 cases), multivariable 1 (reference) 0.98 (0.85, 1.14) 1.02 (0.88, 1.18) 1.00 (0.86, 1.16) 1.07 (0.92, 1.24) 0.31 0.75  Rectum   Men (230 cases), multivariable 1 (reference) 0.96 (0.63, 1.45) 0.97 (0.64, 1.49) 1.10 (0.73, 1.67) 0.75 (0.47, 1.19) 0.34   Women (307 cases), multivariable 1 (reference) 1.05 (0.73, 1.49) 0.99 (0.69, 1.43) 0.92 (0.64, 1.34) 0.85 (0.58, 1.24) 0.27   Pooled (537 cases), multivariable 1 (reference) 1.01 (0.77, 1.32) 0.99 (0.75, 1.30) 1.00 (0.76, 1.32) 0.81 (0.60, 1.08) 0.15 0.87 Flavanones  Colorectal   Men (1061 cases)    Age adjusted 1 (reference) 1.05 (0.86, 1.28) 1.08 (0.89, 1.31) 0.99 (0.82, 1.21) 0.87 (0.71, 1.06) 0.08    Multivariable 1 (reference) 1.10 (0.90, 1.34) 1.17 (0.96, 1.43) 1.12 (0.91, 1.36) 1.02 (0.83, 1.25) 0.98   Women (1458 cases)    Age adjusted 1 (reference) 0.88 (0.74, 1.04) 0.90 (0.76, 1.06) 0.82 (0.69, 0.96) 0.84 (0.72, 1.00) 0.05    Multivariable 1 (reference) 0.90 (0.76, 1.07) 0.96 (0.81, 1.13) 0.89 (0.75, 1.05) 0.93 (0.78, 1.10) 0.48   Pooled (2519 cases), multivariable 1 (reference) 0.99 (0.82, 1.19) 1.05 (0.86, 1.28) 0.99 (0.79, 1.23) 0.96 (0.84, 1.10) 0.62 0.62  Colon   Men (831 cases), multivariable 1 (reference) 1.07 (0.85, 1.35) 1.18 (0.94, 1.47) 1.16 (0.92, 1.45) 1.01 (0.80, 1.28) 0.90   Women (1151 cases), multivariable 1 (reference) 0.87 (0.72, 1.05) 0.90 (0.74, 1.08) 0.91 (0.75, 1.09) 0.93 (0.77, 1.13) 0.77   Pooled (1982 cases), multivariable 1 (reference) 0.96 (0.78, 1.17) 1.02 (0.78, 1.33) 1.02 (0.80, 1.29) 0.96 (0.83, 1.12) 0.92 0.77  Rectum   Men (230 cases), multivariable 1 (reference) 1.15 (0.75, 1.75) 1.14 (0.75, 1.74) 0.94 (0.61, 1.47) 1.00 (0.64, 1.56) 0.66   Women (307 cases), multivariable 1 (reference) 1.04 (0.72, 1.49) 1.21 (0.85, 1.72) 0.82 (0.56, 1.21) 0.92 (0.63, 1.34) 0.34   Pooled (537 cases), multivariable 1 (reference) 1.08 (0.82, 1.42) 1.18 (0.90, 1.54) 0.87 (0.65, 1.17) 0.95 (0.71, 1.27) 0.33 0.69 Flavan-3-ols  Colorectal   Men (1061 cases)    Age adjusted 1 (reference) 0.94 (0.77, 1.15) 0.97 (0.80, 1.18) 1.00 (0.83, 1.21) 1.03 (0.85, 1.25) 0.48    Multivariable 1 (reference) 0.99 (0.81, 1.21) 1.05 (0.86, 1.28) 1.09 (0.89, 1.33) 1.12 (0.92, 1.36) 0.18   Women (1458 cases)    Age adjusted 1 (reference) 0.89 (0.75, 1.05) 0.88 (0.74, 1.03) 0.89 (0.76, 1.05) 0.99 (0.84, 1.16) 0.40    Multivariable 1 (reference) 0.93 (0.79, 1.11) 0.93 (0.79, 1.10) 0.94 (0.80, 1.11) 1.04 (0.88, 1.23) 0.25   Pooled (2519 cases), multivariable 1 (reference) 0.96 (0.84, 1.09) 0.98 (0.86, 1.11) 1.00 (0.87, 1.15) 1.07 (0.95, 1.21) 0.09 0.65  Colon   Men (831 cases), multivariable 1 (reference) 1.07 (0.85, 1.34) 1.12 (0.89, 1.41) 1.16 (0.93, 1.46) 1.20 (0.96, 1.50) 0.13   Women (1151 cases), multivariable 1 (reference) 0.92 (0.77, 1.11) 0.95 (0.79, 1.14) 0.85 (0.70, 1.02) 1.02 (0.85, 1.23) 0.52   Pooled (1982 cases), multivariable 1 (reference) 0.98 (0.85, 1.13) 1.02 (0.87, 1.19) 0.99 (0.72, 1.35) 1.09 (0.93, 1.27) 0.16 0.39  Rectum   Men (230 cases), multivariable 1 (reference) 0.77 (0.51, 1.18) 0.85 (0.56, 1.28) 0.89 (0.59, 1.35) 0.90 (0.60, 1.35) 0.97   Women (307 cases), multivariable 1 (reference) 0.97 (0.67, 1.42) 0.85 (0.58, 1.25) 1.39 (0.98, 1.98) 1.13 (0.79, 1.63) 0.20   Pooled (537 cases), multivariable 1 (reference) 0.88 (0.66, 1.16) 0.85 (0.64, 1.13) 1.13 (0.73, 1.75) 1.02 (0.78, 1.34) 0.25 0.50 Anthocyanins  Colorectal   Men (1061 cases)    Age adjusted 1 (reference) 0.83 (0.69, 1.01) 0.92 (0.76, 1.10) 0.84 (0.69, 1.02) 0.78 (0.64, 0.94) 0.03    Multivariable 1 (reference) 0.88 (0.73, 1.07) 0.99 (0.82, 1.20) 0.94 (0.77, 1.14) 0.88 (0.72, 1.08) 0.36   Women (1458 cases)    Age adjusted 1 (reference) 0.92 (0.78, 1.09) 1.01 (0.86, 1.19) 0.91 (0.77, 1.08) 0.96 (0.81, 1.13) 0.71    Multivariable 1 (reference) 0.96 (0.81, 1.14) 1.08 (0.92, 1.28) 0.99 (0.84, 1.18) 1.08 (0.90, 1.28) 0.35   Pooled (2519 cases), multivariable 1 (reference) 0.93 (0.82, 1.05) 1.04 (0.92, 1.18) 0.97 (0.85, 1.10) 0.98 (0.81, 1.19) 0.98 0.19  Colon   Men (831 cases), multivariable 1 (reference) 0.92 (0.74, 1.14) 0.98 (0.79, 1.21) 1.00 (0.81, 1.25) 0.85 (0.67, 1.07) 0.26   Women (1151 cases), multivariable 1 (reference) 0.96 (0.79, 1.15) 1.06 (0.88, 1.28) 0.98 (0.81, 1.19) 1.04 (0.86, 1.27) 0.57   Pooled (1982 cases), multivariable 1 (reference) 0.94 (0.81, 1.08) 1.02 (0.89, 1.18) 0.99 (0.86, 1.15) 0.95 (0.77, 1.16) 0.76 0.23  Rectum   Men (230 cases), multivariable 1 (reference) 0.74 (0.48, 1.13) 1.01 (0.67, 1.50) 0.70 (0.45, 1.10) 0.99 (0.65, 1.50) 0.83   Women (307 cases), multivariable 1 (reference) 1.00 (0.69, 1.45) 1.17 (0.81, 1.68) 1.04 (0.71, 1.52) 1.04 (0.71, 1.52) 0.35   Pooled (537 cases), multivariable 1 (reference) 0.88 (0.66, 1.17) 1.09 (0.83, 1.43) 0.87 (0.60, 1.27) 1.10 (0.83, 1.45) 0.40 0.63 1 Multivariable relative risks were adjusted for age (in months), pack-years of smoking before age 30 y (0, 1–4, 5–10, or >10 pack-years), history of colorectal cancer in a parent or sibling (yes or no), history of endoscopy (yes or no), regular aspirin use (yes or no), BMI (in kg/m 2 ; <25, 25 to <30, ≥30), physical activity (low, medium, high), alcohol consumption (0 to <5, 5 to <10, 10 to <15, or ≥15 g/d), total calories (quintiles), energy-adjusted total vitamin D intake (quintiles), total calcium intake (quintiles), red meat intake (quintiles), and processed meat intake (quintiles). P -het, P value for heterogeneity by sex; Q, quintile. When examining subsites of CRC, no association between flavonoid subclasses and risk of colon cancer (neither proximal nor distal colon cancer; data not shown) or rectal cancer was observed in both cohorts. The associations were not materially altered after additional adjustment for Alternate Healthy Eating Index–2010 score or Dietary Approaches to Stop Hypertension diet scores (data not shown). When we investigated potential effect modification in the association between flavonoid subclass intake and risk of CRC by age, smoking, alcohol intake, and physical activity, no statistically significant interactions were observed (all P-interaction > 0.05). Associations between flavonoid subclasses and risk of CRC were not modified by time—that is, no substantially different results were obtained when flavonoid intake at beginning of follow-up (Supplemental Table 6) or with a latency of 0–4 y, 4–8 y, or 8–12 y (Supplemental Table 7) was investigated. The association between intake of flavonols, of which onions are a main food source, and risk of CRC was not substantially different when follow-up was started in 1990 (the first year onions were specifically asked for in the FFQs) (data not shown). We further explored the association between main food sources of flavonoids and risk of colorectal, colon, and rectal cancer (Supplemental Table 8). Dietary intake of blueberries (main food source of anthocyanins), oranges (main food source of flavones and flavanones), or tea (main food source of flavonols and flavan-3-ols) was not associated with risk of CRC in men or women. BODY.DISCUSSION: In this study comprising 2 large cohorts with >1,347,000 person-years of follow-up and 2519 CRC cases, we observed no association between habitual intake of any flavonoid subclass in relation to risk of CRC after multivariable adjustment. Thus, these findings do not support the hypothesis that dietary flavonoid intake is associated with a lower risk of CRC. To our knowledge, this is the largest prospective study relating habitual intake of various subclasses of flavonoids to risk of CRC. Epidemiologic data investigating flavonoid intake in relation to CRC risk are conflicting. Inverse associations between flavonols, flavan-3-ols, and anthocyanins and risk of CRC have been observed in case-control studies (40). However, because flavonoids originate largely from plant-based foods and beverages (i.e., foods generally considered healthy), differential recall of these foods by CRC cases compared with healthy control participants is a likely source of bias in these studies. Our findings do not support the findings from the secondary analysis of the Polyp Prevention Trial, in which flavonol intake was associated with a lower risk of advanced adenoma recurrence (16). If flavonols truly influence the development of adenomas, we would expect that early flavonol intake should have a stronger association with risk of CRC than recent intake. However, the latency analysis gave only weak support for flavonol intake 12–16 y before diagnosis being inversely associated with risk of CRC. Our findings also do not confirm the previously observed associations between flavan-3-ol intake and lower risk of rectal cancer (15) or urinary flavan-3-ols and lower risk of colon cancer (17), but they are in line with a meta-analysis including 3 prospective studies in which no statistically significant association between flavan-3-ol intake and risk of CRC was observed (40). Furthermore, the lack of association between dietary intake of flavonols, flavones (10, 12, 14), and flavanones (41, 42) and risk of CRC observed here is in line with previous prospective studies. Our results showed no clear association between anthocyanin intake and risk of CRC. In 2 previous prospective studies (41, 42), no associations between anthocyanins and risk of CRC were observed. It has been suggested that colonic microbial catabolites of anthocyanins may mediate biological activities, including anticarcinogenic properties (4). Because the catabolic yields differ largely among individuals depending on the composition of the gut microflora, it is possible that in an epidemiologic study such as the present one, a reduced risk of CRC could have been observed only in individuals with a certain microflora. The growing research on the gut microbiome may enable future epidemiologic studies to take this into account. Of the major flavonoid food sources, tea, oranges, and blueberries were not associated with risk of CRC in our study. This is in line with a recently published meta-analysis on tea (43) intake in relation to risk of CRC and previous prospective studies investigating citrus fruit intake and risk of CRC (44, 45). We are not aware of any prior prospective study investigating blueberry intake in relation to CRC. Although our study and the majority of prospective studies do not support an inverse association between dietary intake of flavonoid subclasses and risk of CRC, the evidence from experimental studies remains biologically plausible. In in vitro studies, flavonoids, especially flavan-3-ols and flavonols from tea, have been shown to inhibit growth of human colon cancer cell lines (46). In addition, it has been shown that flavan-3-ols from apples can reactivate silenced tumor suppressor genes in CRC cells (47). However, the evidence on protective effects of flavonoids against colon carcinogenesis is less conclusive in animal studies (47). It remains unclear whether anticarcinogenic effects of flavonoid subclasses observed in experimental studies play a relevant role in the etiology of CRC in humans because the flavonoid concentrations used in in vitro studies or doses used in in vivo studies can hardly be reached through habitual dietary intake (3). For example, studies investigating the effect of dietary intake of the flavonol quercetin on tumor formation in mice typically used a quercetin concentration of 2% in the diet (48). Also, many human intervention studies investigating the effect of flavonoid intake on intermediary markers such as antioxidant biomarkers or carcinogenesis markers such as DNA damage used experimental doses that are difficult to attain with habitual diet (e.g., daily doses of 50–500 mg quercetin) (49). For comparison, median intakes of flavonols were 18 mg/d in the HPFS and 15 mg/d in the NHS. Bioavailability studies have shown that a 50-mg dose of quercetin leads to plasma concentrations of 0.75–1.5 μmol/L quercetin (49), which is 10-fold lower than the typical dose of 10 μM used in in vitro studies with colon cancer cell lines (2). However, because a substantial proportion of dietary flavonoids reaches the colon (4), it is expected that at least some of the biological activity, including potential anticarcinogenic properties, may act locally through colonic metabolites, but these effects are difficult to quantify. Our study has several limitations. First, the FFQ was not specifically designed to investigate flavonoid intakes. Therefore, some foods rich in flavonoids (e.g., herbs) were missing from the FFQ, and foods with varying flavonoid contents may have been subsumed within one food item (e.g., apples and pears). Furthermore, flavonoid contents may vary depending on species, seasonal variation, maturity, and various agricultural, food-processing, and storage practices. These factors may have contributed to misclassification of flavonoid intakes, which may have led to an attenuation of observed associations, and thus may have obscured the detection of weak associations. Another limitation lies in the multiple comparisons that were made during this hypothesis-driven data analysis. We also acknowledge that our findings are not necessarily generalizable to other populations. Strengths of our study include the prospective study design; the repeated dietary intake assessment, which allowed for estimation of long-term intake as well as investigation of different latencies; and the large number of cases, which enabled us to investigate CRC individually according to location. Furthermore, the detailed data on potentially confounding factors are a strength and turned out to be important, because some of the associations that were observed in age-adjusted models were largely attenuated in the fully adjusted models. In conclusion, our study findings do not support the hypothesis that diets high in flavonoids may reduce risk of CRC. The lack of association may be related to the low habitual intake of flavonoids, interindividual variation of colonic microbial catabolite production, and/or misclassification of flavonoid intake. Epidemiologic studies employing biomarkers of flavonoids may help elucidate whether the anticarcinogenic effects of flavonoids that have been observed in experimental studies may also be relevant in humans with habitual dietary intake.

TITLE: Evaluation of Aggregometery Parameters and Efficacy of Plavix versus Clopidex in Patients Suffering from Ischemic Heart Disease: A Randomized Double Blind Clinical Trial ABSTRACT.BACKGROUND:: Ischemic heart disease is the leading cause of death in most societies. In a pathophysiologic point of view, it chiefly results from the formation of thrombus in coronary arteries which could not be only prevented by aspirin. Many of clinical trials have shown the long-term benefits of antiplatelet drugs in reducing the risk of thrombotic accidents. ABSTRACT.OBJECTIVES:: Clopidogrel is a thienopyridine derivative used to prevent platelets from adhering together by direct inhibition of Adenosine diphosphate (ADP), the major factor behind platelets aggregation. Sanofi-Aventis and Bristol-Myers are companies that produce Clopidogrel by the name of Clopidogrel bisulfate. Its trade name is Plavix, nonetheless in Iran it is distributed under the name of Clopidex by Exir Company. In this study we are to compare Plavix and Clopidex in terms of efficacy as well as aggregometry parameters like ADP and PRP (Platelet Rich Plasma). ABSTRACT.PATIENTS AND METHODS:: This is a double blind clinical trial in which we had two groups of patients suffering from Ischemic heart disease who were selected by inclusion criteria. Group A (36 patients) took Plavix (75 mg/d) and group B (36 patients) used clopidex (75 mg/d) both for 30 days. The aggregometry parameters also consisted of PRP and ADP that were run on the patients before and after the study. Finally, a comparison of aforementioned tests, quality of life, lab parameters and compliance in both groups was provided. ABSTRACT.RESULTS:: In groups A and B, the mean levels of PRP before the study were 348000 and 340000/μL respectively. The ADPs were also 73/76 and 68/07 μM that showed no significant difference (P > 0.05).The Means of ADP5 in group A before and after the study were 66.40 and 43.84 μM respectively that there was significant difference (P = 0.001). The Means of ADP5 in group B before and after the study were 58.04 and 40.16 μM respectively that there was significant difference (P < 0.001).The Means of ADP20 in group A before and after the study were 73.76 and 54.97 μM respectively which showed significant difference (P < 0.001). The Means of ADP20 in group B before and after the study were 68.07 and 52.49 μM respectively which showed significant difference (P = 0.001). Difference of ADP5 between group A and B was not significant (P = 0.495). Difference of ADP20 between group A and B was not significant (P = 0.721). The Means of PRP in group A before and after the study were 348000 and 335000/ μL respectively that there was no significant difference (P = 0.66). The Means of PRP in group B before and after the study were 340000 and 336000/ μL respectively that indicated no significant difference (P = 0.81). Difference of PRP between group A and B was not significant (P = 0.563). ABSTRACT.CONCLUSIONS:: Our findings suggested that both drugs significantly lessen the ADP level; even so there was no significant difference between two groups in PRP and ADP factors. BODY.1. BACKGROUND: Ischemic heart disease is a condition which is characterized by an insufficient supply of blood and oxygen to myocardium. The most common cause of myocardial ischemia is atherosclerotic disease of epicardial coronary arteries that engenders reduction in myocardial blood flow, that is, inadequate perfusion of myocardium through coronary arteries. The most prevalent cause of death across industrial and developing countries is escalation of ischemic heart disease (1). Epidemiology of therapeutic regimens in acute myocardial infarction (AMI) indicates substantial increases in the use of thrombolytic therapy (2). The activation of platelets is controlled by a variety of receptors. In fact, receptors' stimulation triggers two subsequent processes. First is the activation of internal signaling pathway and second is the binding of platelet to the adhesive protein of other thrombocytes. Several transmembrane receptors are found on the platelets, including ADP, PRP, prostaglandin, Lipid and chemokine. Adenosine receptors are responsible for transduction of ADP-induced signaling events through the binding of adenosine receptors on the platelet surface (1). Platelet inhibition seems a sure way for the prevention and treatment of ischemic heart disease and to step this way. BODY.2. OBJECTIVES: Clopidogrel has markedly proved safe and efficient (3). Myocardial infraction is the most common and best-known type of heart ischemic diseases. Each year more than 32 million cases of MI are diagnosed (4). Thrombosis is the major reason for MI in patients with atherosclerosis in coronary arteries. Studies indicate that MI can shorten mean life of people, aged 60 and over, by 8-10 years (5). Although Aspirin is a chosen drug for patients with coronary intervention, adding an adenosine diphosphate (ADP) receptor antagonist (Clopidogrel) can provide greater protection from thrombotic complications (6). ADP (adenosine diphosphate) has a receptor on the platelet membrane which can be irreversibly blocked when taking an oral anti-platelet drug named clopidogrel by patient (7). Aggregation of platelets by ADP, on the grounds of a research study, can be significantly reduced via clopidogrel (P < 0.01) (8). PRP (Platelet Rich Plasma) is considered as an autologous biotherapy which is based on platelet-healing proprties. When platelets are activated, they release proteins (cytokines and growth factors) which improve regenerative process (9). Clopidogrel is produced by the name of Clopidogrel bisulfate by Sanofi-Aventis and Bristol-Myers Companies and then distributed to the market under the trade name of Palvix. In Iran this drug is produced by Exir Company under the trade name of Clopidex. As Plavix costs are very expensive in Iran, Exir, company produced it at much lower price. Because ADP and PRP are counted as the main factors of platelets' aggregation; hence, in this study we strive to compare the aggregometery parameters of the aforemenstioned drugs looking into sample population. BODY.3. PATIENTS AND METHODS: This is a double blind clinical trial study that was done on patients with Ischemic Heart Disease in Baqiyatallah Hospital which is a specialized, referral hospital with 700 bed in 2012 in Tehran, Iran. The patients were selected on the basis of definite diagnosis of the disease along with qualifying for inclusion criteria and then were randomly divided into two drug groups we have totally 50 patients in each group and according to the pilot study, sample size was determined 35 patients in each group, using Alfa = 0.5, Beta = 0.9. The first group (35 patients) was given Clopidex tablets (75 mg/d) made by Exir Pharmaceutical Company and the second (35 patients) took Plavix tablets (75 mg/d) made by Sanofi-Aventis company, both for one month The rest patients were excluded from study because of non-appropriate following of drug order dose or not taking blood sampling on time. All the patients were examined by a cardiologist at the beginning of the study, one month and two months later. For all the patients, clinical and demographic data were gathered using specified questionaries. Biochemical blood tests (complete blood count, fasting blood sugar, cholesterol, triglyceride, Cratinin, Blood Urea Nitrogen) and aggregometry tests altogether were done before and after study. Besides, 10 cc of vein blood was taken from patients for detection of aggregometry parameters like ADP and PRP. Platelet aggregometry was determined through measuring OD (Optical Density) of mixed PRP after adding the agonist to aggregometer cell. Most aggregometers can be standardized and calibrated by putting patients' PRP in the holder place of cell which indicates 0% of light transition; and if putting PPP in it, 100% of light transition occurs. Increase in light transition from 0% to 100% in recording curve or digital displayer reflects that aggregometer is reading. When the agonist is added to PRP, platelets start aggregating which results in the rise of light transition. This light is recorded and used as platelet activation standard.This study was approved by ethical committee of Baqiyatallah University and got Iranian registry of clinical trial (IRCT) code of 201011025073N1. Inclusion criteria of the patients made up of: Ischemic Heart Disease or ischemia, cooperation of patients in experiment and adherence to appropriate drug use. Exclusion criteria includes :Existence of any hemorrhagic diseases, for example peptic ulcer and intracranial hemorrhage, Existence of accompanied diseases interfering with drugs taken, history of allergy to plavix or Clopidex, any contraindication for taking Palvix or Clopidex (Thrombocytopenia, liver disorders, Neutropeni), Those of patients who needed some mechanical hemodynamic support like using balloon pump. All patients were asked to sign a consent form for participation. The Helsinki principals were considered in this study. All side effects were explained to patients and all drugs and lab tests were free of charge. Patients were free to stop taking drugs at any time during the test as well. Finally the results were compared together and statistically analyzed by SPSS software. Data were analyzed with SPSS version 17 (SPSS Inc., Chicago, Ill., USA). Continuous variables are presented as the mean ± Standard Deviation (SD) and Median, Inter Quartile Range (IQR), whereas categorical data are presented as frequency and percentages. Independent t tests or Mann-Whitney Test were used for continuous variables. In this study, the probability value of 0.05 or less (P≤0.05) was set to know the significance level. BODY.4. RESULTS: In this study, group a received plavix and group B received clopidex. The investigated aggregometery parameters in this study were PRP (Platelet rich plasma) and ADP (Adenosine diphosphate). Demographic data such as age and sex of patients in this study had no significant difference between group A and B (Table 1). In groups A and B, the mean levels of PRP before the study were 348000 and 340000/μL respectively. The ADPs were also 73/76 and 68/07 μM that showed no significant difference (P > 0.05).The Means of ADP5 in group A before and after the study were 66.40 and 43.84 μM respectively that there was significant difference (P = 0.001). The Means of ADP5 in group B before and after the study were 58.04 and 40.16 μM respectively that there was significant difference (P < 0.001).The Means of ADP20 in group A before and after the study were 73.76 and 54.97 μM respectively which showed significant difference (P < 0.001). The Means of ADP20 in group B before and after the study were 68.07 and 52.49 μM respectively which showed significant difference (P = 0.001) (Table 2). Difference of ADP5 between group A and B was not significant (P = 0.495). Difference of ADP20 between group A and B was not significant (P = 0.721) (Table 3). The Means of PRP in group A before and after the study were 348000 and 335000/ μL respectively that there was no significant difference (P = 0.66). Table 1. The Comparison Demographic Parameters between two Groups Groups A B P value No. (%) Mean ± SD No. (%) Mean ± SD Sex 0.813 Female 19 (52.8%) - 21 (58.3%) Male 17 (47.2%) - 15 (41.7%) Age - 64.14 ± 10.92 60.94 ± 13.51 0.274 Table 2. The Comparison Aggrigometery Parameters between two Groups Before and After the Study a , b Group Before After IQR P value Mean ± SD Median IQR Mean ± SD Median A PRP 348000 ± 87374.12 3.62E5 108000 335000 ± 67609.66 3.40E5 118000 0.666 ADP20 73.76±18.22 3.13E5 109000 54.97 ± 16.20 3.03E5 151000 < 0.001 ADP5 66.40 ± 23.75 77.65 18.2 43.84 ± 17.92 75.00 22.3 0.001 PT Count 214000 ± 66073.28 57.30 20.85 204000 ± 52438.43 57.30 31.6 0.910 B PRP 340000 ± 88077.30 70.00 19.05 336000 ± 87315.42 63.60 36.4 0.815 ADP20 68.07 ± 15.53 49.80 31.85 52.49 ± 22.21 40.00 29.5 0.001 ADP5 58.04 ± 19.29 1.99E5 85500 40.16 ± 18.52 1.94E5 64000 < 0.001 PT Count 201000 ± 63190.72 2.04E5 87000 194000 ± 48448.50 1.91E5 41000 0.999 a Abbreviations: ADP, Adenosine Diphosphate; PRP, Platelet-rich Plasma b All data are expressed as Mean ± SD or No. (%), P value base on Wilcoxon Signed Ranks Test Table 3. The Comparison Difference of Aggrigometery Parameters between two Groups a , b Groups A B P value Mean ± SD Median Range Mean ± SD Median Range PRP 18000 ± 96153.17 4000.00 458000 9875.00 ± 102813.88 16000 458000 0.563 ADP20 19.81 ± 24.04 21.40 83.00 17.32 ± 23.82 21.40 82.80 0.721 ADP5 23.10 ± 33.02 15.50 107.30 19.00 ± 24.71 15.40 101.10 0.495 PT Count 13700 ± 93252.32 6000.00 365000 11500 ± 90876.73 3000.00 365000 0.995 a Abbreviations: ADP, Adenosine Diphosphate; PRP, Platelet-rich Plasma b All data are expressed as Mean ± SD, P value base on Mann-Whitney test The Means of PRP in group B before and after the study were 340000 and 336000/ μL respectively that indicated no significant difference (P = 0.81). Difference of PRP between group A and B was not significant (P = 0.563) (Table 3). The platelet count in group A showed no significant differences before and after the study (P = 0.910) and in group B the situation was the same either (P = 0.999). The differences between two groups was also the same (P = 0.994) (Table 3). The quality of life based on VAS standard, before and after study in group A and B had a significant difference (P < 0.05), but the difference between two groups was non-significant (P > 0.05). There were no significant differences between two groups with regard to the laboratory parameters (PT, PTT, FBS, BUN, Cr, TG, and Cho) (P > 0.05). There was not seen any significant difference between two groups as to compliance and adverse drug reaction, (P > 0.05). BODY.5. DISCUSSION: Ischemic Heart disease is a condition characterized by inadequate supply of blood and oxygen to myocardium. Epidemiology of therapeutic regimens for acute myocardial infarction (AMI) indicates considerable increases in the use of thrombolytic therapy (2). The activation of platelets is controlled by a variety of receptors. Several transmembrane receptors are found on platelets, namely ADP and PRP. Many of clinical trials have shown the long-term benefits of antiplatelet drugs for decrease in the risk of thrombotic accidents. Clopidogrel is a thienopyridine derivative which prevents platelets from adhering together by direct inhibition of adenosine diphosphate, that is, the factor behind platelets' aggregation (10). Our findings showed that PRP parameter in group A and B had no significant difference before and after the study (P = 0.666) and the difference of PRP between groups A and B was not significant. The ADP parameter with concentrations of 5 and 20, was significantly reduced in both groups after the study, Even so there was no significant difference between groups A and B. The ADP in concentration of 5, was significantly reduced in group A, after the study (P = 0.001) and in group B the situation was the same either (P < 0.001), but the differences between groups A and B were non-significant (P = 0.495) (Table 3). The ADP in both concentration (5 and 20) in group A and B showed no significant difference. In a randomized double blind clinical trial that was performed to evaluate the efficacy and safety of Clopidogrel and aspirin in decreasing the risk of ischemic stroke and MI, it turned out that the long term use of Clopidogrel in patients with atherosclerotic vascular disease is more effective than aspirin. However there was no significant difference in safety (10). In a research study, 197 patients who had undergone coronary artery bypass graft (CABG) were devided into two groups. 102 patients took Clopidogrel (75 mg/d) and 95 patients used Clopidogrel (75 mg/d) plus Aspirin (100 mg/d). There was no significant difference in graft patency between two groups (P > 0.05) (11). In another study, 70 randomly chosen patients with ischemic stroke were divided into two drug groups. One group received aspirin and the other received aspirin plus Clopidogrel. Platelets studies were performed on both groups. Treatment with Clopidogrel plus aspirin had a distinctively better platelet inhibition activity compared to aspirin itself in patients after ischemic stroke (12). To find out the spread of aspirin resistance, a clinical study on patients who had acute coronary syndrome was done. The function of platelets was analyzed through platelet function analyzer (PFA) on 100 patients in terms of collagen or ADP (Col/ADP) and collagen or epimephrine (Col/Epi). The results showed that 19% (n = 20) of the patients put up resistance to aspirin (13). In another study Joanna Fong et al. evaluated the rate of biochemical response of aspirin and clopidogrel. The results suggested that biochemical nonresponse to antiplatelet drugs may occur in ischemic heart disease and different factors can affect this response (8). Milionis et al. compared the effect of aspirin and clopidogrel on patients who had ischemic heart stroke for the first time. They were given aspirin (n = 880) and clopidogrel (n = 348). In the initial 6 months of treatment through these drugs, the distinction of survival was obvious; it was 93.8% for aspirin and 97% for clopidogrel. In addition, the collection of cardiovascular accidents was lower in patients treated with clopidogrel (n = 60, 17.2%) vs. those with aspirin (n = 249, 28.3%) (P < 0.0001) (14). In a research study, a novel dynamic layer-by-layer (d-LbL) biointerface on a nano-scale was developed which functioned as an anti-coagulation surface. It was utilized as a biologically-active substrate for platelet adhesion and aggregation. Totally, PRP + ADP was more effective at increasing platelet aggregation (15). Boris Shenkman et al. studied the Unresponsiveness to antiplatelet drugs like clopidogrel and aspirin in acute coronary syndrome patients (ACS). In a laboratory study 404 patients were selected. On days 1 and 4 the first group (n = 114) was evaluated with PA, and then a patient with a decrease less than 10% in ADP induced after treatment with clopidogrel, was called a non-responding (NR) to clopidogrel. This value is correlated with the aggregation of ADP-induced more than 70%. Besides, a patient who had a value for AA-induced aggregation more than 60% was described a non-responding (NR) to aspirin. On day 4 the second group was evaluated by both previous methods and the results were respectively: less than 2.8% and more than 3.4% for clopidogrel and aspirin NR. The NR incidence for clopidogrel and aspirin was 22% and 27% respectively (16). In another study, the clopidogrel or aspirin resistance of patients with acute coronary syndrome (ACS) was ascribed to metabolic syndromes (MS). The factors using for this aim were platelet-rich plasma (PRP), collagen and adenosine diphosphate (ADP). High levels of resistance to antiplatelet drugs were determined in MS patients. ACS patients without MS also showed this resistance to a significant degree (17). In 2009 Massie BM et al. carried out the treatment applying warfarin and clopidogrel or aspirin during a period of 30 months. 1587 patients who had heart failure within the past 3 months were enrolled. Final results indicated that warfarin is not superior to aspirin and clopidogrel (18). Our findings suggested that both drugs significantly lessen the ADP level; even so there was no significant difference between two groups in PRP and ADP factors. This research study suggests that aggregometry parameters indicate no significant differences in patients who receive Plavix or clopidex.

TITLE: The effect of household heads training on long-lasting insecticide-treated bed nets utilization: a cluster randomized controlled trial in Ethiopia ABSTRACT.BACKGROUND: Long-lasting insecticide-treated bed nets (LLITN) have demonstrated significant impact in reducing malaria-related childhood morbidity and mortality. However, utilization of LLITN by under-five children is not satisfactory in many sub-Saharan African countries due to behavioural barriers. Previous studies had focused on the coverage and ownership of LLITN. The effect of skill-based training for household heads on LLITN utilization had not yet been investigated. A cluster-randomized trial on the effect of training of household heads on the use of LLITN was done in Ethiopia to fill this knowledge gap. ABSTRACT.METHODS: The study included 22 (11 intervention and 11 control) villages in southwest Ethiopia. The intervention consisted of tailored training of household heads about the proper use of LLITN and community network system. All households in each group received free LLITN. Data were collected at baseline, six and 12 months of the follow up periods. Utilization of LLITN in the control and intervention villages was compared at baseline and follow up periods. ABSTRACT.RESULTS: A total of 21,673; 14,735 and 13,758 individuals were included at baseline, sixth and twelfth months of the project period. At the baseline survey, 47.9% of individuals in the intervention villages and 68.4% in the control villages reported that they had utilized LLITN the night before the survey. At the six month, 81.0% of individuals in the intervention villages and 79.3% in the control villages had utilized LLITN. The utilization of LLITN in all age groups in the intervention villages was increased by 17.7 percentage point (95% CI 9.7-25.6) at sixth month and by 31.0 percentage point (95% CI 16.9-45.1) at the twelfth month. Among under-five children, the LLITN utilization increased by 31.6 percentage point (95% CI 17.3-45.8) at the sixth month and 38.4 percentage point (95% CI 12.1-64.7) at the twelfth months of the project period. ABSTRACT.CONCLUSION: Household level skill-based training has demonstrated a marked positive effect in the utilization of LLITN. The effect of the intervention steadily increased overtime. Therefore, distribution of LLITN should be accompanied by a skill-based training of household heads to improve its utilization. ABSTRACT.TRAIL REGISTRATION: Australian New Zealand Clinical Trials Registry (ACTR number: ACTRN12610000035022). BODY.BACKGROUND: Long-lasting insecticide-treated bed nets (LLITN) have demonstrated a significant impact in the reduction of malaria-related childhood morbidity and mortality [1-5]. However, the utilization of LLITN is greatly affected by behavioral factors in countries south of the Sahara. Previous studies had given much emphasis on the coverage and ownership of LLITN [5-7]. Nonetheless, all owned LLITN are not being used practically. A study conducted in the two biggest regions of Ethiopia showed that 91% of households owned at least one LLITN but only 65% of the LLITN had been utilized the night before the survey [6]. A more recent study in Ethiopia has documented a significant reduction of LLITN utilization [7], which indicates the need for sustainable mechanism of improving LLITN utilization. In Africa, the utilization of LLITN is affected by practical and technical difficulties related to fixing of the net above the mat and the design of the house [8,9]. Previous studies have also documented that people tend to use LLITN during the rainy season and peak malaria transmission periods [10]. To resolve the technical difficulties and improve LLITN utilization, the community should have access to information and skill-based trainings. Previous studies have not addressed behavioural aspect of LLITN utilization [6-8]. As far as could be ascertained, no other study has addressed the impact of household heads training on the proper utilization of LLITN. In this study, the hypothesis was that empowering household heads on the proper use of LLITN through training will increase its utilization by all age groups including under-five children. BODY.METHODS.STUDY AREA: The study was conducted in a malaria endemic area in Gilgel Gibe Field Research Centre (GGFRC). The GGFRC, with a total population of 50,000, was established in 2005 and is located 260 Km south-west of Addis Ababa, the capital city of Ethiopia [9,11]. The study area lies between latitudes 7°42'50"N and 07°53'50"N and between longitudes 37°11'22"E and 37°20'36"E, at an altitude of 1,734-1,864 metres above sea level. It has annual rainfall of between 1,300 and 1,800 mm with a mean annual temperature of 19°C. The main socio-economic activities of the local communities are mixed farming involving the cultivation of staple crops and husbandry [11]. According to previous studies, the prevalence of malaria in the area among under five children ranged from 8.3 to 10.5% [9,11]. A possible ecological change due to the construction of a hydroelectric dam might have contributed for the high prevalence of malaria in the area [9,11]. BODY.METHODS.STUDY DESIGN AND SAMPLING: The study utilized a cluster-randomized trial. The primary endpoint of the trial was differences in utilization of LLITN among under-five children between the control and intervention groups. Secondary endpoints included fever and malaria prevalence among under-five children. The study population consisted of heads of households, under-five children and pregnant women in twenty-two Gots (villages). Heads of the households were selected to be trained on the proper use of LLITN. The major rationale of selecting heads of the households is that they are the decision makers in all matters (including health issues) within a household. To avoid contamination, villages in the field research centre were stratified into North and South of the reservoir of the dam. Gots in the north direction of the reservoir were selected randomly as intervention whereas those villages in the south direction of the dam were selected to be control groups and both groups are within the same distance (10-15 Km) from the reservoir. A total of 22 Gots (11 intervention and 11 control villages) were included as mentioned in detail elsewhere [9]. BODY.METHODS.THE INTERVENTION AND DATA COLLECTION PROCEDURES: The intervention and data collection procedure is published elsewhere [9]. In brief, the study had three phases: preparatory, intervention and evaluation phases. Baseline survey was undertaken from February to March, 2009. After the baseline survey, the intervention phase was started in May 2009 and continued for one year. The intervention consists of tailored training of household heads on the proper use of LLITN and establishing community network system. Nine village residents were identified and given training of trainers (TOT) about the proper utilization of LLITN for five days. The village residents gave training to all heads of the households in each Got using posters and manuals prepared in local language. After a five-day training, demonstration on use of LLITN was undertaken in the rural tukul (huts commonly built out of mud and wood walls, and hay roofs) houses in the study villages. Community network system was also established in the intervention villages to make the intervention sustainable. The network system consisted of regular communication among district health office, trained village residents and the researchers [9]. A malaria focal person at the district health office, village residents and the researchers had regular monthly meeting to discuss utilization of LLITN and challenges, additional need of LLITN and anti-malarial drugs. The focal person at the district health office was responsible to monitor and supervise the activities of the trained village residents. The investigators had also regular independent supervision at randomly selected villages. The village residents report their activities to the malaria control programme and investigators regularly. The investigators, the malaria control team, the trained village residents and the Health Extension Workers (HEW) have regular monthly monitoring meeting to discuss progress of activities and challenges. Health extension workers are female health workers who completed high school and trained for one year to give minimum health packages to the community at the health post and household level. During the baseline survey, seventy-seven percent of households had at least one LLITN. After the training, 12,500 LLITN were distributed to the control and intervention Gots. LLITN were obtained from UNICEF Ethiopia and households were given LLITN based on family size. All households in both intervention and control groups were given at least two LLITNs, however for families with large size (≥ 8), three LLITN were provided. In total 4,135 households (2,105 interventions and 2,030 controls) were included in the distribution. As of May 2009, each household in the intervention villages has been visited by the trained village residents monthly to check for the proper use of LLITN and the occurrences of malaria. The proper use of LLITN was evaluated using observation and checklist. The checklist consisted of age and sex of each household member and questions related to proper utilization of LLITN. The proper hanging of the net in the four angles of the bed/mattress using locally made wooden materials, status of the net (new or old), and the proper placement of the net under the mattress or bed were checked in each visit. The intervention and control villages were monitored using the same checklist to monitor the utilization of LLITN. The trained village residents also collect information on the occurrence of malaria/fever using questionnaire. Mass blood investigation was made at baseline, 6 and 12 months of the project to assess malaria and anaemia. A total of eight (four among women and four among men) focus group discussions (FGD) were conducted in the control and intervention villages at the baseline and 12th months of the project period. The FGD participants were selected purposively in consultation with the Kebele (small administrative units) leaders. Individuals who were supposed to have adequate information were included in the FGD. The FGD were conducted by experienced investigators using a check-list and all the FGD were tape recorded. BODY.METHODS.DATA MANAGEMENT AND STATISTICAL ANALYSIS: Data were entered into computer, edited, cleaned, and analysed using SPSS-16 and STATA version 10. Both unadjusted and adjusted effect sizes for the trial endpoints are presented. The adjusted results were presented by taking into account potential confounders such as age and sex. Indirect standardized proportion ratios were compared between the intervention and control villages at 6- and 12-months of the project period by taking into account baseline cluster size, sex and age. The indirect standardized figures, as well as baseline proportions were then compared using a t-test. Multivariate analysis was conducted using complex surveys logistic regression in STATA. A p-value of less than or equal to 0.05 was considered to be significant for all tests. The presentation has followed the CONSORT statement guidelines for cluster randomized trials [12]. Analysis of the qualitative data was done manually. All focus group discussions' (FGD) notes and tap records were translated from Afan Oromo (local language) to English. The translated texts were read by the investigators several times and mutually exclusive and meaningful categories/themes were developed. The data were then coded according to the category system. Data belonging to each category were retrieved, assembled and viewed to get meaningful interpretation based on the objectives of the study. The interpreted qualitative data were presented together with the quantitative results to triangulate the findings. Individuals' quotes were used to illustrate important findings. BODY.METHODS.ETHICAL CONSIDERATIONS: The proposal was approved by Jimma University and the WHO ethical committee. Written consent was obtained from caretakers of under-five children. BODY.RESULTS.BASELINE CHARACTERISTICS: A total of 21,673; 14,735, and 13,758 individuals in the 22 villages were included at baseline sixth and twelfth months of the project periods (Table 1). The utilization of LLITN was assessed among 4,034; 2,967, and 2,796 under-five children at baseline, sixth and 12th months respectively. At the baseline survey, perceived utilization of LLITN by all age groups in the control villages (68.4%) was higher than the intervention villages (47.9%) (p = 0.001). Similarly, utilization of LLITN by under-five children in the control villages (76%) was higher than the intervention villages (52.1%) (p = 0.000). The detailed baseline result was published elsewhere [9]. After the baseline assessment, more than 12,500 LLITN were distributed for all villages equally and its utilization was then assessed by the trained village residents. Table 1 Baseline characteristics of participants and outcome variables Variable Baseline 6 months 12 months Control, n (%) Intervention, n (%) Control, n (%) Intervention, n (%) Control, n (%) Intervention, n (%) Age in months  5 1670 (18.0) 2364 (19.1) 1288 (19.0) 1679 (21.1) 1299 (18.9) 1497 (21.7)  5-14 2767 (29.9) 3683 (29.8) 2312 (34.1) 2609 (32.8) 2360 (34.4) 2085 (30.2)  ≥ 15 4824 (52.1) 6321 (51.1) 3175 (46.9) 3672 (46.1) 3199 (46.6) 3326 (48.1) Total 9261 12368 6775 7960 6858 6908 Sex  Male 4703 (50.8) 6184 (50.0) 3418 (50.5) 3876 (48.7) 3437 (50.1) 3360 (48.7)  Female 4562 (49.2) 6192 (50.0) 3357 (49.5) 4084 (51.3) 3427 (49.9) 3534 (51.3) Total 9265 12376 6775 7960 6864 6894 LLITN utilization by all age group the previous night  Yes 6338 (68.4) 5928 (47.9) 5365 (79.3) 6448 (81.0) 3718 (71.7) 4577 (97.1)  No 2929 (31.6) 6450 (52.1) 1397 (20.7) 1511 (19.0) 1471 (28.3) 135 (2.9) Total 9267 12378 6762 7959 5189 4712 LLITN utilization by under-five children the previous night  Yes 1269 (76.0) 1232 (52.1) 978 (76.2) 1536 (91.5) 624 (61.2) 1195 (97.6)  No 401 (24.0) 1132 (47.9) 306 (23.8) 143 (8.5) 396 (38.8) 29 (2.4) Total 1670 2364 1284 1679 1020 1224 BODY.RESULTS.SIX MONTHS OUTCOMES: The LLITN utilization rate in all age groups has increased in both arms (79.3% in the control villages versus 81.0% in the intervention villages) (Table 1). This gave an effect size (mean difference) of 17.7 percentage-points (95% CI 9.7-25.6, p = 0.000). The utilization of LLITN increased among under-five children in both arms at the sixth month (76.2% in the control villages vs. 91.5% in the intervention villages). Among children under five, an effect size of 31.6 percentage-points (95% CI 17.3-45.8, p = 0.001) was observed (Table 2). Table 2 Utilization of LLITN in the control and intervention villages in south-west Ethiopia Variable Baseline 6 months 12 months Effect size (difference of differences) £ At 6 month At 12 months Control , n (%) Intervention , n (%) Control , n (%) Intervention , n (%) Control , n (%) Intervention , n (%) ¥ Uadj percentage; Adj (95% CI) P -value Uadj percentage , Adj (95% CI) P -value LLITN utilization by all age groups the previous night  Yes 6338 (68.4) 5928 (47.9) 5365 (79.3) 6448 (81.0) 3718 (71.7) 4577 (97.1) 22.2%; 17.7 (9.7, 25.6) 0.000 45.9% ; 31.0 (16.9,45.1) 0.000  No 2929 (31.6) 6450 (52.1) 1397 (20.7) 1511 (19.0) 1471 (28.3) 135 (2.9) Total 9267 12378 6762 7959 5189 4712 LLITN utilization by under-five children the previous night  Yes 1269 (76.0) 1232 (52.1) 978 (76.2) 1536 (91.5) 624 (61.2) 1195 (97.6) 39.2%; 31.6 (17.3, 45.8) 0.001 60.3%; 38.4 (12.1, 64.7) 0.009  No 401 (24.0) 1132 (47.9) 306 (23.8) 143 (8.5) 396 (38.8) 29 (2.4) Total 1670 2364 1284 1679 1020 1224 Had fever in the last two weeks  Yes 709 (17.4) 700 (8.3) 1590 (23.5) 534 (6.7) 858 (12.5) 165 (2.4) - 7.7% ; -15.0 (-20.8, - 9.3) 0.000 - 1.0; , -2.1 (-5.6, 1.4) 0.224  No 3369 (82.6) 7773 (91.7) 5174 (76.5) 7405 (93.3) 5998 (87.5) 6744 (97.6)  Total 4078 8473 6764 7939 6856 6909 Sought treatment within 24 hours of onset of fever  Yes 388 (55.1) 419 (60.5) 485 (30.8) 152 (30.5) 128 (14.9) 27 (16.9) - 5.7%; -2.2 (-17.2, 12.7) 0.763 - 3.4; , -6.7 (-23.7, 10.2) 0.419  No 316 (44.9) 273 (39.5) 1088 (69.2) 346 (69.5) 729 (85.1) 133 (83.1)  Total 705 692 1574 498 857 160 ¥ Uadj = unadjusted effect size, adj = adjusted effect size for age, clustering and sex, CI = Confidence interval, £ Difference of difference at 6 month = (follow up at six months minus baseline) intervention- (follow up at six months minus baseline) control, £ Difference of difference at 12 month = (follow up at 12 months minus baseline) intervention- (follow up at12 months minus baseline) control. Negative value shows reduction of the respective outcome measures. LLITN = Long-lasting insecticide-treated bed nets BODY.RESULTS.TWELVE MONTHS OUTCOMES: The LLITN utilization rate in all age groups had increased from 81.0% at the sixth month to 97.1% at the twelfth month in the intervention villages and it had decreased from 79.3% to 71.7% in the control villages. This gave a mean difference of 31.0 percentage-points (95% CI 16.9-45.1, p = 0.000). The LLITN utilization rate among under-five children was increased from 91.5% at the sixth month to 97.6% at the 12th months of the follow up period. The effect size of the LLITN utilization rate in under-five children was 38.4 percentage-points (95% CI 12.1-64.7, p = 0.009) (Table 2). BODY.RESULTS.COMMUNITY PERCEPTION ON THE UTILIZATION OF LLITN AND BURDEN OF MALARIA: The community in the intervention villages perceived that malaria has declined as a result of the distribution of the free LLITN and the continuous training given to them. "In our village, the burden of malaria is declining since free bed nets are given to us and we are using them properly based on the training given to us", a woman at one of the intervention village. There was a difference in the skill and knowledge of the FGD participants concerning the use of bed nets at the baseline and 12th month of the project period. At baseline, most participants did not know how to hang the nets properly. On the contrary, at the 12th month, most FGD participants in the intervention villages clearly described how to properly use the bed nets. A 50 years-old farmer at the intervention villages confidently described the proper use of LLITN as follows: "We dig four holes in the four directions of our bed and put four strong sticks in the holes. We then stretched the bed net over the bed/mattress and tie its four angles on the sticks. We always make sure that the edges of the bed nets are properly put under the mattress so as to prevent the entry of mosquitoes during the night." In the intervention villages, priority concerning utilization of LLITN was given for Sebiyi (pre-school children) and pregnant women. However, some FGD participants mentioned that some households gave priority for the head of the household and wives. On the other hand, all the FGD participants in the control villages described that the burden of malaria is still very high particularly among children and women. Lack of knowledge and skill to use LLITN, inability of the insecticide spay to kill the mosquitoes and ever increasing of the population of mosquito in the area were the three major reasons for the high burden of malaria in the control villages. Proper use of LLITN in the control villages is greatly affected by lack of training of the community and health education materials such as posters. As a result of lack of skill and knowledge, most people use the LLITN for other purposes such as bed sheets and cloaks for bags or other properties. A male farmer participant in the control villages angrily described the burden of malaria and lack of proper utilization of LLITN as follows: "I have never witnessed a decline of malaria in our village. It is affecting and killing our children every time. Most people in our villages do not know how to use the bed nets given to them." BODY.DISCUSSION: This study investigated the effect of training of households on the LLITN utilization. In the presence of serious challenges to sustain LLITN utilization, such types studies are important to guide programme planners and policy makers. Unlike previous studies, which focused on LLITN ownership, this study combined ownership and skills oriented training to empower the community to maximize LLITN utilization. In light of declining trend of LLITN utilization in Ethiopia, the experience of this study may help decision makers to adopt similar community empowering strategies particularly for people residing in malaria risk areas. The main finding of this study indicates that training of household heads has significant impact on LLITN utilization. Specifically, training of households heads on LLITN use increased the utilization of LLITN use by 17.7 percentage-points in the immediate term in high malaria transmission season and the effect was sustained and pronounced (31.0 percentage-points) in low malaria transmission season. This significant increase occurred despite equivalent distribution of LLITN in both the intervention and control villages. This reaffirms previous reports that ownership doesn't guarantee utilization [13]. The utilization of LLITN in the three surveys on the project was much higher than reports from elsewhere in the country [7,14,15]. The continuous demographic surveillance system and the health education messages by the Gilgel Gibe Field Research centre staffs and the Jimma University graduate programme students might have contributed for the high utilization of LLITN compared to other sites. However, the intervention Gots are much better in utilization of LLITN compared to the control groups. Malaria is prevalent in the area due to the possible ecological disruption as a result of the construction of the hydroelectric dam [11]. The community members also witnessed the high burden of malaria in all the seasons particularly in the control villages. The burden of malaria and anaemia in the control and intervention villages is presented elsewhere [16]. LLITN utilization in both intervention and control villages increased at the six months survey. This period is rainy and of high malaria transmission season where previous reports indicated more frequent use of nets [10]. However, it is interesting that the intervention had significant impact on utilization at the sixth months of the project. Importantly, the impact of the intervention is more pronounced among under-five children with an effect size of 31.6 and 38.4 percentage-points at the sixth and 12th months of the project period. This may imply that the training has also brought change in prioritizing vulnerable groups. Nonetheless, one should realize that such priority might come because of distribution of multiple LLITN per household. In any case, the findings of this study indicate that intervention had significant impact on utilization of LLITN utilization by under-five children. During the twelve month survey, which is dry, hot and of low malaria transmission season, the utilization has decreased from 79.3% to 71.7% in the control groups and increased in the intervention villages from 81.0% to 97.1%. One encouraging observation here is that the intervention increased utilization over extended period with different weather conditions and malaria transmission seasons. The decline in the control villages may further prove seasonal variation of LLITN use [10]. Such consistent improved utilization of LLITN after a year were not observed in other interventions intended to increase utilization. For instance, studies looking at the effect of incentives on changing health behaviours have found that incentives are effective in the short-term, but often fail to influence long-term behavior [13,17]. This highlights the importance of skilled-based education which has long-lasting effect on changing behaviour and sustaining the desired outcomes. After LLITN were owned by households, ensuring utilization has been a challenge. Different methods have been tried to address major barriers through individual level health education and mass communications [18,19]. The underlying assumption in such initiatives is that providing information about the risks associated with malaria and the benefits of LLITN will lead to higher perceived value of LLITN and thus increased use of LLITN. Such approaches, however, may not be the most efficient means of ensuring LLITN utilization. Incentives have also been tried for increasing LLITN utilization. Nonetheless, incentive induced utilization was shown to last for short period [12]. Studies have documented the low utilization rates in sub-Saharan Africa are related to technical difficulties [8,9]. Therefore, interventions focusing on skill-based training such as the one employed in this study are very important. The qualitative findings also confirmed that the intervention was beneficial to ensure proper utilization of LLITN and identify priority groups such as under-five children and pregnant women. The community acceptance of the intervention and acceptance with local authorities implies the applicability of the intervention in other settings. Shortly after completion of the research, local health authorities implemented the intervention in the control and other surrounding villages. The government of Ethiopia has deployed 30,578 health extension workers (HEWs) serving almost all villages in rural areas [20,21]. The HEWs are widely involved in diagnosis and treatment of malaria and health education in the community [21,22]. The skilled-based training of household heads described in this study can be linked to the activities of the HEW to make it sustainable. This study has utilized a rigorous study design to assess the impact of skill based training on the utilization of LLITN in programme settings. However, the study has some limitations. Firstly, self-reported use of LLITN might lead to over reporting. Second, the sample size in the 6th and 12th months is lower than the baseline and this might introduce information bias. The long-term sustainability and the cost-effectiveness of such interventions is not clearly known. Lastly, information bias might be introduced during recording and reporting of data regarding LLITN utilization and presence of fever. BODY.CONCLUSION: Community empowerment through training of household heads on the use of long lasting insecticide treated nets (LLITN) was found to be an effective intervention in increasing the utilization of LLITN in both low and high transmission seasons. The effect of the intervention was more visible among under-five children. Adoption of similar LITTN use mobilization strategies is recommended in high risks areas, such as water development projects. Integrating such skill-based training in the existing health care setting would make it sustainable. Further research is suggested to evaluate the long-term impact of the intervention and its cost-effectives. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: AD conceived the study and was involved in the design, coordination, field supervision, analysis and drafted the manuscript. AAR, TD and FT were involved in the data analysis and reviewed the article. LS and FA participated in the design, field supervision and report writing. ZB and MS were involved in field supervision and writing of the qualitative report. AZ was involved in the laboratory quality control and field supervision. SB and KD involved in organizing the data, interpreting the findings and drafted and reviewed the article. All authors read and approved the manuscript.

TITLE: Critical weight loss is a major prognostic indicator for disease-specific survival in patients with head and neck cancer receiving radiotherapy ABSTRACT.BACKGROUND:: Pre-treatment weight loss (WL) is a prognostic indicator for overall survival (OS) in head and neck cancer (HNC) patients. This study investigates the association between WL before or during radiotherapy and disease-specific survival (DSS) in HNC patients. ABSTRACT.METHODS:: In 1340 newly diagnosed HNC patients, weight change was collected before and during (adjuvant) radiotherapy with curative intent. Critical WL during radiotherapy was defined as >5% WL during radiotherapy or >7.5% WL until week 12. Differences in 5-year OS and DSS between WL groups were analysed by Cox's regression with adjustments for important socio-demographic and tumour-related confounders. ABSTRACT.RESULTS:: Before radiotherapy, 70% of patients had no WL, 16% had ⩽5% WL, 9% had >5–10% WL, and 5% had >10% WL. Five-year OS and DSS rates for these groups were 71%, 59%, 47%, and 42% (P<0.001), and 86%, 86%, 81%, and 71%, respectively (P<0.001). After adjustment for potential confounders, >10% WL before radiotherapy remained significantly associated with a worse OS (HR 1.7; 95% CI 1.2–2.5; P=0.002) and DSS (HR 2.1; 95% CI 1.2–3.5; P=0.007). The 5-year OS and DSS rates for patients with critical WL during radiotherapy were 62% and 82%, compared with 70% and 89% for patients without critical WL (P=0.01; P=0.001). After adjustment, critical WL during radiotherapy remained significantly associated with a worse DSS (HR 1.7; 95% CI 1.2–2.4; P=0.004). ABSTRACT.CONCLUSION:: Weight loss both before and during radiotherapy are important prognostic indicators for 5-year DSS in HNC patients. Randomised studies into the prognostic effect of nutritional intervention are needed. BODY: Patients with head and neck cancer are predominantly at risk for malnutrition due to anorexia, sequelae of the treatment that hamper food intake (e.g., xerostomia or dysphagia) and metabolic alterations as a result of inflammation, which can be induced by the tumour or the therapy (Van Cutsem and Arends, 2005; Baracos, 2006; Richey et al, 2007; Silver et al, 2007). Malnutrition is a subacute or chronic state in which a combination of varying degrees of undernutrition and inflammatory activity has led to a change in body composition and diminished function (Soeters et al, 2008). Weight loss is one of the main symptoms of malnutrition. Weight loss is a frequently observed problem among patients with head and neck cancer (Jager-Wittenaar et al, 2007; Ehrsson et al, 2010). Malnutrition before treatment may be seen in as high as 63% of patients, and its prevalence is associated with the location of the tumour (Nayel et al, 1992; Argiris et al, 2004; Unsal et al, 2006; Jager-Wittenaar et al, 2007; Capuano et al, 2010, 2008; Langius et al, 2010). During treatment, many patients develop treatment-related toxicities, of which dysphagia caused by mucositis is one of the most prominent. These acute toxicities result in discomfort and difficulties with eating. During radiotherapy, the prevalence of malnutrition rises to 41–88% (Nayel et al, 1992; Unsal et al, 2006; Langius et al, 2010). Sequelae of radiation therapy and weight loss may continue for several weeks after completion of radiotherapy (Langius et al, 2010; Mehanna et al, 2010). It has been demonstrated that weight loss before treatment is a prognostic indicator for overall survival (Brookes, 1985; Pugliano et al, 1999; Van Bokhorst-de van der Schueren et al, 1999; Nguyen and Yueh, 2002; De Cassia Braga et al, 2003; Argiris et al, 2004; Van Den Broek et al, 2004; Pedruzzi et al, 2008). As the association between weight loss before or during radiotherapy and disease-specific survival in patients with head and neck cancer is unclear, we set out to investigate this. BODY.MATERIALS AND METHODS.STUDY POPULATION: A consecutive cohort of head and neck cancer patients treated by radiotherapy between January 2000 and January 2009 (n=1799) was investigated for inclusion in this study. Main inclusion criterion was curative radiotherapy, either as sole treatment (with or without chemotherapy) or postoperatively. Patients younger than 18 years, patients with a tumour of the ear, skin, or oesophagus, and those with previous cancer history were excluded (Figure 1). The final study population was composed of 1340 patients. This study was approved by the Medical Ethics Committee of the VU University Medical Center Amsterdam. BODY.MATERIALS AND METHODS.RADIOTHERAPY: All patients were treated on 6-MV linear accelerators (Varian Medical Systems, Inc., Palo Alto, CA, USA), immobilised in the supine position by the use of individually designed facial masks. From January 2000, conventional three-dimensional conformal radiotherapy was applied until the clinical introduction of parotid gland sparing IMRT in October 2004. In the primary irradiated patients, the tumour and lymph-node metastases were treated with 2 Gy per fraction up to a total dose of 70 Gy. A dose of 46 Gy in daily fractions of 2 Gy (or an equivalent dose of 35 daily fractions of 1.55 Gy) was given to the elective nodal areas. Patients treated with postoperative radiotherapy received 2-Gy daily fractions at the primary site and nodal metastases to a total dose of 56 or 66 Gy, depending on the surgical margin status and the presence of extranodal spread. Again the elective dose was 46 Gy in 2-Gy daily fractions (or an equivalent dose of 28 fractions of 1.8 Gy or 33 fractions of 1.65 Gy). In the case of concomitant chemoradiation, three cycles of cisplatin 100 mg m−2 were given on days 1, 22, and 43. From the start of radiotherapy, patients received dietary counselling to achieve individual nutritional requirements. If nutritional requirements could not be reached by regular food products, then energy-enriched oral nutritional supplements and/or enteral tube feeding by nasogastric tube or percutaneous endoscopic gastrostomy were subscribed. BODY.MATERIALS AND METHODS.DATA COLLECTION: All data were prospectively collected. At baseline, patient and tumour characteristics were recorded. Weight loss before radiotherapy was recalled by the radiotherapist and, based on the equation (current weight−usual weight)/usual weight × 100%, categorised into four groups: no weight loss, ⩽5% weight loss, >5–10% weight loss and >10% weight loss. Body weight was measured at the start of radiotherapy (±7 days) and weekly thereafter until the eighth week, and at 12 weeks after the start of radiotherapy (labelled as 'during radiotherapy'). Body weight was measured by wearing light indoor clothing and shoes on a digital electronic scale (Seca (Hamburg, Germany), Alpha 770) to the nearest 0.1 kg. Weight was corrected for clothing and shoes by subtracting 2.0 kg for men and 1.3 kg for women (Frank and Dunlop, 2000). BODY.MATERIALS AND METHODS.DEFINITIONS: Critical weight loss was defined as body weight loss of >5% from the start of radiotherapy until week 8 or >7.5% until week 12 according to the international consensus statement (White et al, 2012). Because we earlier observed that acute radiotherapy induced toxicity and weight loss arise after 2 weeks of radiotherapy (Langius et al, 2010), the time span for critical weight loss during radiotherapy was expected to be the last month of radiotherapy. Overall survival was defined as the time elapsed between the start of radiotherapy and the date of death of any cause, or if the patient was still alive, and 5 years after the start of radiotherapy. Disease-specific survival was defined as the time elapsed between the start of radiotherapy and the date of death due to cancer, or if the patient was still alive, and 5 years after the start of radiotherapy. Patients who were lost to follow-up within 5 years were censored at their last date of follow-up. In the analysis of disease-specific survival, deaths due to causes other than head and neck cancer were treated as censored observations at the time of death. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: To test differences in patient, tumour, and treatment characteristics between survivors and non-survivors, χ2 tests were used regarding gender, tumour location, TNM stage (Edge et al, 2010), baseline WHO performance score, weight loss before radiotherapy, treatment modality, and radiotherapy on the neck nodes, and independent t test was used regarding age. Survival curves were generated using the method of Kaplan–Meier. The log-rank test was used to examine the difference in overall and disease-specific survival between weight loss groups. Cox's proportional hazard models were used to build multivariate (i.e., adjusted) models. Relevant factors influencing both weight loss and the survival period were selected a priori, based on the literature. Potential confounder variables for the association between weight loss before radiotherapy and overall and disease-specific survival were age, gender, tumour location (oral cavity, oropharynx, nasopharynx, hypopharynx, larynx, nasal cavity and paranasal sinuses, major salivary glands, and unknown primary), TNM stage (I, II, III, and IV), prior head and neck surgery, and baseline WHO performance score (0, 1, and 2/3). For the association between weight loss during radiotherapy and overall and disease-specific survival, we additionally adjusted for weight loss before radiotherapy (no weight loss, ⩽5% weight loss, >5–10% weight loss, and >10% weight loss), treatment modality (RT alone, RT+surgery, and RT+chemotherapy±surgery), and radiotherapy on the neck nodes (none, unilateral, and bilateral). We attempted to adjust for comorbidity by the Adult Comorbidity Evaluation-27 (ACE-27) test (Paleri and Wight, 2002), but the scores were available only in patients receiving chemoradiotherapy. Therefore, a subgroup analyses was performed to analyse the effect of comorbidity on the association between weight loss and overall and disease-specific survival. Interaction between weight loss and gender or age with respect to overall and disease-specific survival was investigated, but both were no effect modifiers. Proportional hazard assumptions for each model was investigated and confirmed by testing the constancy over time of the log-hazard ratio for each model. P-values of <0.05 were considered as statistically significant. Analyses were performed using the SPSS software (version 20.0.0; 2011 IBM Corporation, New York, NY, USA). BODY.RESULTS: The majority of patients (70%) were male. The mean age was 61.4±12.0 years, and ranged from 19 to 96 years. Tumours were mainly located at the larynx and oropharynx. Eighteen percent of patients had stage I tumours, 21% stage II, 18% stage III, whereas 43% had stage IV tumours. Slightly more than half of the patients received a combined modality treatment (Table 1). Four-hundred and seventy-one patients (35%) died within 5 years. No significant difference was found between the survivors and the non-survivors regarding gender. There were significant differences between the survivors and the non-survivors regarding age, tumour location, TNM stage, weight loss before radiotherapy, WHO performance score, treatment modality, and radiotherapy on the neck nodes (Table 1). BODY.RESULTS.WEIGHT LOSS BEFORE RADIOTHERAPY: Before radiotherapy, 70% of patients had no weight loss, 16% had ⩽5% weight loss, 9% had >5–10% weight loss, and 5% had >10% weight loss (Table 1). Five-year overall survival rates for these groups were 71%, 59%, 47%, and 42%, respectively (log rank: P<0.001; Figure 2). Five-year disease-specific survival rates for these groups were 86%, 86%, 81%, and 71%, respectively (log rank: P<0.001). The unadjusted Cox's regression analysis (Table 2) showed that weight loss before radiotherapy (of any category) was significantly associated with a worse overall survival. In addition, we found that the two most severe weight loss categories (>5–10% and >10% weight loss) were significantly associated with a worse disease-specific survival. After adjustment for all previously mentioned potential confounders, >10% weight loss before radiotherapy remained significantly associated with a worse overall (HR 1.7; 95% CI 1.2–2.5; P=0.002) and disease-specific survival (HR 2.1; 95% CI 1.2–3.5; P=0.007) (Table 2). In the subgroup of chemoradiotherapy, comorbidity nearly affected the association between weight loss before chemoradiotherapy and overall survival (>10% weight loss HR 3.3, 95% CI 1.7–6.7, P=0.001; >5–10% weight loss HR 2.3, 95% CI 1.3–4.2, P=0.005; ⩽5% weight loss HR 1.8; 95% CI 1.0–3.2; P=0.041). Comorbidity was not significantly associated with disease-specific survival. BODY.RESULTS.WEIGHT LOSS DURING RADIOTHERAPY: Critical weight loss during radiotherapy was observed in 57% of patients. Mean weight loss during radiotherapy was 4.1 (±4.7) kg, which corresponded to 5.4 (±6.1)% of body weight. On average, patients with critical weight loss lost 9.0 (±4.8)% of their body weight. Patients with critical weight loss had lower 5-year overall survival rates than patients without critical weight loss during radiotherapy (survival rates: 62% vs 70% log rank: P=0.01). However, the adjusted association was no longer statistically significant (HR 1.1; 95% CI 0.9–1.4; P=0.295) (Table 3). The 5-year disease-specific survival rate for patients with critical weight loss was 82%, compared with 89% for patients without critical weight loss (HR 1.7; 95% CI 1.2–2.3; P=0.001; Figure 3). After adjustment for all confounders, disease-specific survival was still significantly worse for patients with critical weight loss during radiotherapy (HR 1.7; 95% CI 1.2–2.4; P=0.004) (Table 3). In the subgroup of chemoradiotherapy, all patients who died because of head and neck cancer had critical weight loss during therapy. Therefore, no further subgroup analyses were possible. Patients who were excluded because of missing baseline weight (Figure 1), had a shorter overall survival time compared with the included patients (log rank: P=0.009). Disease-specific survival was not significantly different. BODY.DISCUSSION: Weight loss as a consequence of (chemo)radiotherapy is a common problem in patients with head and neck cancer (Langius et al, 2013). However, little is known about its prognostic effect on disease-specific survival. This study shows that critical weight loss during radiotherapy is independently associated with a 1.7 times higher risk of dying of head and neck cancer. Moreover, this large study demonstrates that weight loss before radiotherapy is also independently associated with almost two-fold risk of dying. The association between pre-treatment weight loss and disease-specific survival has been investigated only once in a subgroup of patients with head and neck cancer (Regueiro et al, 1994). In that study, weight loss before radiotherapy was an independent predictor for disease-specific survival in patients with cancer of the oropharynx, with a hazard ratio of 2.3. We found comparable results in this mixed group of head and neck cancer patients, and this finding therefore can now be extended to the entire group of patients with head and neck cancer. Recently, two studies investigated the impact of weight loss during radiotherapy on survival (Pai et al, 2012; Cho et al, 2013). In the study of Pai et al (2012), weight loss during radiotherapy was an independent prognostic factor for locoregional control, but not for survival. Cho et al (2013) found weight loss of ⩾10% during and 1 year after treatment as an independent prognostic factor for disease-free survival (hazard ratio 2.2), but not for overall survival, in patient with cancers of the oral cavity and oropharynx). Our results are in line with those of Cho et al (2013). We examined the association of critical weight loss during radiotherapy and 5-year overall and disease-specific survival. In the unadjusted analysis, we found that critical weight loss during radiotherapy was significantly associated with a worse 5-year overall survival, but this association disappeared after adjusting for other relevant prognostic factors. However, critical weight loss during radiotherapy was an independent prognostic factor for 5-year disease-specific survival. The observation that patients with critical weight loss during radiotherapy have worse disease-specific survival suggests that treatment is less effective in this patient group. The direct effect of radiotherapy on cancer cell death through irreparable DNA damage is intensified by stimulating an anti-tumour immune response (Vu et al, 2010; Kwilas et al, 2012; Tong et al, 2012). The poor response to treatment might be due to an impaired immune response as a consequence of insufficient food intake in the malnourished patients. The immune system is highly dependent upon an adequate availability of amino acids (Li et al, 2007) and specific vitamins, minerals, and trace elements (Wintergerst et al, 2007; Mora et al, 2008). Deficiency of nutrients alters the immune response, even when the deficiency state is relatively mild (Chandra, 2002). We attempted to link critical weight loss and immunity in our own series. It was previously demonstrated that a severe deficiency of peripheral blood iNKT cells in patients with head and neck cancer was significantly related to poor clinical outcome, suggesting their critical contribution to anti-tumour responses in head and neck cancer patients (Molling et al, 2007). We post hoc analysed the relation between weight loss and iNKT cells and found that patients with >5% weight loss had significantly lower numbers of T cells (722±306 vs 1162±580T cells per μl in patients with ⩽5% weight loss, P=0.021), and more often a low (<25th percentile) iNKT cell level compared to patients with ⩽5% weight loss (55% vs 15% of patients, P=0.004). Although this analysis strengthened our hypothesis, further investigation of the impact of malnutrition on immune response in cancer patients is clearly warranted. Weight loss during radiotherapy frequently occurs in patients with head and neck cancer. In our study, the prevalence of critical weight loss during and shortly after radiotherapy was 57%. Several different definitions are used to define malnutrition or severe weight loss (Meijers et al, 2010). Our cutoff points for critical weight loss during radiotherapy were based on the international consensus statement of the Academy of Nutrition and Dietetics and the American Society for Parenteral and Enteral Nutrition (White et al, 2012). Lower or higher cutoff points could probably decrease or increase the hazard ratios. Although we did not reach weight stabilisation during radiotherapy with our nutritional policy, critical weight loss can be avoided. Several studies in patients with head and neck cancer showed that nutritional therapy can be effective in stabilising body weight during radiotherapy (Isenring et al, 2004; Macia et al, 1991). Our results may cautiously suggest that preventing malnutrition by nutritional therapy might exert a positive influence on cause-specific survival. However, intervention studies are needed to answer the important question if prevention of weight loss indeed has an impact on survival. Most of the previous studies on the association of pre-treatment weight loss and overall survival performed only unadjusted analyses. The drawback of unadjusted survival analyses is that confounders may disturb the relation between weight loss during radiotherapy and survival. Thus, significant differences in the unadjusted analyses may result from other confounding variables, such as tumour location and disease stage. A strength of the present study is that herein adequate adjustment for relevant prognostic factors was possible. Some limitations of this study should be acknowledged. First, we had to exclude 172 patients (13%) for the analysis of weight loss during radiotherapy, and these patients had a worse overall survival compared with the included patients. It is unknown whether the exclusion of those patients has affected our results, that is, the absence of an association between critical weight loss during radiotherapy and overall survival. Second, the dose distribution of radiotherapy may influence the amount of weight loss during radiotherapy. Given the number of patients we did not delineate organs at risk for weight loss, but we used target volume in general (local vs unilateral neck vs bilateral neck) as a surrogate for dose to the swallowing structures. In another analysis (unpublished data), we found that this provides useful information with regard to the dose distribution to the most important organs at risk. Third, despite the adjustment for important confounding variables, we were not able to adjust for comorbidity in the entire group of patients. We used the WHO performance score. In the subgroup of patients receiving chemoradiotherapy, results of the ACE-27 test (Paleri and Wight, 2002) were available and used for a subanalysis. In contrast to what we expected, comorbidity did not alter the effect of weight loss on survival. Fourth, just as in other studies, pre-treatment weight loss had to be recalled because patients were newly referred to the hospital. We suppose that patient might have underreported their weight loss, especially patients with the smallest amounts of weight loss who where therefore classified into the reference group. This might have lead to underestimation of the prognostic effects on survival. On the other hand, it has been demonstrated that self-reported weight is a highly sensitive method to define malnutrition (Haverkort et al, 2012). The weight loss categories in our study had large intervals, so small differences in weight loss estimation would not have lead to other classification. We therefore deem that the results might marginally be influenced by the recall method, and if so, the extent of the association between weight loss before radiotherapy and survival is probably underestimated. In conclusion, weight loss both before and during radiotherapy are important prognostic indicators for 5-year disease-specific survival in patients with head and neck cancer. Randomised studies into the prognostic effect of nutritional intervention during radiotherapy are needed.

TITLE: Oral paracetamol versus oral ibuprofen for treatment of patent ductus arteriosus ABSTRACT.OBJECTIVE: This study was performed to investigate the safety and efficacy of oral paracetamol versus oral ibuprofen in the treatment of patent ductus arteriosus (PDA) in premature infants. ABSTRACT.METHODS: Premature infants with PDA with a gestational age of ≤32 weeks or birth weight of ≤1500 g were included in this randomized study. ABSTRACT.RESULTS: A total of 120 premature infants fulfilled the inclusion criteria. Of these 120 infants, 34 fulfilled the treatment criteria and 22 were finally randomized. We found no significant difference in the mortality or primary closure rates between the two groups. We also found no significant difference in the short-term neonatal outcomes. ABSTRACT.CONCLUSIONS: Either oral paracetamol or oral ibuprofen can be used safely and effectively to treat PDA in premature infants. BODY.INTRODUCTION: Hemodynamically significant patent ductus arteriosus (hsPDA) is a major risk factor for mortality and morbidity in very-low-birth-weight infants.1 The standard of care is to treat all cases of hsPDA. The methods of treatment, which medications to use, and treatment timing continue to be subjects of research.2 Intravenous indomethacin and intravenous ibuprofen are both widely used for PDA treatment.3,4 In Jordan, however, as in many other low-resource countries, these two medications are not available. Studies from these countries have investigated oral preparations of paracetamol5–8 and oral and rectal forms of ibuprofen.9–13 Many studies have shown that oral ibuprofen is both safe and effective in treating PDA.10–13 Furthermore, recent reports on the use of oral paracetamol are promising.5–8 Paracetamol inhibits prostaglandin synthetase activity by acting at the peroxidase segment of the enzyme.14 Peroxidase is activated at a 10-fold lower concentration of peroxide than cyclooxygenase. The peroxide concentration is decreased in certain neonatal morbidities that are accompanied by hypoxemia. Hypothetically, under these conditions paracetamol should be a more effective drug than cyclooxygenase inhibitors.15 Paracetamol is also the only option when a patient has a contraindication for ibuprofen use.16 Oral paracetamol is a safe and readily available medication that is much less expensive than the intravenous preparation. Few studies have compared the safety and efficacy of oral preparations of ibuprofen and paracetamol in treating PDA.17–19 To help fill this knowledge gap, we conducted the present study to evaluate the incidence of PDA in our population and compare oral ibuprofen and oral paracetamol for the treatment of PDA in premature infants. BODY.MATERIALS AND METHODS: This randomized parallel study was conducted in the Neonatal Intensive Care Unit of Jordan University Hospital, Amman, Jordan, from March 2015 to October 2016. The study was approved and funded by the Deanship of Scientific Research at the University of Jordan and is registered in the ISRCTN registry under the number ISRCTN12302923 DOI 10.1186. All procedures performed in this study were in accordance with the ethical standards of and granted ethical approval by the institutional review board of Jordan University Hospital (reference number 108/2014/IRBJ). The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments. BODY.MATERIALS AND METHODS.PATIENTS’ CHARACTERISTICS: All premature infants with a gestational age of ≤32 weeks or birth weight of ≤1500 g were included. The exclusion criteria were as follows: ductal-dependent congenital heart diseases, major congenital malformation, grade 3 to 4 intraventricular hemorrhage, renal impairment (defined as a creatinine concentration of >1.5 mg/dl), pulmonary hemorrhage, thrombocytopenia of <60,000/mm3, and an elevated alanine transaminase concentration. Echocardiography was performed for all included infants from 3 to 5 days of age or when they showed symptoms of PDA, whichever occurred earlier. PDA was considered hemodynamically significant if two of the following criteria were present: wide pulse pressure (defined as systolic blood pressure − diastolic blood pressure of >1⁄2 systolic blood pressure), reversal of flow in the descending aorta by Doppler echocardiography, left atrial dilatation determined by a left atrial:aortic root ratio of >1.4:1.0 on M-mode echocardiographic evaluation from the parasternal view, or left ventricular dilatation. We did not include the size of the ductus as a criterion because it is relative to the size of the infant. Symptoms of PDA were metabolic acidosis, increased respiratory demands not explained by respiratory distress syndrome or its complications (usually after a period of improvement), decreased urine output, delayed capillary refill, and newly onset persistent mottling.20,21 To fulfill the treatment criteria, infants were required to either demonstrate hsPDA by echocardiography or signs indicating the presence of symptomatic PDA. The parents of newborns who fulfilled these criteria were subsequently approached for informed consent. Informed consent was obtained from the parents of all individual participants included in the study. BODY.MATERIALS AND METHODS.RANDOMIZATION AND TREATMENT PROTOCOL: The qualifying preterm infants were randomized by computer to receive either oral paracetamol or oral ibuprofen. Randomization numbers were placed inside sequentially numbered opaque envelopes. The following laboratory investigations were conducted within 24 hours before treatment was initiated and within 24 hours after treatment was finished: complete blood count, platelet count, creatinine, and alanine transaminase. A head ultrasound was also performed before and after treatment. The oral paracetamol group received 10 mg/kg/dose followed by 1 to 2 ml of 0.9% saline every 6 hours for 3 days. Regarding ibuprofen treatment protocol, previous studies used a loading dose of 10 mg/kg17,18 or 20 mg/kg19 on the first day and then half of this dose for the first and second days of treatment. We used the same dose for the 3-day course to minimize errors. The oral ibuprofen group received 10 mg/kg/dose followed by 1 to 2 ml of 0.9% saline once daily for 3 days. Echocardiography was repeated within 24 hours of the last treatment. Response to treatment was defined as resolution of symptoms with either complete closure of the PDA or a very small hemodynamically insignificant PDA evident by echocardiography. In these cases, echocardiography was repeated before discharge. If no response was found after the first course of treatment, a second course of treatment with the same drug was given for another 3 days. If no response as seen after two courses, a third course of treatment was started using the other drug. If three courses of medical treatment failed, surgery was performed only if the PDA was causing ventilation difficulties. Primary closure was defined as response to treatment after a 3-day course of the assigned drug. Secondary closure was defined as response to treatment after two courses or a total of 6 days of treatment with the assigned drug. Tertiary closure was defined as response to treatment after switching to the other drug in the trial after failure of two courses of treatment. During the study period, all infants received the same fluid and enteral feeding protocol. They all started at 80 ml/kg/day, which was increased daily in increments of 20 ml/kg/day. If the PDA required treatment, fluid administration was kept at a maximum of 120 ml/kg/day until the end of the treatment. Feeding was started on day 1 or 2 at 20 ml/kg/day according to the availability of breast milk. It was increased to 20 ml/kg/day, but was not progressively increased during the treatment period. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: SPSS version 21 (IBM Corp., Armonk, NY, USA) was used to conduct the statistical analyses. Numerical data are represented by mean ± standard deviation. Categorical data are represented by their respective rates or proportions. A t-test was used to compare means, and the chi-square test was used to compare proportions. P values of <0.05 were considered statistically significant. BODY.RESULTS: In total, 128 premature infants were included. Eight of them died before day 3 of life and did not show signs of PDA. The 120 surviving premature infants were screened, and 22 fulfilled the treatment and randomization criteria as shown in Figure 1. All infants who were screened because of PDA-related symptoms were found to have hsPDA by echocardiography. Figure 1.Study flow diagram The treatment group was divided into two subgroups: the paracetamol group and the ibuprofen group. The mean gestational age was 28 weeks in both groups. Apart from a lower Apgar score in the first minute of life in the paracetamol group, there were no statistically significant differences in the demographic characteristics between the two groups (Table 1). Table 1. Demographic characteristics of infants included in the study Paracetamol group (n=13) Ibuprofen group (n=9) P value Gestational age (wks) 28 (23–32) 28 (25–35) Birth weight (g) 1059±386 1192±269 0.861 Small for age 3 3 0.595 Cesarean section 9 7 0.658 Inborn 12 9 0.394 Male sex 11 6 0.323 Multiple gestation 4 6 0.096 Apgar score  First minute 5 7 0.013*  Fifth minute 7 9 0.104 Data are presented as mean (range), mean ± standard deviation, or number of infants. All infants had a gestational age of ≤32 weeks or birth weight of ≤1500 g. * P <0.05 The mortality rate was 23% in the paracetamol group and 22% in the ibuprofen group. The primary closure rate was 69% in the paracetamol group and 78% in the ibuprofen group. Other neonatal outcomes are shown in Table 2. A summary of three previously published randomized trials on the present study topic as well as the current study is presented in Table 3. Table 2. Comparison of premature infant mortality and neonatal morbidity in the paracetamol and ibuprofen groups Paracetamol group (n=13) Ibuprofen group (n=9) P value Mortality 3 2 0.962 Primary closure 9 7 0.658 Secondary closure 3 1 Tertiary closure 0 1 RDS 12 6 0.125 Surfactant therapy 9 6 0.898 Pulmonary hemorrhage 1 1 0.783 BPD 1 0 0.394 MV 9 5 0.512 Sepsis 7 4 0.664 NEC 3 2 0.962 ROP 0 0 IVH 7 2 0.137 Grade 1 5 2 Grade 2 2 0 Grade 3 0 0 Grade 4 0 0 PVL 0 0 Data are presented as number of patients. RDS: respiratory distress syndrome, BPD: bronchopulmonary dysplasia, MV: mechanical ventilation, NEC: necrotizing enterocolitis, ROP: retinopathy of prematurity, IVH: intraventricular hemorrhage, PVL: periventricular leukomalacia Table 3. Comparison between previously published studies that investigated the use of oral paracetamol and oral ibuprofen in treatment of PDA in premature infants and the current study Study China 15 Turkey 16 Iran 17 Jordan Method Randomized Randomized Exclusion performed after randomization Randomized Sample size 160 90 150 22 Gestational age (wks) ≤34 ≤30 <37 ≤32 Birth weight (g) NA ≤1250 NA ≤1500 Paracetamol dose 15 mg/kg/dose for 3 days 15 mg/kg/dosefor 3 days 15 mg/kg/dose for 3 days 10 mg/kg every 6 h for 3 days Ibuprofen dose 10 mg/kg (day1)5 mg/kg (days 2–3) 10 mg/kg (day 1) 5 mg/kg (days 2–3) 20 mg/kg (day 1) 10 mg/kg (days 2–3) 10 mg/kg/day for 3 days Rate of any PDA NA 82% NA 42% Rate of hsPDA NA 46% NA 28% Primary closureParacetamol vs. ibuprofen 81.2% vs. 78.8% 72.5% vs. 77.5 82.1% vs. 75.8% 69.0% vs. 78.0% MortalityParacetamol vs. ibuprofen 12.5% vs. 15.0% 7.5% vs. 5.0% NA 23.0% vs. 22.0% PDA: patent ductus arteriosus, hsPDA: hemodynamically significant patent ductus arteriosus, NA: not available BODY.DISCUSSION: Only a few studies to date have compared oral paracetamol and oral ibuprofen for the treatment of PDA in premature infants. Prior to the present study, three randomized studies were published.17–19 Although our study examined a relatively lower number of patients, it was conducted using very strict diagnostic and treatment criteria. Echocardiography was performed based on symptoms or age. When infants were asymptomatic, they were screened between days 3 and 5 of life. Screening asymptomatic newborns before day 3 of life can lead to unnecessary treatment of a potentially spontaneously closing ductus. Additionally, screening should not be performed later than day 5 of age because this might affect the response to therapy. However, later detection of PDA in asymptomatic newborns might also reflect an insignificant ductus that may not require treatment. Because symptoms were not considered in the previously published studies, we surmise that echocardiography was performed for screening regardless of age. Table 3 summarizes the differences between the present study and the three previously published studies. In the present study, the rate of any PDA was 42% and the rate of hsPDA was 28%; both are lower than previously reported rates.17–19 This difference may reflect the timing of the screening and treatment criteria. None of the infants who did not fulfill the treatment criteria had complications related to PDA. The mortality rate was similar in the two treatment groups. Deceased neonates were smaller and less mature, and most of them had a PDA that failed to close after the first course of treatment (Table 4). Table 4. Comparison between survivors and nonsurvivors among premature infants included in the study Nonsurvivors (n=5) Survivors (n=17) P value Gestational age (wks) 26.0±1.5 29.0±1.9 0.0042 Birth weight (g) 776±192 1212±301 0.0066 Primary closure 1 15 0.0026 Data are presented as mean ± standard deviation or number of patients. Primary closure was accomplished in most patients in both treatment groups. Because most PDAs were closed after a 3-day course of the assigned drug, it might be more advisable to treat PDA for 3 days and then confirm failure of treatment before extending the duration of therapy to avoid further side effects of the medication. Tertiary closure was accomplished in one patient randomized to the ibuprofen group whose PDA failed to close after two courses of ibuprofen and closed after switching to paracetamol. Failure of a PDA to close after a 6-day course of either medication should not discourage treating physicians from trying the other medication before sending the infant to surgery. Data on the safety of paracetamol have been reported, and little evidence of side effects has been found.21 None of the infants in the present study showed any signs of hepatic toxicity. Ibuprofen has milder vasoconstrictive side effects than other nonsteroidal anti-inflammatory drugs. Mild elevation of blood urea nitrogen and insignificant gastrointestinal symptoms were reported in a previous study.22 In our study, the included neonates did not show any significant elevation in the blood urea nitrogen concentration or any gastrointestinal side effects. There were no differences in the neonatal complications between the paracetamol and ibuprofen groups as shown in Table 2. Long-term safety data of paracetamol and ibuprofen are scarce. A follow-up study of one trial investigating the neurodevelopmental outcomes of children who received oral ibuprofen and oral paracetamol at 18 to 24 months of age showed no difference between the two groups.23 We conclude that both oral ibuprofen and oral paracetamol are safe and effective in treating PDA in premature infants. Implementing fluid and feeding protocols might help to decrease both the complications of the PDA and the side effects of the treatment medication. This study included a small number of patients. Multicenter studies are needed to recruit adequate numbers of patients within a reasonable period of time.

TITLE: ACE Inhibition and Endothelial Function: Main Findings of PERFECT, a Sub-Study of the EUROPA Trial ABSTRACT.BACKGROUND: ACE inhibition results in secondary prevention of coronary artery disease (CAD) through different mechanisms including improvement of endothelial dysfunction. The Perindopril-Function of the Endothelium in Coronary artery disease Trial (PERFECT) evaluated whether long-term administration of perindopril improves endothelial dysfunction. ABSTRACT.METHODS: PERFECT is a 3-year double blind randomised placebo controlled trial to determine the effect of perindopril 8 mg once daily on brachial artery endothelial function in patients with stable CAD without clinical heart failure. Endothelial function in response to ischaemia was assessed using ultrasound. Primary endpoint was difference in flow-mediated vasodilatation (FMD) assessed at 36 months. ABSTRACT.RESULTS: In 20 centers, 333 patients randomly received perindopril or matching placebo. Ischemia-induced FMD was 2.7% (SD 2.6). In the perindopril group FMD went from 2.6% at baseline to 3.3% at 36 months and in the placebo group from 2.8 to 3.0%. Change in FMD after 36 month treatment was 0.55% (95% confidence interval −0.36, 1.47; p = 0.23) higher in perindopril than in placebo group. The rate of change in FMD per 6 months was 0.14% (SE 0.05, p = 0.02) in perindopril and 0.02% (SE 0.05, p = 0.74) in placebo group (0.12% difference in rate of change p = 0.07). ABSTRACT.CONCLUSION: Perindopril resulted in a modest, albeit not statistically significant, improvement in FMD. BODY.INTRODUCTION: ACE inhibition has been shown to reduce morbidity and mortality among a variety of patients, i.e. patients with congestive heart failure,[1–3], survivors of a myocardial infarction [4, 5], a-symptomatic patients with left ventricular dysfunction [6, 7], patients with acute myocardial infarction [8–11], post-myocardial infarction patients [6, 7, 12–14] and older high risk patients with documented coronary heart disease without heart failure and, in some, no left ventricular dysfunction [15]. Recently, the EUROPA trial expanded the evidence by showing that ACE inhibition reduces cardiovascular event risk by 20% among a relatively low risk population of patients with stable coronary heart disease without apparent heart failure [16]. In contrast, the PEACE trial study did not show a benefit of ACE inhibition over placebo [17]. Several mechanisms may explain the beneficial effects of ACE inhibition on morbidity and mortality [18]. One of them is the effect of ACE inhibition on shifting the balance between vasoconstrictive forces (angiotensin II) and vasodilatative forces (bradykinin and nitric oxide) towards vasodilatation [18]. Also, ACE inhibition may protect against endothelial apoptosis. These mechanisms lead to an improvement in endothelial dysfunction. The PERFECT study, as a sub-study to the EUROPA trial, was designed to verify the above mentioned pathophysiologic concept [19]. In the PERFECT study, B-mode ultrasonography of the brachial artery was be used to assess endothelial function in response to ischaemia (reactive hyperaemia) as measured by flow mediated vasodilatation (FMD). The present paper describes the main findings of the PERFECT study. BODY.METHODS.DESIGN: The rationale and design of the PERFECT study has been detailed elsewhere [19]. In short, the PERFECT study is a study nested within the EUROPA trial. The EUROPA trial is a three year double-blind, multi-centre, placebo-controlled randomised study that aims at studying the effect of the ACE-inhibitor perindopril on morbidity and mortality in over 12,000 patients with stable coronary artery disease without clinical heart failure [16]. The PERFECT study was designed as a parallel group randomised placebo controlled trial to determine the effect of perindopril (8 mg/day) on brachial artery endothelial function, as assessed by FMD. After a run-in period of 4 weeks with 4 mg/day perindopril, participants were randomised to perindopril or placebo. The recruitment for the PERFECT study started in May 1998. Both the EUROPA trial and the PERFECT study finished recruitment in June 1999. In the PERFECT study 333 patients were recruited in 20 European centers (8 in Czech Republic, 1 in Germany, 2 in Greece, 4 in Netherlands, 4 in Poland and 1 in Sweden). Endothelial function, measured as FMD was assessed at the baseline visit, just before the run-in period. Follow-up B mode ultrasound for FMD assessment were performed at 6, 12, 24 and 36 months after randomisation. In addition, an endothelial independent vasodilatation test (NTG) using nitroglycerine sublingually, was performed at baseline and at 36 months after randomisation. The NTG measurements were performed without withholding of used medications. BODY.METHODS.STUDY POPULATION: The population enrolled in the PERFECT study is similar to that enrolled in the EUROPA trial [16]. In short, the main inclusion criteria are 18 years of age or above; documented coronary artery disease, not scheduled for re-vascularisation and informed consent obtained. Documented coronary heart disease includes a history of previous myocardial infarction (confirmed by ECG demonstrating Q waves in two contiguous leads and/or changes in cardiac enzymes more than or equal to twice the normal values) of at least 3 months prior to the selection visit, or, a history of PTCA or CABG of at least 6 months prior the selection visit, or, abnormal findings at coronary angiography (angiographical evidence of ≥70% narrowing of ≥1 major coronary artery according to visual analysis). Also, men with a history of chest pain documented by either a positive exercise test or by the development of regional wall motion abnormalities during stress echocardiography or nuclear scintigraphy or perfusion defects during scintigraphy perfusion imaging could be included. The main exclusion criteria were clinical signs of heart failure requiring treatment; systolic blood pressure <100 mmHg; uncontrolled treated hypertension with systolic blood pressure >180 mmHg and/or a diastolic blood pressure >100 mmHg; use of ACE inhibitors or angiotensin II receptor inhibitors within 1 month prior to the first selection visit; renal failure with serum creatinine >150 μmol/l; serum potassium >5.0 mmol/l; liver disease with liver enzymes >3 times upper normal values; a history of stroke or cerebral transient ischaemic attacks within the preceding 3 months and known intolerance to ACE inhibitors. All patients who participated in the main EUROPA study and had been enrolled in study centres that participated in the PERFECT study, were additionally invited to participate in the PERFECT study. All those patients that were willing and gave informed consent were considered as PERFECT participants. Since refusal rates for PERFECT were assumed to be independent of the treatment assignment, it was expected that the PERFECT participants would resembles closely the EUROPA participants. BODY.METHODS.PERFECT STUDY MEASUREMENTS: The study outcomes were (1) absolute change in FMD percentage of the brachial artery between the 36 month measurement and the baseline measurement, (2) absolute change in FMD percentage of the brachial artery between the 6 month measurement and the baseline measurement, (3) Rate of change in FMD in 36 months. BODY.METHODS.ASSESSMENT OF BRACHIAL ARTERY FLOW MEDIATED VASODILATATION (FMD): Preceding the start of the PERFECT study, a detailed training programme was implemented to ensure standardisation of the FMD measurements across all centers. Based on the performance during and after the training programme all sonographers entered the certification procedure. Ultrasound examinations were performed using a Duplex scanner with a >7 MHz or 7–10 MHz linear array transducer, with on line ECG recording. The examinations were recorded on (S-)VHS videocassette and sent to a core laboratory for off line reading. The details of the measurement have been detailed elsewhere [19]. In short, patients were studied in supine position. Three ECG leads were attached. The arm was placed in a specifically developed splint to reduce arm movements during the procedure as much as possible and to allow for fixation of the ultrasound transducer. The blood pressure cuff was placed just below the elbow, and below the ultrasound transducer. After a 10 min rest, the brachial artery at the elbow was visualised using a ultrasound machine with a >7 MHz or 5–10 MHz linear array transducer. When a satisfactory longitudinal optimal image of the brachial artery was obtained, the position of the transducer was fixed in the holder. Three B-mode images showing the lumen diameter were frozen on the R-wave of the ECG to provide information for off-line measurement of the 'baseline' lumen diameter for the ischemia test. Then the blood pressure cuff was inflated to suprasystolic levels (50 mmHg above) for a four-minute period. During that time the sonographer checked whether the optimal image was steady. After deflation of the blood pressure cuff, ultrasound examination continued for 5 min. Every 15 s a B-mode image was frozen on the R-wave of the ECG (end-diastole) for off-line lumen diameter measurements [20, 21]. At baseline and at month 36 the effect of nitroglycerine was evaluated after the ischemia test using a similar procedure as described above. When a satisfactory longitudinal optimal image of the brachial artery was obtained, three B-mode images showing the lumen diameter were frozen on the R-wave of the ECG to provide information for off-line measurement of the 'baseline' lumen diameter for the nitroglycerine test. Then 400 μg of nitroglycerine were sublingually administered. The ultrasound examination continued for another 5 min. Every 15 s a B-mode image was frozen on the R-wave of the ECG (end-diastole) for off-line lumen diameter measurements. The off line reading of the PERFECT Study ultrasound examinations was done using Brachial Tools®, version 3.2.6 (Medical Imaging Applications, Iowa, USA). In short, first the frozen images on videotape are digitised and put into a time sequence. Then the reader manually identifies the part of the brachial artery in which the boundaries are good and stable visualised in all these images. Next, the Brachial Tools® software detects the boundaries through all these images and provides numerical data on lumen diameters of all the images. The diameter at baseline was based on the average of the three frozen images. FMD was estimated as [maximal lumen diameter after ischemia − diameter at baseline]/diameter at baseline. The nitroglycerine response (flow independent vasodilatation) was estimated as [maximal lumen diameter after nitroglycerine − diameter at baseline]/diameter at baseline. BODY.METHODS.SAMPLE SIZE CONSIDERATIONS: At the start of the preparations of the PERFECT study in 1998, an estimate of what was considered a clinically relevant difference in FMD from baseline varied between 1.8 and 6.7% in the published literature. In a study among subjects who underwent coronary angiography, the positive predictive value of FMD less than 3% in predicting coronary endothelial dysfunction was 95% [22]. It had been argued that in clinical trials the number of subjects needed should be such that at least a 2% difference in FMD is detectable [23]. In an earlier study performed by our group, among 32 healthy volunteers the mean FMD was 7.7% (SD 5.0) [24]. Since the present study is a multi-centre which generally increases the variability in the FMD measurements compared to single-centre studies, the sample size calculation is to be considered conservative. With a 90% power, a two-sided alpha of 5%, an absolute difference in FMD of 2.0% can be assessed with a sample size of 131 subjects in each arm of the trial. With an anticipated 10% drop-out rate in the EUROPA trial, a total of 288 subjects needed to be randomised. BODY.METHODS.DATA ANALYSIS: General characteristics of the study population were given according to the intention to treat principle (ITT). The ITT population comprised patients who had a 6 month FMD measurement for the analyses on 6 month change in FMD. For 36 month change the ITT population comprised patients with 36 month FMD data. For the rate of change analyses, the ITT population comprised of all those patients who had at least one 6 month baseline FMD value. Those patients who had no follow-up FMD measurements or those who had inadequate measurements were excluded for the statistical analyses. Absence to compliance of treatment was not a reasons to exclude patients from the analyses. For each patient the difference in FMD at 6 month and baseline was calculated (FMD value at 6 months − FMD value at baseline). A similar approach was used for 36 month difference in FMD. Next, linear regression analyses were applied to study whether the 6 month or 36 month differences in FMD differed between treatment groups using difference in FMD as dependent variable and treatment assignment as independent variable. No adjustments were additionally made since the treatment assignment was achieved using a randomisation procedure. These analyses were carried out using SPSS statistical package (version 12.0). This analytic approach had a priori been decided upon although we recognise that for the 36 month effects the reduction in sample size due to the absence of 36 month measurements could be considerable. To evaluate differences in rate of change of FMD between treatment groups a random effects model was applied using SAS statistical software (version 8.1). Apart from the assumption that the change in FMD is linear over time, we made no assumptions for the intercept (random) of the model and for correlation structure (unstructured) between repeated FMD measurements. The primary model FMD percentage as outcome, and had as independent variables assigned treatment group, visit time from baseline, and the interaction term time × group assignment. Rate of change was estimated per 6 month intervals. The advantage of using a random effects model above the earlier mentioned analytic approaches is that all FMD measurements that have been performed during the study will be used in the analyses and thereby increasing the power. Although this is a randomised controlled trial and therefore in principle adjustment for other variables is not needed, we did perform analyses where adjustments were made for study center and for those factors that were imbalanced at baseline (gender, chest pain, hypertension). Reproducibility of the FMD measurement was assessed using the information of the FMD measurement at baseline and at the 6 month visit in the placebo group. Overall reproducibility, i.e., reflecting the combined variability due to between and within sonographer, and between and within reader and between visit aspects, was evaluated using Intraclass correlation coefficient. BODY.RESULTS: The general characteristics of the 333 randomised participants are given in Table 1. Overall the characteristics, including the brachial artery measurements were well balanced between treatment groups except for some factors. The perindopril group comprised more men and more patients with a history of hypertension. In contrast, chest pain was less common. Out of these 333 subjects information on FMD was considered too poor in 20 subjects to be evaluated in the study. These subjects (9 in the perindopril group) were therefore not used in the further analyses with FMD. Figure 1 depicts the distribution of the FMD measurements at baseline (top) and the nitroglycerine response measurements (bottom) among the participants. Follow-up measurement of FMD at 6 month visit, 12 month visit, 24 months and 36 month visits were done in 325, 324, 319, and 301 patients, respectively. Of those, the FMD scans indicated of sufficient quality were 310, 292, 293 and 268, respectively. For NTG measurements at baseline and end of study these figures were 330, 286, 304 and 258, respectively. Data on the reproducibility of the FMD measurement was based on 145 repeat scans within a 6 month time frame, and combined the effects of variance due to the assessment (technical and biological) and due to the offline measurement. The latter has been documented earlier [19], and showed an Intraclass correlation coefficient of 0.65 for the ischemia test. The overall reproducibility was moderate with an Intraclass correlation of 0.12 (p = 0.25). Restriction to centers with higher reproducibility findings did not materially affect the main findings, therefore results pertaining to all participants are presented. Table 1General characteristics of the PERFECT study population by assigned treatmentCharacteristicPerindopril (n = 167)Placebo (n = 166)Age (years)60.88.960.09.6Female gender (%)2112Medical history (%) Previous myocardial infraction59.963.6 Previous PTCA40.142.2 Previous CABG36.534.9 Abnormal CAG76.072.9 History of chest pain and positive exercise test9.615.7 Previous TIA/stroke4.23.0 Peripheral arterial disease10.29.0 Diabetes mellitus16.815.7Current drug use (%) Platelet Inhibitors94.095.2 Lipid lowering62.963.9 Beta blockers78.473.5 Calcium antagonists32.927.1 Nitrates40.136.7 Diuretics12.013.3SBP (mmHg)13716.013514.8DBP (mmHg)80.98.080.28.0History of hypertension (%)53.941.6Heart rate (bpm)66.69.965.78.0Height (cm)171.98.0173.47.5Weight (kg)82.312.382.912.4Current smoking (%)11.412.0Total cholesterol (mmol/l)5.10.845.20.93HDL (mmol/l)1.260.321.280.36LDL (mmol/l)3.020.813.030.78Hypercholesterolaemia (%)69.566.9Brachial artery measurements FMD (%)2.622.642.872.58 Baseline diameter FMD (mm)5.020.805.050.92 Maximal diameter FMD (mm)5.1470.795.190.94 NTG response (%)9.475.99.055.8 Baseline diameter NTG (mm)5.050.795.080.93 Maximum diameter NTG (mm)5.520.805.510.90Values are means with standard deviations or percentages. Significance testing, excluding whether a difference is due to chance is useless, since all potential differences were due to chance because of the randomisation processNTG Nitroglycerin, FMD flow mediated dilatation, LDL low density lipoprotein, HDL high density lipoprotein, SBP systolic pressure, DBP diastolic pressure, TIA transient ischemic attack, CAG coronary angiogram, CABG coronary artery bypass grafting, PTCA percutaneous coronary angioplastyFig. 1Distribution of FMD values and NTG values at baseline in the PERFECT study participants In Fig. 2 the FMD response over time is presented. An improvement is seen in the perindopril group, starting after 12 months, whereas in the placebo group the trend is towards no change over time. Fig. 2Mean FMD (standard errors) by visit and assigned treatment group (perindopril dotted; placebo solid) The mean change in FMD between baseline and 36 months was 0.91% (SD 3.77) in the perindopril group and 0.35% (SD 3.63) in the placebo group. The change in FMD of the brachial artery between the 36 month measurement and the baseline measurement was 0.55 % (95% confidence interval −0.42 1.47; p value 0.23) higher in the perindopril group compared to placebo. This statistically non-significant difference constitutes a relative improvement in FMD of 20% (=0.55/2.74). This analysis was based on measurements available at both baseline and 36 month of 256 participants. Adjustment for center or for imbalances at baseline did not materially affect the magnitude, direction or significance of the intention-to-treat estimate. The change in flow-mediated vasodilatation of the brachial artery between the 6 month measurement and the baseline measurement between treatment groups was −0.12% (95% CI −0.87, 0.85; p value 0.97). This analysis was based on measurements available at both baseline and 6 months of 294 participants. Adjustment for center or for imbalances at baseline did not materially affect the magnitude, direction or significance of the intention-to-treat estimate. The rate of change in endothelial function per 6 months as estimated by a random effect model using data of all participants was 0.14% (SE 0.05, p = 0.02) in the perindopril group and 0.02% (SE 0.05, p = 0.74) in the placebo group (Fig. 3) The difference in rate of change in FMD between the treatment arms was 0.12% (p = 0.07). Adjustment for center or for imbalances at baseline did not materially affect the magnitude, direction or significance of the intention-to-treat estimate. Fig. 3Rate of change in flow-mediated vasodilatation (FMD) per 6 month. P value for the difference between treatment arms No difference in nitroglycerine response results at 36 months was found between perindopril and placebo. Mean values (SD) were 8.2% (5.4) and 8.8% (5.4), respectively. BODY.DISCUSSION: The PERFECT study was designed to evaluate whether long-term administration of perindopril improves endothelial dysfunction in patients with stable CAD and without clinical heart failure. Our findings, analysed in two ways, indicate that long-term use of perindopril is compatible with an improvement in FMD, although these findings were not statistically significant. The rate of change in endothelial function per 6 months showed a significant improvement over time in the perindopril group, where the comparison with placebo showed that these improvements bordered on statistical significance (p of 0.07). The mechanisms underlying the beneficial effects of ACE inhibition are complex, and include inhibition of angiotensin II production with, subsequently, a reduction of its negative effects on the vascular system. Through coupling to the angiotensin II type 1 receptor, angiotensin II leads to vasoconstriction, vascular inflammation with an increase in pro-inflammatory genes, adhesion molecules and macrophage recruitment, an increased uptake and oxidation of LDL by endothelial cells and oxyradical production leading to endothelial dysfunction. Furthermore, it stimulates sympathetic activation and aldosterone release, adding to vasoconstriction and endothelial dysfunction. ACE inhibitors may positively affect endothelial dysfunction by decreasing angiotensin II production alone. However, equally or possibly more important is their effect on bradykinin. Through coupling to the bradykinin B2 receptor bradykinin results in NO and endothelial-derived hyperpolarizing factor. In addition it promotes prostacyclin release. As a consequence, bradykinin is not only a strong vasodilator, it also inhibits, through NO production, vascular smooth muscle cell growth and migration, improves endothelial function, inhibits the expression of cell adhesion molecules, prevents platelet adhesion, and restores the fibrinolytic balance by tPA production. As such, bradykinin effectively counteracts the deleterious effects of angiotensin II. The improvement of endothelial function with ACE inhibition is bradykinin-dependent [25]. Of importance, not all ACE inhibitors increase bradykinin production to a similar extent. This depends, amongst other factors, on tissue affinity of the ACE inhibitor. Perindopril is one of the few ACE inhibitors with profound tissue affinity [26]. It increases bradykinin significantly more than enalapril despite similar effects on circulating angiotensin II [27]. Also, perindopril increases bradykinin levels at lower doses than needed to reduce angiotensin II levels [28]. Indirectly, the pivotal role of bradykinin on endothelial function is suggested by the observation that perindopril, but not an angiotensin receptor antagonist telmisartan, improves endothelium-dependent vasodilatation, after long-term treatment despite similar anti-hypertensive effects [29]. The PERTINENT study, another sub-study of EUROPA, provides further evidence in support for the improvement in endothelial function observed in the PERFECT study [50]. PERTINENT studied the effect of 8 mg perindopril versus placebo after one year treatment on plasma markers of atherosclerosis, neurohormonal activation and apoptosis. A significant reduction in angiotensin II and cytokine levels was found together with a reduction in apoptosis. Of importance, bradykinin increased significantly accompanied by an increase in ecNO synthase in perindopril but not in placebo treated patients. These data from a similar patient population as in PERFECT provide evidence that pivotal mechanisms in endothelial function are improved by perindopril treatment and could lead to an improvement in endothelial function, as indicated in PERFECT. There have been several studies performed on the effect of ACE inhibition and endothelial function as measured by FMD of the brachial artery (Table 2) [30–42]. Most studies were single centre studies, had a limited number of subjects enrolled (varying from 9 to 46), and were of short duration (from postprandial effect to 6 months). The designs applied were mostly cross-over trials. Overall, the effects varied from beneficial effects on FMD response to no effect at all without consistency in patient groups. That severely limits direct comparison with our results. In PERFECT the first FMD measurement after randomisation was at 6 months. No difference in FMD was found at 6 months between treatment groups. Our study is unique in that we studied long term effects of intervention on FMD. A recent review of studies on the long term effect of ACE inhibition on endothelial function, as assessed by a variety of measurements (vascular response and/or plasma markers) concluded that for CAD patient results are consistently showing an improvement in endothelial function [43]. This is substantiated by the results from trials showing that coronary endothelial dysfunction improves after 6 months of treatment with ACE inhibitors [44]. Table 2Studies performed on the effect of ACE inhibition and endothelial function as measured by FMD of the brachial arteryAuthorPublication yearStudy designType of patientFollow-upInterventionControl armFindingsYavuz [31]2003RCTEssential hypertensives6 monthsEnalapril (n = 12)Losartan (n = 9)FMD improved under both treatmentsTezcan [32]2003Case controlEssential hypertensives versus healthy controls6 monthsEnalapril (n = 9)FMD improved under enalaprilGhiadoni [33]2003RCT (before/after)Essential hypertension6 monthsNifedipine (n = 28) amlodipine (n = 28) atenolol (n = 29) nebivolol (n = 28) telmisartan (n = 29) perindopril (n = 28)FMD improved under perindopril onlyEllis [34]2002RCTStable heart failure 1 monthAce inhibition plus candersatan (n = 14)Ace inhibition plus placebo (n = 14)No difference in FMDBae [35]2001RCTAngiographically CADPostprandialLisinoprilHigh fat/low fat/fenofibrateNo effect of FMDCheetham [36]2001Cross over RCTType II diabetes4 weeksLosartan (n = 12)PlaceboImprovement of FMDSchalwijk [37]2000Case controlType II diabetes/healthy controls5 weeksQuinaprilPlaceboNo difference in FMDAnderson [38]2000Cross over RCTStable CHD patients8 weeksQuinapril (n = 20) enalapril (n = 20) losartan (n = 20) amlopidine (n = 20)FMD improved only under quinaprilMcFarlane [39]1999Cross over RCTType I diabetes12 weeksPerindopril (n = 20)TriamtereneNo effect on FMD of perindoprilKoh [40]1999Before/after comparisonCHD8 weeksQuinapril (n = 9)Improvement in FMDWilmink [41]1999Cross over RCTHealthy volunteers2 weeksQuinapril (n = 30) losartan (n = 30)No improvement in basal FMD , improvement in postprandial FMD reductionMullen [42]1998RCTInsulin diabetes24 weeksEnalapril (n = 46)Placebo (n = 45)No effect on FMDRCT Randomised controlled trial, CHD coronary heart disease, FMD flow mediated vasodilatation Some aspects of the study need some consideration to appreciate the findings. Firstly, the initial sample size estimation was based on assumptions that came from cross-sectional studies, since longitudinal data were not available. When we perform a posterior power calculation based on the availability of 240 subjects using actually observed data on the difference between baseline and 36 months in FMD in the control arm (0.35% with a SD 3.63), we had a 80% power to detect a difference in change from baseline between the perindopril group and the placebo group of 1.3%, assuming a two sided alpha of 0.05. Our power to detect a 2% difference, as was originally proposed, was even higher. However, our power to detect the currently observed difference of 0.55% was only 20%, suggesting that we can not really exclude the fact that perindopril may have an effect on endothelial function. Secondly, it has been documented that the value of FMD measurement may be affected considerably by life style habits, such as recent physical activity, smoking, and food intake, technical aspects of the measurement, such as cuff location, and concomitant drug treatment, such as statins [45–48]. Furthermore, the reproducibility of the FMD measurement in situations where lifestyle factors were controlled optimally, may be moderate with coefficients of variation between 25–50%, indicating that within person variability may be considerable [49]. This in general leads to attenuation of the magnitude of the relations under study. Although in PERFECT a uniform training and FMD methodology was used, the study was a multicenter trial, which compared to single center studies generally increases variability as shown by our reproducibility findings. This was one of the reasons we applied a random effect model to increase the power of the study to detect meaningful differences between treatment arms. The baseline FMD value in our population was low, similar to that found in other studies in CAD patients [48], and may indicate the presence of endothelial dysfunction in this group. The effect of ACE inhibition on FMD over time was modest, although significant. This may be partially attributed to the fact that most of our patients were using multiple drugs that improve endothelial function as well, for example statins [49]. This may have reduced our ability to demonstrate the full benefit of ACE inhibition per se. In conclusion, appreciating the variability in the FMD measurement and the widespread use of other drugs that may affect FMD, our findings are compatible with the notion that the beneficial effects of perindopril on cardiovascular morbidity and mortality in the EUROPA study may be at least partly explained by an improvement in endothelial function. Participating PERFECT centers CZECH REPUBLIC: CESKY KRUMLOV: J. Florian, MD (principal investigator), V. Kuchar, MD; BRNO: B. Semrad, MD (principal investigator), J. Ziembova, MD, J. Schildberger, MD; PILSEN, H. Rosolova, MD (principal investigator), V. Jankovych, MD; PRAQUE: R. Spacek, MD (principal investigator), P. Stanka; PRAQUE: J. Hradec, MD (principal investigator), J. Malik, MD; TABOR: J. Charouzek, MD (principal investigator) V. Jirka, MD; PRAGUE: P. Jansky (principal investigator), Koelbel, MD; SLANY: G. Marcinek, MD (principal investigator), M. Votypka-Pecha; BRNO: L. Groh (principal investigator), I. Hofirek, L. Nechvatal. GERMANY: MUNICH: C. von Schacky, MD, PhD (principal investigator), S. Stoerk, MD, P. Markov. GREECE: ATHENS: Gialavos, MD, (principal investigator), A. Androulakis, MD; ATHENS: J. Lekakis, MD, C. Papamichael, MD. NETHERLANDS: HARDERWIJK: R. Dijkgraaf, MD (principal investigator), Y.Jansen-Timmer, H. Bralts; HENGELO: J.J.J. Bucx, MD (principal investigator until 01/01/2000), H. Droste, MD (principal investigator from 01/01/2000), G. Assink; EMMEN: L.F.M. van de Merkhof, MD (principal investigator), R. Vinke; ROTTERDAM: van Nierop, MD (principal investigator), I.M. Toonder, E. Korten. POLAND: KRAKOW: A.Gackkowski, MD (principal investigator); KRAKOW: T. Zielinsky, MD (principal investigator), T. Rywik, MD; KATOWICE: A.M. Wnuk-Wonjar, MD (principal investigator), C. Vita; KATOWICE: M. Tendera, MD (principal investigator), M. Kazmierski; SWEDEN: MALMO: J. Persson, MD (principal investigator), G. Osting; Endothelial function core laboratory Vascular Imaging Center Utrecht, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands. Michiel Bots, MD, PhD; Ronald P. Stolk, MD, PhD; Rudy Meijer, Dicky Mooiweer-Bogaerdt ; Frank Leus, Brigitte Wernert, Eefje Spithoven, (http://www.juliuscenter.nl/research facilities/vascular imaging center) EUROPA Executive Committee Bertrand M, Ferrari R, Fox K, Remme W.J, Simoons M. EUROPA Steering Committee Aldershville J, Denmark; Hildebrandt P, Bassand JP, France; Cokkinos D, Greece; Toutouzas P, Greece, Eha J, Estonia; Erhardt L,Sweden; Erikssen J, Noway; Grybauskas R, Lithuania; Kalnins U, Latvia; Karsch K, Sechtem U, Germany; Keltai M, Hungary; Klein W, Austria; Luescher T, Switzerland; Mulcahy D, Ireland; Nieminen M, Finland; Oto A, Ozsaruhan O, Turkey; Paulus W, Belgium; Providencia L, Portugal; Remme W J, the Netherlands; Riecansky I,Slovakia; Ruzyllo W, Poland; Santini U, Tavazzi L, Italy; Soler-Soler J, Spain; Widimsky P, Czech Republic.

TITLE: Preoperative conventional chemoradiotherapy versus short-course radiotherapy with delayed surgery for rectal cancer: results of a randomized controlled trial ABSTRACT.BACKGROUND: There still is no evidence which neoadjuvant therapy regimen for stage II–III rectal cancer is superior. The aim of this study was to compare results achieved after long-course chemoradiotherapy (CRT) with short-term radiotherapy (RT) followed by delayed surgery. ABSTRACT.METHODS: A randomized trial was carried out between 2007–2013. One hundred fifty patients diagnosed with stage II–III rectal cancer were randomized into one of two neoadjuvant treatment arms: conventional chemoradiotherapy (CRT) and short-term radiotherapy (RT) followed by surgery after 6–8 weeks. Primary endpoints of this trial were downstaging and pathological complete response rate. Secondary endpoints were local recurrence rate and overall survival. ABSTRACT.RESULTS: The pathological complete response was found in 3 (4.4%) cases after RT and 8 (11.1%) after CRT (P = 0.112). Downstaging (stage 0 and I) was observed in 21 (30.9%) cases in RT group vs. 27 (37.5%) cases in CRT group (P = 0.409). Median follow-up time was 39.7 (range 4.9–79.7) months. 3-years overall survival (OS) was 78% in RT group vs. 82.4% in CRT group (P = 0.145), while disease-free survival (DFS) differed significantly – 59% in RT group vs. 75.1% in CRT group (P = 0,022). Hazard ratio of cancer progression for RT patients was 1.93 (95% CI: 1.08–3.43) compared to CRT patients. ABSTRACT.CONCLUSION: Three-years disease-free survival was better in CRT group comparing with RT group with no difference in overall survival. ABSTRACT.TRIAL REGISTRATION: http://clinicaltrials.gov identifier NCT00597311. January 2008. BODY.BACKGROUND: Preoperative conventional chemoradiotherapy (25 × 2 Gy + 5-Fu) with delayed surgery and short-term radiotherapy (5 x5Gy) with immediate surgery are the most common regimens of treatment, causing reduction of local recurrence rate for patients with resectable rectal cancer [1–7]. Two meta-analysis [8, 9] and review [10] showed no differences between these regimens in terms of survival, local recurrence, morbidity, mortality, resectability and the rate of sphincter preservation; however, pathological complete response and toxicity were higher after neoadjuvant chemoradiation. Radu et al. [11] were the first to present the hypothesis that short-term radiotherapy and delayed surgery could give similar results as conventional CRT. Stockholm III trial reported similar results of complete response, rate of complications and toxicity after preoperative long-course or short-course radiotherapy with delayed surgery [12]. The approach of short-term radiotherapy with delayed surgery has also been evaluated in prospective randomized trial (http://clinicaltrials.gov identifier: NCT00597311) which was carried out between 2007–2013 in the Hospital of Lithuanian University of Health Sciences. Comparison was mainly based on the rates of downstaging after short-term radiotherapy or conventional chemoradiotherapy followed by surgery after 6–8 weeks. Initial results of this trial including the first 83 patients were published previously in 2012 [13]. This is a secondary evaluation of the treatment arms including the same 83 and the total required 150 patients. BODY.METHODS: The study was approved by Kaunas Regional Committee of Ethics of Biomedical Research (Protocol No. 137⁄2006). The inclusion, exclusion criteria and initial results have been reported previously [13]. Completely informed consent was obtained from the patients. Digital examination, endoscopy with biopsy, chest X-ray, abdominal ultrasound and blood analysis were performed. T and N stages were assessed using endorectal ultrasound (ERUS), pelvic computerized tomography (CT) and magnetic resonance imaging (MRI) and patients with resectable stage II or III (T3 N0, T4 N0, Tx N+) rectal cancer were included and blindly randomized into one of the two arms. ERUS, pelvic CT and MRI were repeated before the surgery for re-evaluation of T and N stage and maximal wall thickness. The investigator was not blinded to the pre-treatment ERUS results. The problem of clinical staging was that ERUS was not technically possible for all patients because of localization and size of the tumor and MRI was not made for the first 40 patients. One hundred fifty patients diagnosed with resectable stage II or III rectal cancer were randomized to two neoadjuvant treatment arms: conventional chemoradiotherapy (CRT; 50 Gy in total administered during a period of 5 weeks, 2 Gy per fraction and two cycles of 5-FU/Leucovarin, 400 mg/m2 of 5-fluorouracil i/v in combination with leucovorin 20 mg/m2 i/v for 1–4 days on the first and on the fifth week) and short-term radiotherapy (RT; 5 fractions of radiotherapy, 5 Gy per fraction, administered each day for 5 days, a dose of 25 Gy in total). Patients were assessed with respect of possible adverse effects of neoadjuvant treatment, along with filling QLQ questionnaires. Data regarding toxicity will be presented in another article. Both groups underwent surgery in a period of 6 to 8 weeks after a termination of preoperative treatment. Type of surgery was chosen depending on the site of lesion in images of ERUS, pelvic CT and MRI, but final decision was made during the surgery and it was one of the following: abdominoperineal resection, Hartman's procedure, proctectomy with coloanal anastomosis, or anterior rectal resection. Surgery was performed by the same 6 surgeons specializing in coloproctology. Total mesorectal excision was mandatory during surgery. Adjuvant Chemotherapy of 5-FU (400 mg/m2 i/v and Leucovorin (20 mg/m2 i/v) 1–5 days for 4 cycles every 4 weeks was started within 8 weeks after surgery. BODY.METHODS.FOLLOW-UP: Patients were examined every 3–6 months during the first 2 years after surgery, and once per year for the following 5 years at least. Evaluation consisted of physical examination, blood tests, abdominal ultrasound, chest radiography and colonoscopy every 6–12 months. CT and MRI were performed in cases of suspected local recurrence or metastasis. No patient was lost during the follow up. BODY.METHODS.STATISTICAL ANALYSIS: The primary endpoint of this trial was downstaging and pathological complete response rate. In order to calculate the sample size it was assumed that the downstaging rate after chemoradiotherapy would be 40%. To detect a 20% difference between the groups using the χ2 test and a significance level of 0.05, the study should include 74 patients (37 patients in each group). Secondary endpoints were local recurrence and overall survival. Secondary endpoints analysis was planned to be performed after recruitement of 150 patients. The χ2 test was used for comparison of proportions and the Mann–Whitney U test for comparison of continuous variables. Actuarial curves were calculated by the Kaplan–Meier method and were compared by the log rank test. The Cox's proportional hazards model was used to calculate the hazard ratios and 95% confidence intervals (c.i.) in the univariable analysis. All the tests were two sided. Calculation of time intervals was started from the date of randomization. Patients who had not undergone primary tumour excision or who had distant metastases detected before or during surgery were considered as treatment failures at the time of randomization and were excluded from the study. When calculating the rate of permanent stoma, diverting stoma or stoma after Hartmann's procedure were excluded, if stoma was reversed later. BODY.RESULTS: Amongst 150 randomly assigned patients 10 were excluded from the study due to protocol violation. All eligible patients were included in the statistical analysis (Fig. 1). Patients' characteristics were similar between the two treatment groups (Table 1).Fig. 1CONSORT diagram for patients randomized in the trial Table 1Patients main characteristicsVariableRT n = 68CRT n = 72 P Age (years)65.6 (SD = 9.5)63.14 (SD = 10.1)0.141Gender (%) Male43 (63.2)50 (69.4)0.437 Female25 (36.8)22 (30.6)ASA1 (%)1 (1.5)1 (1.4)ASA2 (%)32 (48.5)31 (43.1)0.808ASA3 (%)33 (50)40 (55.5)Period from the end ofneoadjuvant therapy till surgery (days)48.03 (SD = 12.5)47.14 (SD = 8.6)0.622Clinical stage (%) II16 (23.5)15 (20.8)0.701 III52 (76.5)57 (79.2)Tumor distance from anal verge (%) < 5 cm34 (50)30 (41.7) 5–10 cm29 (42.6)37 (51.4)0.575 11–15 cm5 (7.4)5 (6.9) BODY.RESULTS.SURGERY: RT and CRT groups were comparable regarding the type of surgery and morbidity. Results are presented in Table 2.Table 2Type of surgery and complications according to treatment groupsVariableRT (%) n = 68CRT (%) n = 72 P Radical surgery57 (83.8)64 (88.9)0.382Non-radical11 (16.2)8 (11.1) Postive distal margin (R+)20 Positive circumferential margin (CRM ≤1 mm)88 Positive both margins (R and CRM)10Postoperative hospital stay (days)10.06 (SD = 6.9)9.15 (SD = 3.7)0.355Sphincter saving surgery47 (69.1)52 (72.2)0.687Permanent stoma27 (39.7)25 (34.7)0.542Defunctioning stoma41 (60.3)47 (65.3)Anterior resection with anastomosis35 (51.5)40 (55.6)0.959Proctectomy with coloanal anastomosis6 (8.8)7 (9.7)Hartman's procedure6 (8.8)5 (6.9)Abdominoperineal resection21 (30.9)20 (27.8)Complications24 (35.3)19 (26.8)0.277 Anastomotic4 (5.8)5 (6.9) Wound7 (10.2)6 (8.3) Stoma4 (5.8)1 (1.4) Intrasurgical2 (3.3)3 (4.3) Other7 (10.2)4 (5.9)Reoperations1 (1.5)4 (5.6)0.235 BODY.RESULTS.PATHOLOGY: A histopathological complete response was found in 3 (4.4%) cases after RT and 8 (11.1%) after CRT (P = 0.112). Downstaging (stage 0 and I) was observed in 21 (30.9%) cases in RT group vs.27 (37.5%) cases in CRT group (P = 0.409). There were more cases with early pathological stage (pT0, pT1) in the CRT group and more cases with pT3 disease in the RT group, however, the difference was not statistically significant (P > 0.05). The positive lymph-nodes were found in 25 (36.8%) cases in the RT group and in 18 (25%) cases of CRT group (P > 0.05). The majority of patients had a moderately differentiated (G2) tumour (83.6% and 79.7% in the RT and CRT groups, respectively). Groups were comparable in terms of circumferential and distal margin, tumor size, vascular and lymphatic invasion (Table 3).Table 3Results of postoperative pathological examinationVariableRT (%) n = 68CRT (%) n = 72 P ypT03 (4.4)8 (11.1)0.112ypT13 (4.4)5 (6.9)ypT218 (26.5)19 (26.4)ypT344 (64.7)36 (50)ypT404 (5.6)ypN043 (64.2)54 (75)0.318ypN119 (27.9)14 (19.4)ypN26 (8.9)4 (5.6)Differentiation G16 (9)5 (7.8)0.306 G256 (83.5)51 (79.7) G35 (7.5)8 (12.5)ypL1 (lymphatic)23 (37.1)22 (31.9)0.530ypV1 (vascular)19 (31.1)15 (21.7)0.233Distal resection margin (mm)29.83 (SD = 15.1)31.62 (SD = 17.1)0.538Circumferential resection margin (mm)5.02 (SD = 4.44)7 (SD = 7.19)0.193Tumor size (mm)26.22 (SD = 12.22)25.13 (SD = 12.48)0.622 BODY.RESULTS.LONG-TERM ONCOLOGICAL RESULTS: Median follow-up time was 39.7 (range 4.9–79.7) months. During the time of observation 31 patients had died: 17 (25%) in RT group and 14 (19.4%) in CRT group (P > 0.05). Hazard ratio of death for RT patients compared to patients in CRT group was 1.64 (95% CI: 0.8–3.43). Cancer progression was observed in 16 (25%) cases in RT group vs. 13 (18.3%) cases in CRT group (P > 0.05). The rate of local recurrence between the groups was: 2 (3.1%) cases in RT group vs. 4 (5.6%) in CRT group (P > 0.05). During the follow-up distant metastases developed in 14 (21.9%) cases after RT and in 9 (12.7%) cases after CRT (P > 0.05). Hazard ratio of distant metastases for RT patients compared to CRT patients was 2.2 (95% CI: 0.95–5.10). Causes of death and cancer progression are summarized in Table 4.Table 4Causes of death and cancer progressionVariableRT (%) n = 68CRT (%) n = 72 P Death17 (25)14 (19.4)0.429 related to cancer1212 not related to cancer30 unknown22Progression of rectal cancer16 (25)13 (18.3)0.345 local recurrence alone03 (4.2) distant metastases alone12 (18.8)8 (11.3) local recurrence with distant metastases in liver and lungs2 (3.1)1 (1.4) metastatic tumor in peritoneal cavity2 (3.1)1 (1.4) Three- years OS was 78% in RT group vs. 82.4% in CRT group (P = 0,145), while DFS was 59% in RT group vs. 75.1% in CRT group (P = 0,022) (Fig. 2). Hazard ratio of cancer progression (distant and local) for RT patients compared to CRT patients was 1.93 (95% CI: 1.08–3.43).Fig. 2Overall and disease free survival rates for the trial patients according to treatment received BODY.DISCUSSION: Currently there is no optimal neoadjuvant treatment regimen for locally advanced rectal cancer yet. According to ESMO clinical practice guidelines of 2013, conventionally fractionated chemoradiotherapy (25 × 2 Gy) with delayed surgery or short-course radiotherapy (5 ×5 Gy) with surgery in 1 week for resectable rectal cancer are recommended [14]. These recommendations are based on the results of well known trials [1], but the problem persists that these two treatment options are quite different, resulting in different treatment policies among countries or even specialists in the same country. Two meta-analyses revealed no differences between these regimens in terms of the rates of survival, local recurrence, morbidity, mortality, resectability and the rate of sphincter preservation, and only pathological complete response and toxicity were higher after neoadjuvant chemotherapy [8, 9]. Bujko et al. compared neoadjuvant short-course radiotherapy (RT) followed by surgery within 7 days with conventional long-course chemoradiotherapy (CRT) and found that 4 years overall, disease-free survival rates and local recurrence rate did not differ significantly between the groups [6]. Complete response rate was higher in CRT group: 16.1% vs 0.7%, tumor involvement of the circumferential margin was 4% after chemoradiotherapy vs 13% after short-course radiotherapy (P < 0.05), but the toxicity (incidense of III-IV grade adverse effects) rate was also significantly higher 18.2% vs 3.2% in CRT vs RT group, respectively. Retrospective data from Radu et al. showed that pathological complete response and local disease control have been similar between short-term radiotherapy with delayed surgery (6–8 weeks) and long-course chemoradiotherapy, with low rates of toxicity in both groups [11]. Stockholm III trial compared short-course radiotherapy (5 × 5 Gy) with long-course radiotherapy (25 × 2 Gy) without chemotherapy both with delayed surgery and reported 12.5% complete response rate after short-course RT compared with 5% after long-course RT (P < 0.05) [12]. On the contrary, we found that complete response rate was 4.4% after RT vs. 11.1% after CRT (P > 0.05) and downstaging (pathological stage 0 and I) was observed in 30.9% cases in RT group vs. 37.5% cases in CRT group. According to Stockholm III trial results, pathological stage 0 and I was found in 45% cases after short-course and in 30% cases after long-course RT. These differences could be explained as follows: chemotherapy was added to long-course radiotherapy according the protocol of our trial. Disease progression was observed in 25% of cases in RT group vs. 18.3% of cases in CRT group. The rate of local recurrence between groups was: 3.1% of cases in RT group vs 5.6% of cases in CRT group, respectively. 3 year overall survival was comparable between the groups: 82.4% after CRT vs. 78% after RT, but disease-free survival was significantly better after CRT (75.1%) than after RT (59%). Distant metastases (undetected preoperatively) were found intraoperatively for 5 patients (6.7%) after short-course radiotherapy and 3 (4%) patients after chemoradiotherapy. These patients were excluded from the analysis of long-term results. The question if this distant spread was missed during primary investigation is open, because abdominal ultrasound (not CT) as a routine method of investigation was used according our trial protocol, but it could also be the result of early cancer progression, and in that case DFS would be slightly worse. Local recurrence rate for resectable rectal cancer after surgery with TME is quite low. The TME approach increases the likelihood of clear circumferential resection margins which corresponds to decreased rates of pelvic recurrence [15]. The majority of rectal cancer deaths is likely to be associated with distant metastases, not from local recurrence. This could explain why no survival benefit was found in the majority of trials comparing various regimens of neoadjuvant treatment for rectal cancer [2]. Chemotherapy controlling distant progression of the disease could be beneficial for these patients, but FFCD study did not prove any advantage for the addition of 5-Fu to RT in terms of DFS or OS [7]. There is no discussion that low-risk rectal cancer patients for whom imaging allows safe R0 resection should go for initial surgical treatment, while preoperative chemoradiotherapy should be administered for patients with high risk of local recurrence (threatened resection margins), [16]. A major concern is a large group of patients with intermediate-risk rectal cancer (T3, >3 mm CRM), for whom neoadjuvant chemoradiotherapy appears to be potential overtreatment, but preoperative systemic chemotherapy controlling distant spread could be beneficial. A pilot trial from Memorial Sloan Kettering Cancer Center reported promising results of selective use of chemoradiation for patients with intermediate-risk rectal cancer. Results from this pilot study served as a background to initiate the currently undergoing PROSPECT trial [16]. Despite discussions most authors agree that careful staging and individualized treatment approach including selective combination of surgery, chemo- or radiotherapy, should be recommended for patients with rectal cancer. BODY.CONCLUSION: According to the results of the randomized controlled trial, 3-years DFS was better in CRT group compared with RT group with no difference in OS. Surgical recovery and perioperative morbidity were similar between the groups.

TITLE: Efficacy of a Probiotic‐Prebiotic Supplement on Incidence of Diarrhea in a Dog Shelter: A Randomized, Double‐Blind, Placebo‐Controlled Trial ABSTRACT.BACKGROUND: Diarrhea is the most frequent morbidity affecting kenneled dogs in animal shelters. Diarrhea impacts animal welfare and the finances of the shelter as they must treat, clean, and house affected animals until recovered. ABSTRACT.HYPOTHESIS/OBJECTIVES: Supplementing dogs entering an animal shelter with a probiotic‐prebiotic, known as a synbiotic, will decrease the incidence of diarrhea. ABSTRACT.ANIMALS: Seven hundred and seventy‐three dogs entering an animal shelter in the United Kingdom. ABSTRACT.METHODS: A prospective double‐blind, randomized, placebo‐controlled trial. ABSTRACT.RESULTS: Statistical difference was found between the groups across 3 measures of diarrhea incidence. First, the mean percentage of scored days per dog that were scored as diarrhea throughout their stay was 2.0% in the synbiotic group and 3.2% in the placebo group (P = .0022). Second, the occurrence of diarrhea within the first 14 days' stay was 18.8% in the synbiotic product group and 27.2% in the placebo group (P = .0008). Third, the occurrence of ≥2 consecutive days of diarrhea within the first 14 days' stay was 4.6% in the synbiotic product group and 8.0% in the placebo group (P = .0300). ABSTRACT.CONCLUSIONS AND CLINICAL IMPORTANCE: Supplementing healthy dogs entering an animal shelter with a synbiotic supplement significantly decreased the incidence of diarrhea in this trial. Animal shelters can use synbiotic supplements to improve animal welfare and decrease costs involved in cleaning and housing animals as well as potentially decreasing veterinary intervention. BODY: AbbreviationsCFUcolony‐forming unitUKASUnited Kingdom Accreditation Service Diarrhea is defined as an increase in fecal water content, which usually leads to changes in fecal volume, fluidity, and frequency of defecation.1 Kenneling increases the likelihood of dogs developing diarrhea,2 and diarrhea is reported as the most common disease of dogs housed in animal shelters.3 Diarrhea has a direct negative impact on the welfare of kenneled dogs and also may lead to extended time periods in the animal shelter as affected animals cannot be rehomed until they are producing normal feces. In addition, there is a financial burden on the animal shelter resulting from delayed rehoming, veterinary interventions, and additional cleaning. Probiotics are live microorganisms that, when delivered in adequate amounts, confer a health benefit to the host.4, 5 Prebiotics are ingredients selectively fermented in the gastrointestinal tract to allow specific changes, in the composition, activity, or both in the gastrointestinal microbiota, conferring benefits upon host well‐being and health.6 When a probiotic and a prebiotic are given together, the combination is called a synbiotic, suggesting a synergistic relationship between the 2. There are several proposed mechanisms to the underlying antagonistic effects of probiotics on various microorganisms, including the following: modification of the gut microbiota, competitive adherence to the epithelial mucosa, strengthening of the gut epithelial barrier, and modulation of the immune system to convey an advantage to the host.7 These proposed probiotic mechanisms of action could have a clinically relevant impact on treatment and prevention of acute diarrhea.8 Two previous clinical trials have investigated the use of probiotic supplementation for the prevention of diarrhea in kenneled dogs. In 1 trial that used different doses of probiotic, significantly fewer supplemented dogs passed unacceptable (loose or diarrheic) feces in the first week of relocation to a kennel facility and during their total stay, when compared to the control group.9 Another study failed to obtain statistically significant evidence to accept their hypothesis that kenneled dogs fed a probiotic would be less likely to have diarrhea of ≥2 days duration than those given placebo,10 and this study frequently is referenced in review articles about probiotics11, 12, 13, 14, 15, 16, 17, 18 when discussing the efficacy of probiotics on gastrointestinal disease in dogs. Yet of the 182 dogs recruited in the trial, only 2 (1 in each group) had diarrhea of ≥2 days' duration.10 With this incidence of diarrhea in the placebo group (1.25% of dogs), 18,608 dogs would have been required to detect a 33% reduction in ≥2 days' diarrhea, with a significance level of 0.05 and a power of 80%1. Therefore, it is not possible to draw conclusions about probiotic efficacy in kenneled dogs from this trial because of the low incidence of diarrhea. The aim of our study was to test the hypothesis that prophylactic treatment of dogs in an animal shelter with a synbiotic supplement would decrease the incidence of diarrhea compared with a control group. BODY.MATERIALS AND METHODS: A prospective, randomized, double‐blind placebo‐controlled trial was designed for dogs entering Wood Green, The Animals Charity, Godmanchester, for which ethical approval was obtained from the University of Cambridge Ethics and Welfare Committee (reference CR93). All dogs entering the animal shelter were considered for inclusion in the trial. Animals were excluded if they left the shelter before kennel assignment and randomization (i.e., strays that were reclaimed by their owners). All dogs entering the shelter were examined by a veterinarian, and all dogs with abnormal clinical findings suggesting preexisting disease or with a history of chronic or recurrent diarrhea were excluded from the trial (see Fig 1). Figure 1Flow diagram of dogs through the trial phases. On admission to the animal shelter, dogs were housed in an isolation block for 7–10 days before being moved to a main kenneling site that could hold a maximum of 100 dogs. Dogs were fed twice daily by a variety of donated foods or a prescribed diet if the veterinarian deemed doing so necessary. During kennel assignment, dogs were randomized to receive either synbiotic or placebo capsules by taking a randomly selected colored plastic disc from an opaque bag. The bag contained fifty identical plastic discs with equal numbers of 2 different colors; each color assigned to a specific group. Once a counter was selected, it was placed back in the bag. When 2 dogs entered the shelter as a pair, they shared a kennel and both animals were assigned to the same treatment group. The synbiotic and placebo capsules were produced by Protexin Veterinary and matched for color, size, texture, and packaging. They were differentiated only by capsule A or capsule B printed on the blister foil. The active capsule was a commercially available product containing Enterococcus faecium NCIMB 10415 4b1707, 2 × 109 colony‐forming units(cfu)/capsule, and 46.4 mg/capsule Preplex® prebiotic, a combination of fructo‐oligosaccharide (FOS) and acacia (gum arabic)2 . The placebo contained 180 mg maltodextrin, a complex carbohydrate commonly used as an inert additive. One capsule was opened and sprinkled on each dog's food once daily, at the evening meal. Supplementation began at the first evening meal after randomization and continued throughout the dog's stay. All staff at the animal shelter and the monitor were blinded to capsule contents. The unblinding key was held by a third party with no other role in the trial. Dogs had their fecal consistency score recorded on a daily health sheet throughout the duration of their stay at the shelter. Any results from the day of randomization were not included in analysis because it was not possible to know whether these feces were passed before or after the first dose in the evening meal. Before the trial started, staff received training on how to score feces either: hard, normal, soft, diarrhea, or no feces passed. The score was attained by comparing the feces passed by the dog to a scoring system with a descriptor and a representative picture: HardHard. Dry and crumbly. Kickable. NormalWell‐formed. Firm but not hard. Easy to pick up. SoftShaped but wet feces. Lose their shape as they are picked up. DiarrheaWatery feces with no shape. The softest fecal reading of the day's feces was used as the fecal score for the day for each animal. When dogs shared a kennel, a single fecal score of the most liquid feces was assigned to both dogs when defecation was not witnessed. Frequency of defecation was not recorded because kenneled animals were not under constant supervision. Dogs that had <1 day of fecal scoring were excluded from analysis because no data were available. Dogs left the trial if they were adopted, fostered, euthanized, or at the request of the veterinarian. The dog's date of entry into the trial, date of exit from the trial, sex, breed, group allocation, and daily fecal score all were recorded. The average number of days with fecal scores recorded for dogs in each group was statistically compared by a Mann‐Whitney U‐test. A chi‐square test was used to evaluate distribution of sexes to each group which was not statistically different. Samples of both synbiotic and placebo capsules were removed from the trial site at the end of the trial and sent to a United Kingdom Accreditation Services (UKAS)‐accredited laboratory for culture. The primary outcome measure, the incidence of diarrhea, was analyzed by comparing the synbiotic and placebo groups' mean percentage of days scored as diarrhea out of the total number of days with fecal scores recorded throughout each dog's stay by a Wilcoxon rank sum test. The secondary outcome measure, the occurrence rate of ≥1 day of diarrhea and ≥2 days of diarrhea by day 14 of the animal's stay, was plotted on a Kaplan‐Meier survival curve and analyzed by log rank tests. Statistical analyses were performed by an independent medical statistician3 by a commercially available software package4 . A power calculation was performed before the trial. Animal shelter staff estimated that 30% of the population of dogs experienced at least 1 episode of diarrhea during their stay. To detect a change in incidence of diarrhea from 30% with the placebo to 20% for the treatment group with a significance level of 0.05 and power of 80%, 582 dogs were required for the trial5 . Fecal scores were recorded on each dog's daily health sheets, and these sheets could not be collected for analysis until after the animal had left the shelter. Therefore, it was not possible to count dogs as they completed the trial. For this reason, a year of recruitment was estimated, based on previous dog intake numbers, to be sufficient to obtain the 582 dogs required by the power calculation. BODY.RESULTS: A total of 773 dogs completed the trial to analysis. Figure 1 includes a description of the dogs' inclusion or exclusion from the trial. There were 399 dogs with 8,904 days of fecal scores recorded in the synbiotic product group and 374 dogs with 8,411 days of fecal scores recorded in the placebo group (Table 1). Forty‐two dogs entered as part of a pair and shared a kennel; 13 pairs were assigned to the synbiotic group and 8 pairs to the placebo group. The number of days with a fecal score recorded for a single dog ranged from 1 to 168 days (median, 16.5 days) for the synbiotic group and 1–148 days (median, 16 days) for the placebo group. By a Mann‐Whitney U‐test to compare the median number of days with recorded fecal scores, the distribution of the synbiotic and placebo groups did not differ significantly (Z score, 0.3123; P = .7565)6 . Table 1 Overview of each day's fecal score for dogs in the synbiotic group and the placebo group throughout their shelter stay Synbiotic 399 Dogs Placebo 374 Dogs Total 8904 (100.0%) 8411 (100.0%) Normal 7872 (88.4%) 7152 (85.0%) Hard 34 (0.4%) 42 (0.5%) Soft 820 (9.2%) 959 (11.4%) Diarrhea 178 (2.0%) 258 (3.1%) John Wiley & Sons, Ltd Overall, 55.37%, 428 of 773 dogs were male. The synbiotic group was comprised of 53.13%, 212 of 399 male dogs, and in the placebo group, 57.75%, 216 of 374 dogs were male. The difference between sexes in groups was compared by a 2‐tailed chi‐square test and was found not to be statistically significant (P = .1965)7 . The 7 most common breeds recorded for both groups were the same, with the 4 most common appearing in the same order of occurrence: crossbreeds (124 synbiotic group, 120 placebo group), Staffordshire Bull Terriers (40 synbiotic group, 54 placebo group), Jack Russell Terriers (39 synbiotic group, 32 placebo group), and Lurchers (26 synbiotic group, 30 placebo group). Seven dogs in each group received novel protein diets for food‐related dermatology conditions, whereas 3 dogs in the synbiotic group and 4 in the placebo group received weight loss diets. The mean percentage of days scored as diarrhea of the total number of scored days during each dog's stay was 2.0% in the synbiotic group and 3.2% in the placebo group (P = .0008). Table 1 shows an overview of each day's fecal score for dogs in the synbiotic group and placebo group. Table 2 shows a summary of the number of dogs with ≥1 day scored as diarrhea throughout their overall stay at the shelter, and by week for the first 4 weeks. Table 2 : Summary of the number of dogs with ≥1 day scored as diarrhea, throughout their overall stay at the shelter and by week, for the first 4 weeks Synbiotic Placebo Overall Number of dogs with data 399 374 Number of dogs with diarrhea 90 123 % 23% 33% Week 1 Number of dogs with data 399 374 Number of dogs with diarrhea 40 65 % 10% 17% Week 2 Number of dogs with data 328 304 Number of dogs with diarrhea 32 38 % 10% 13% Week 3 Number of dogs with data 226 223 Number of dogs with diarrhea 15 26 % 7% 12% Week 4 Number of dogs with data 147 130 Number of dogs with diarrhea 9 21 % 6% 16% John Wiley & Sons, Ltd Kaplan‐Meier survival curves were used to compare the temporal pattern of first appearance of diarrhea between the intervention groups (Fig 2), evaluated by the log rank test. By day 14, 18.8% of dogs in the synbiotic group and 27.2% in the placebo group had experienced at least 1 episode of diarrhea (P = .0022). By day 14, 4.6% of dogs in the synbiotic group had experienced diarrhea lasting ≥2 days, compared with 8.0% in the placebo group (P = .0300). Figure 2Plot of Kaplan‐Meier Estimate of time to first diarrhea. Dogs with ≥1 day's data. No adverse events or effects were reported in either intervention group. The UKAS‐accredited laboratory reported the synbiotic capsules to contain 1.58 × 1010 cfu/capsule and placebo capsules <18 cfu/capsule of bacteria in the Enterococcus genus. BODY.DISCUSSION: The objective of our trial was to test the effects of an orally administered prophylactic synbiotic supplement on the incidence of diarrhea in dogs entering an animal shelter. The results indicated that supplementation with the synbiotic significantly decreased diarrhea incidence across all 3 measures in the trial. The first measure of incidence used mean percentage of days per dog that were scored as diarrhea throughout their stay instead of the overall group percentage of days scored as diarrhea. This approach was used to prevent data from being skewed in the case of a few long‐stay animals with persistent diarrhea being assigned to the same group. Survival analysis, a collection of statistical procedures for data analysis in which the outcome variable of interest is time until an event occurs,19 was used for the remaining 2 measures of diarrhea incidence. Fourteen days was chosen before commencing the trial as the time point for Kaplan‐Meier survival analysis because it was the median number of days stay at the shelter in the year preceding data collection. Sixteen was the median number of days stay at the shelter for our sample. Kaplan‐Meier survival analysis method was used to compare the percentage of dogs that had experienced at least 1 episode of diarrhea in each group within the first 14 days. The percentage of dogs in each group that had had diarrhea lasting ≥2 days by day 14 also was compared. This number was of interest because when diarrhea lasted ≥2 consecutive days, the dogs would be scheduled for a veterinary evaluation and treatment would be prescribed. A trial that used the probiotic species Bifidobacterium animalis, which is commercially available in the United States of America, also found that significantly fewer probiotic‐supplemented dogs passed unacceptable feces (loose or diarrheic) during the first week of relocation, and during their overall stay at a kennel facility, when compared to controls.9 This trial randomized 134 young Labrador Retrievers or Labrador‐Golden Retrievers to 4 groups: 3 different doses of probiotic and a control group. The dogs in the Bifidobacterium animalis study began their probiotic supplementation 5 weeks before entering the kennels. Enterococcus faecium was selected for the current trial because it currently is the only probiotic species licensed in the European Union for dogs.20 The population of dogs in our study was a mix of ages and breeds, typical of a shelter in the United Kingdom, and were not supplemented before kenneling because it was not realistic for dogs entering an animal shelter. It would be interesting to investigate the effect of synbiotic supplementation in other stressful situations, such as competitions and planned holiday kenneling, to assess the efficacy of the product in these scenarios. A previously performed trial using the probiotic species Enterococcus faecium on dogs in an animal shelter did not find statistical significance to support the hypothesis that the probiotic would decrease the incidence of diarrhea of ≥2 days' duration compared with placebo.10 This trial had a very low incidence of diarrhea lasting ≥2 days (2 dogs in a study population of 182), which was unexpected and possibly a result of short‐stay durations, a consistent diet for all kenneled dogs and lack of stress due to the kenneling facilities. We found a statistically significant effect of Enterococcus faecium in our trial, using the same outcome measure (incidence of diarrhea ≥2 days' duration), in an adequately powered study with a higher incidence of diarrhea. When the same trial was performed in cats, where the incidence of diarrhea ≥2 days' duration in the placebo group was higher (20.7%), a statistically significant decrease in diarrhea ≥2 days' duration was found in the probiotic group (7.4%; P = .0297).10 Limitations to our trial include a lack of medical history for the majority of animals because of the nature of being an animal shelter. This may mean that a predisposition to diarrhea was more prevalent in certain individuals. We believe that this effect was minimized by the randomization process and large sample size. Age was not recorded because of a lack of information given about dogs entering the shelter. Previously, females have been found to have lower odds of developing diarrhea compared with males.2 There were more males than females in our trial, but this difference was not statistically significant (P = .1965). Diet was not kept constant throughout the trial for all dogs, which could have played a role in the development of diarrhea, but this was meant to reflect normal working practice for animal shelters, the majority of which rely on donations to feed their dogs. Again, the large sample size and randomization process should have minimized the effect on our trial. Forty‐two dogs were housed as pairs in our trial and assigned to the same group to decrease the risk of treatment contamination. Given that our trial was carried out in a working animal shelter, it was not possible to alter normal working practices. One hundred and ninety‐one more dogs completed the trial than required by the power calculation (582), and this overage was caused by a specific date as an endpoint rather than counting dogs, which was a more practical solution in a working shelter. A simple 4‐category fecal scoring system (hard, normal, soft, diarrhea) was created for our trial, adapted from the Nestle Purina and Waltham feces scoring systems8 , 9 after staff at the animal shelter expressed concerns over the complexity of the original systems. All staff received training in the 4‐category fecal scoring system and posters were placed in all dog kitchens and admission rooms as a prompt. Fecal scoring is subjective and there is likely to be some intra‐observer and interobserver variability despite having descriptors and representative picture available, but there is evidence to suggest fecal scoring is superior to measuring fecal moisture content and fecal dry matter.21, 22 Animals with a known history of chronic or recurrent diarrhea or abnormalities on clinical examination were excluded from the trial. Therefore, conclusions can only be made about healthy animals entering an animal shelter. It is also not possible to draw any conclusions regarding the efficacy of other potential probiotic strains or prebiotics beyond those used in our trial. Samples of both capsules were cultured at the end of the trial to ensure that no cross‐contamination of the placebo occurred during manufacture or storage, but also to ensure the active capsule contained at least the specification declared on the commercial product, 2 × 109 cfu/capsule. The excess quantity of probiotic was due to overage added by the manufacturer in order to guarantee live bacteria numbers at the end of a 2‐year shelf life. In conclusion, the results of our trial support the hypothesis that supplementing dogs entering an animal shelter with a synbiotic decreases the incidence of diarrhea and provides evidence of the beneficial effect of the prophylactic use of this synbiotic supplement. Decreased diarrhea rates would directly improve the welfare of the dogs and decrease the associated costs of cleaning, treating, and extended stays in animal shelters. The results from our study could be included in a cost‐benefit analysis to establish the net cost of routinely supplementing dogs entering a shelter. There may be an advantage to the use of this synbiotic preparation in decreasing diarrhea triggered by other stressful events such as holiday kenneling and during competitions, but this hypothesis is yet to be tested.

TITLE: That Escalated Quickly—Planning to Ignore RPE Can Backfire ABSTRACT: Ratings of perceived exertion (RPE) are routinely assessed in exercise science and RPE is substantially associated with physiological criterion measures. According to the psychobiological model of endurance, RPE is a central limiting factor in performance. While RPE is known to be affected by psychological manipulations, it remains to be examined whether RPE can be self-regulated during static muscular endurance exercises to enhance performance. In this experiment, we investigate the effectiveness of the widely used and recommended self-regulation strategy of if-then planning (i.e., implementation intentions) in down-regulating RPE and improving performance in a static muscular endurance task. 62 female students (age: M = 23.7 years, SD = 4.0) were randomly assigned to an implementation intention or a control condition and performed a static muscular endurance task. They held two intertwined rings as long as possible while avoiding contacts between the rings. In the implementation intention condition, participants had an if-then plan: "If the task becomes too strenuous for me, then I ignore the strain and tell myself: Keep going!" Every 25 ± 10 s participants reported their RPE along with their perceived pain. Endurance performance was measured as time to failure, along with contact errors as a measure of performance quality. No differences emerged between implementation intention and control participants regarding time to failure and performance quality. However, mixed-effects model analyses revealed a significant Time-to-Failure × Condition interaction for RPE. Compared to the control condition, participants in the implementation intention condition reported substantially greater increases in RPE during the second half of the task and reached higher total values of RPE before task termination. A similar but weaker pattern evinced for perceived pain. Our results demonstrate that RPE during an endurance task can be self-regulated with if-then plans. This finding is particularly important given how frequently RPE is used in exercise science as a correlate of physiological processes that ultimately limit performance. Unexpectedly, participants with implementation intentions reported higher RPE than control participants. This suggests that strategies to self-regulate RPE might have ironic effects that hamper performance, maybe by increasing attention to RPE. This implication is important for exercise physiologists, athletes and coaches. BODY.INTRODUCTION: Physiologists have long sought to find the primary determinant of exercise termination (Marcora and Staiano, 2010). This research has mainly focused on "cardiovascular, respiratory, metabolic, and neuromuscular mechanisms of muscle fatigue" (Marcora and Staiano, 2010, p. 768). Offering a more psychological perspective, Swedish psychologist Gunnar Borg published a paper where he suggested that perceived exertion is "the single best indicator of the degree of physical strain" (Borg, 1982, p. 377). To measure the rating of perceived exertion (RPE), he published the now famous Borg scales (Borg, 1982, 1998). In short, Borg scales are a set of psychophysical self-report scales with different psychometric properties that can be used to assess different aspects of perceived exertion (Borg, 1970, 1982). A substantial body of evidence underlines the claim that RPE is an indicator for physical strain (e.g., Chen et al., 2002; Scherr et al., 2013; Moscatelli et al., 2015). In their meta-analysis, Chen et al. analyzed the association of RPE with different physiological criterion measures of physical exertion. Reported validity coefficients for the relationship of RPE with heart rate, blood lactate, %VO2max, or respiration rate ranged between 0.57 and 0.72. Thus, self-reported RPE does correspond to ones' internal physiological state. Some inconsistencies in the RPE literature notwithstanding (Noble and Robertson, 1996), the importance of RPE as an indicator of physical strain is now generally accepted (Pageaux, 2014). Given the validity of RPE as an indicator of strain, it has been proposed (e.g., Scherr et al., 2013) and utilized (Foster et al., 1996) as a cost effective tool for monitoring training load. For example, Impellizzeri et al. (2004) showed that RPE per training session was a valid indicator of training load in a sample of soccer players. Rehabilitation is another field where RPE is used to monitor training load (Noble and Robertson, 1996; Pena et al., 2003). This underlines the usefulness of RPE measures as indicators of strain, even in applied settings. However, RPE is more than just an indicator of physical strain: according to the psychobiological model of endurance performance RPE limits how long people persist in an endurance task (Marcora, 2008, 2009; Marcora et al., 2008). This proposition is derived from motivational intensity theory (Brehm and Self, 1989; Richter et al., 2016) which constitutes the theoretical basis of the psychobiological model. The theory asserts that people adjust their effort to increasing task difficulty as long as the exertion of effort seems both justified and possible. The psychobiological model applies this reasoning to endurance performance, suggesting that people terminate a strenuous task either after having reached their justified level of effort or when further investment of effort seems impossible. Accordingly, exhaustion and task termination should not be determined by physiological indicators of strain, but rather reflect a deliberate decision to disengage from the task at hand when the justified or possible effort limit is reached (Pageaux, 2014). This theoretical approach represents a marked shift from purely physiological models of (endurance) performance to psychologically grounded models. Empirical support for the propositions of the psychobiological model has accumulated in recent years (e.g., Marcora et al., 2009; Marcora and Staiano, 2010; Blanchfield et al., 2014a,b; but see also, Hureau et al., 2016). In a frequently cited study, participants performed a cycling task at 80% of their aerobic capacity until they were unable to produce the required power (Marcora and Staiano, 2010). Immediately after task termination, maximum voluntary cycling power (MVCP) of participants was measured. MVCP produced by participants was more than 300% of the power participants had to perform in the cycling task they just terminated. This supports the claim that physiological strain (e.g., muscle fatigue) is not the ultimate reason for task termination. However, a very strong association of RPE and task termination was observed, suggesting that RPE was the primary limiting factor of exercise termination. The key property of the psychobiological model is its explicit acknowledgment of psychological factors that determine (endurance) performance (Pageaux, 2014). Indeed, it has been observed that psychological manipulations can have an effect on endurance performance via regulating RPE (Blanchfield et al., 2014a,b). Although research shows that endurance performance can be affected by subtle psychological manipulations that change RPE, the effectiveness of self-regulation strategies for down-regulating RPE and the corresponding effects on performance have not yet been documented. In the present research, we turned to the self-regulation strategy of making if-then plans (so-called implementation intentions; Gollwitzer, 1999, 2014). It is plausible that recreational and professional athletes routinely make plans to regulate their effort and exertion (e.g., planning how to deal with feelings of pain or exhaustion during physical exercise). Furthermore, if-then planning in sport is regularly recommended by institutions, the public press (e.g., Calder, 2009; Gregoire, 2016), and by the scientific community (Achtziger et al., 2008; Brick et al., 2016; McCormick et al., 2016). However, to date it is not clear whether planning how to deal with effort can indeed help people down-regulate their RPE and thereby enhance endurance performance. Implementation intentions are if-then plans in which people link critical situations and goal-directed behaviors: "If I encounter Situation S, then I will perform Behavior B!" This facilitates goal attainment beyond forming mere goal intentions (e.g., "I want to achieve Outcome O/perform Behavior B!") across a variety of domains (e.g., health, academic, interpersonal; Gollwitzer and Sheeran, 2006; Adriaanse et al., 2011b; Belanger-Gravel et al., 2013; Hagger and Luszczynska, 2014). In the domain of endurance performance, for instance, if-then plans could specify how to deal with negative sensations during endurance performance (e.g., Thürmer et al., 2017). Forming implementation intentions promotes goal attainment because the situation specified in the if-part becomes mentally activated and receives attentional and perceptual priority (Wieber and Sassenberg, 2006; Achtziger et al., 2012; Janczyk et al., 2015), making it easy to detect and recognize (Aarts et al., 1999). Additionally, a strong associative link is established between the situation and the goal-directed behavior specified in the then-part that automates behavior (Gollwitzer and Brandstätter, 1997; Brandstätter et al., 2001; Bayer et al., 2009). With implementation intentions, people delegate their action control to situational cues (i.e., they create "instant habits;" Gollwitzer, 1999). This applies to external states as well as to internal, subjective states (e.g., feeling exhausted or stressed; Achtziger et al., 2008; Varley et al., 2011), under the premise that people can recognize these internal states when they occur. Thus, it seems plausible that people can form implementation intentions in which they plan how to deal with sensations of exertion (i.e., RPE) in an endurance task. The corresponding goal-directed behaviors (i.e., to ignore aversive sensations and keep going) should be automatically initiated when people get increasingly exerted. In terms of the psychobiological model, implementation intentions could enhance endurance performance either by delaying the increase of RPE and/or by justifying higher levels of effort. Prior research has observed that people with implementation intentions perceive performance as less straining than participants with mere goal intentions and are therefore more likely to persist when facing increasing difficulty (Freydefont et al., 2016; Legrand et al., 2017). However, even after having formed implementation intentions people disengage from goals that become excessively difficult to attain, for instance when facing significant monetary losses (Legrand et al., 2017, Exp. 3). These findings suggest that implementation intentions can reduce perceived effort of performing goal-directed behaviors, however, without justifying additional effort (i.e., flexible tenacity; Gollwitzer et al., 2008). Taken together, RPE appears to be a crucial limiting factor of performance and is a very influential construct in exercise physiology and psychology (Pageaux, 2016). We investigated whether the self-regulation strategy of forming implementation intentions enables people to down-regulate RPE and thus enhance their endurance performance. We expected a slower increase in RPE in the implementation intention condition compared to a control condition, without differences in the maximum RPE-value. Moreover, we hypothesized that this would translate into better performance in terms of time to failure and/or quality. BODY.METHODS.PARTICIPANTS AND DESIGN: We recruited a sample of 62 female students (age: M = 23.7, SD = 4.0). We determined that this would allow us to detect differences between conditions of d = 0.65 at 95% power, a typical effect size when comparing implementation intentions to a control condition (meta-analysis by Gollwitzer and Sheeran, 2006). Our participants reported to engage in M = 3.4 (SD = 2.2) h of sport per week, 25.9% of which they ascribed to strength training. Four participants reported to be not actively engaged in any sport, while the remaining participants performed their main sport for M = 5.1 (SD = 5.4) years. Only participants with no current or recent injuries of shoulders, arms, or the back were eligible for participation. Further, participants were instructed to refrain from consuming caffeine in the 2 h prior to the study. All participants complied to these restrictions. In a final questionnaire, we asked participants whether they had engaged in an exercise (10 participants in each conditions answered "yes") or consumed alcohol the day before (Three participants in the control and two in the implementation intention conditions answered "yes"). The conditions did not differ with regard to these questions, ps > 0.78. Upon their arrival at the lab, we randomly assigned participants to a control condition (n = 33) or an implementation intention condition (n = 29). Participants signed an informed consent that was approved by the Ethics Committee at the University of Konstanz (approval #24/2016). We compensated participants with 5 Euro or course credit. BODY.METHODS.MEASURES AND APPARATUS.STATIC MUSCULAR ENDURANCE TASK: In cooperation with the scientific engineering service at the University of Konstanz we developed a static muscular endurance task (introduced to participants as the "hot rings task") that allowed us to reliably measure both the duration (i.e., time to failure) as well as the accuracy of performance. We instructed participants to hold two aluminum bars connected by two intertwined rings (length = 46.5 cm; weight of each bar = 55 g) for as long (duration) and with as few contacts between the rings (errors) as possible. Participants stood upright with their arms strapped into a holding device that was connected to the ceiling of the laboratory and comprised a connector element (see Figure 1). Prior to the task, the connector element was locked and the holding device was individually adjusted to participants' height so that their outstretched arms formed a 90° angle with their torso. The connector element was then unlocked at the beginning of the task, causing it to unplug and terminate the task as soon as participants' arms dropped below the preset 90° angle. To continuously track the accuracy of on task performance, a recording box was connected to the bars and registered ring contacts at 50 Hz. Figure 1Schematic illustration of the static muscular endurance task. BODY.METHODS.MEASURES AND APPARATUS.RATINGS OF PERCEIVED EXERTION (RPE) AND PAIN: Every 25 ± 10 s participants reported their perceived exertion (RPE) and pain using Category Ratio 10 (CR10) scales (Borg, 1998, 2004). People can distinguish RPE from other exercise-related sensations (Pageaux, 2016). Care needs to be taken to specify precisely what internal sensations participants should attend to (Pageaux, 2016). To ensure participants' differentiated perceived exertion from pain, we explicitly defined RPE as "the conscious sensation of how hard, heavy, and strenuous a physical task is" (Marcora, 2010). The two scales ranged from 0 ("nothing at all") to 10 ("maximal") or 11 ("even more than max") (Pageaux, 2016). We printed them on separate sheets of paper placed on a wall in front of the participants. BODY.METHODS.PROCEDURE: Upon their arrival in the lab, participants were familiarized with the static muscular endurance task. They were instructed to hold the bars for as long as possible while avoiding contacts between the rings. We adjusted the holding device and informed participants about how to use the CR10 scales for rating their perceived exertion and pain (Pageaux, 2016). Participants then went through a brief demonstration in which they deliberately lowered their arms until the connector element unplugged. After the demonstration was completed, we released participants from the apparatus and provided them with different instructions according to their condition. In the control condition, we instructed participants to repeat the task instructions (i.e., "The task is to persist for as long as possible while avoiding contacts between the rings!"). In the implementation intention condition, we instructed participants to phrase the instructions in terms of a goal intention (i.e., "I want to persist for as long as possible while avoiding contacts between the rings!") and to furnish it with an if-then plan: "If the task becomes too strenuous for me, then I ignore the strain and tell myself: Keep going!" Finally, participants were again strapped into the apparatus and raised their arms into the 90° position. To start the task, we then unlocked the connector element, switched on the recording box, and started a timer to measure time to failure. While participants engaged in the task, they received no verbal feedback and the experimenter remained outside their field of vision to minimize experimenter influence. For the same reason, the verbal prompts for the CR10 scale ratings (i.e., the words "effort" and "pain") were generated using "Text 2 Speech Pro" (Mattos, 2016) and played by the computer. Answers were documented by the experimenters. The same two female experimenters collected all data for this study. The task was terminated as soon as the connector element unplugged and participants were released from the apparatus. The experiment concluded with a final questionnaire comprising six items to measure how strongly participants were committed to perform well in the task (α = 0.78; e.g., "It was important for me to persist for as long as possible in the endurance task," "I did not care how precisely I worked on the endurance task [reversed]") on 7-point Likert scales (1 = does not apply, 7 = fully applies) and demographic questions (e.g., age, gender, physical activity). BODY.RESULTS.DATA ANALYSIS: We report independent t-tests comparing the two conditions and applied Welch-corrections when the assumption of homogenous variances was violated. We augment this by also reporting non-parametric Wilcoxon rank sum tests because values were often not normally distributed. As all dependent variables except time to failure were measured repeatedly during the task, we submitted them to models with Condition (control vs. implementation intention) as between-participants and Time-to-Failure ([0–10%] vs. (10–20%] vs. ...vs. (90–100%]) as within-participants factor. There were, however, empty cells in the design resulting from early quitters, preventing us from using standard mixed ANOVAs. Instead, we turned to mixed-effects models for which empty cells are no issue and do not require removing observations from the dataset. All analyses were run in the statistical software environment R (3.3.1, R Core Team, 2016). We estimated mixed-effects models with lme4 (1.1–12; Bates et al., 2015) and used the Satterthwaite approximation for degrees of freedom implemented in lmerTest (2.0- 32; Kuznetsova et al., 2016) to establish the significance of fixed-effects. Effect sizes were computed with the effsize package (0.6.4; Torchiano, 2016) and plots were created using ggplot2 (2.2.1, Wickham, 2009). BODY.RESULTS.TASK COMMITMENT: Participants in the implementation intention condition (M = 6.0, SD = 0.7) reported lower task commitment than participants in the control condition (M = 6.3, SD = 0.6) after having completed the task, and this difference approached significance, t(60) = 1.87, p = 0.066, g = 0.34, and W = 602.5, p = 0.079. BODY.RESULTS.TASK PERFORMANCE.TIME TO FAILURE: Descriptively, participants in the implementation intention condition (M = 8.5 min, SD = 4.0) performed the endurance task about 1 min longer than control participants (M = 7.5 min, SD = 3.0), but this difference was not significant, t(60) = 1.10, p = 0.274 and W = 408, p = 0.326 (Figure 2A). Figure 2Visualization of results as a function of Condition and Time-to-Failure. Groups did not differ in how long (A) and how accurate (B) they persisted in the static muscular endurance task. Significant between group differences and Time-to-Failure × Condition interactions emerged for RPE (C) and in attenuated form for perceived pain (D) as well. Error bars in (C) and (D) represent standard errors of the mean. BODY.RESULTS.TASK PERFORMANCE.ERROR RATES: We found a significant effect of Time-to-Failure on error rates, F(9, 540) = 4.69, p < 0.001, reflecting that participants committed more errors as the task progressed. Neither the main effect of Condition, F(1, 60) = 0.80, p = 0.374, nor the interaction effect of Condition and Time-to-Failure, F(9, 540) = 0.93, p = 0.494, reached significance, indicating that goal and implementation intention participants did not differ in their susceptibility to committing errors (Figure 2B). BODY.RESULTS.EFFORT AND PAIN RATINGS.RATINGS OF PERCEIVED EXERTION (RPE): We found a significant main effect of Time-to-Failure, F(9, 462.94) = 470.66, p < 0.001, indicating that RPE increased over the endurance task. We also observed a significant interaction of Condition and Time-to-Failure, F(9, 462.94) = 3.77, p < 0.001. As illustrated in Figure 2C, this interaction effect reflects a steeper increase in RPE among implementation intention participants after exceeding a rating of 6–7 on the CR-10 scale. In the final 10% of the task, implementation intention participants (M = 9.4, SD = 1.7) reported significantly higher RPE than control participants (M = 8.2, SD = 2.5), t(56.8) = 2.09, p = 0.026, g = 0.41 and W = 318, p = 0.020. BODY.RESULTS.EFFORT AND PAIN RATINGS.RATINGS OF PERCEIVED PAIN: We found a significant main effect of Time-to-Failure, F(9, 463.29) = 296.19, p < 0.001, indicating that perceptions of pain increased over the endurance task. The interaction effect of Condition and Time-to-Failure approached significance as well, F(9, 463.29) = 1.81, p = 0.064. As illustrated in Figure 2D, this pattern of results reflects a steeper increase in perceived pain among implementation intention participants after exceeding a rating of 5–6 on the scale. In the final 10% of the task, implementation intention participants (M = 8.2, SD = 2.5) reported significantly more intense pain than control participants (M = 6.8, SD = 2.7), t(60) = 2.09, p = 0.041, g = 0.38 and W = 326.5, p = 0.031. BODY.DISCUSSION: In this study, we showed that the self-regulation strategy of forming implementation intentions affects ratings of perceived exertion (RPE) during a static muscular endurance task. Unexpectedly, implementation intention participants who planned to ignore sensations of strain reached higher RPE values before terminating the task while experiencing a significantly faster increase of RPE than control participants. Time to failure and quality of performance was similar between conditions. It thus seems that forming implementation intentions raised the level of effort participants were willing or able to invest but at the same time also increased the rate at which RPE accrued over time. As RPE is among the most frequently assessed concepts in exercise physiology and implementation intentions are a widely recommended and used self- regulation strategy these findings are very important for at least two reasons. BODY.DISCUSSION.PLANS AFFECT RPE: First, this study underlines how readily RPE (which is now widely accepted as a central correlate of physical strain) can be altered by a psychological manipulation. This is important because there is still an ongoing debate regarding which sensory signals contribute to what extent to the perception of effort (Pageaux, 2016). From a peripheral point of view, RPE is a function of afferent signals that originate from peripheral organs involved in a specific task (i.e., high correlations of RPE with heart rate and other physical indicators of strain are consistent with this interpretation). In a particularly convincing study, Amann et al. (2010) showed that partially blocking sensory afferents (by lumbar intrathecal injection of fentanyl) from working muscles led to reduced RPE in a cycling task. Thus, from a peripheral perspective, reduced somatosensory feedback results in a lower perception of effort. From a central point of view, perception of effort is independent from such afferent feedback (Marcora, 2009). To support this claim, Marcora et al. (2008) experimentally dissociated muscle fatigue from the metabolic stress associated with the fatiguing task (metabolic stress in turn stimulated afferent signals) and showed that locomotor muscle fatigue alone led to higher RPE. Thus, maintaining performance when fatigued is associated with increases in central motor command and this is in turn is perceived as effortful. Consequently, from a central point of view, perception of effort is the result of a "conscious awareness of the central motor commands to the locomotor and respiratory muscles (Marcora, 2009, p. 2061)." Our experimental design was not set out to test peripheral and central explanations of RPE against each other. However, our results fit very well into the central explanation: Psychological manipulations can affect attendance to internal and external states (e.g., Pennebaker and Lightner, 1980) and the psychobiological model of endurance performance proposes that psychological manipulations can affect RPE. In our case, participants in the implementation intentions groups were possibly more aware of their sensations while performing the task. This might have caused the increase in perceived exertion. In line with the idea that implementation intentions led to a greater awareness, participants in this group also reported to perceive more pain. BODY.DISCUSSION.IRONIC EFFECTS OF PLANS ON RPE: Second, our results show that the effects of psychological manipulations on RPE and performance are not necessarily straightforward. Contrary to our predictions, implementation intentions participants did not manage to persist longer in the muscular endurance task than goal intention participants. First, they reached higher RPE scores than control participants before disengaging from the task, which could be interpreted as an increased justification of effort. However, this is at odds with the observation that implementation intention participants tended to be less committed to perform well than control participants; moreover, prior research suggests that implementation intentions do not justify additional effort. A cautious explanation might be that participants effectively implemented the "keep going" part of their plan. From this perspective, the higher final RPE scores might be a volitional rather than a motivational effect. Second, RPE rapidly increased among implementation intention compared to control participants, with a steep slope after having passed about the middle of the scale. To illustrate, RPE values in the implementation intention condition exceed those in the control condition by about 1.5 points after 3/4 of the time to failure. Both effects—the higher overall RPE before task termination and the steeper increase—seem to have canceled out each other, rendering time to failure similar between conditions. We did not expect this pattern of results; however, we would like to offer a tentative explanation for our finding. The implementation intention instruction prompted participants to ignore strong sensations of exertion. Contrary to what one would expect under such an instruction, however, implementation intention participants experienced even stronger sensations of exertion than control participants once they had exceeded the middle of the RPE scale. This observation fits well into ironic processing theory (Wegner, 1994), which asserts that mental control is based on two interacting processes: (1) an intentional and effortful operating process that creates thoughts and sensations consistent with a desired state and (2) an automatic and non-conscious monitoring process that searches for mental contents indicating control failure. As long as mental capacity is sufficient, the operating process can successfully ignore or suppress unwanted thoughts and sensations. When mental capacity becomes scarce (e.g., due to physical strain, stress, or cognitive load), however, the operating process is derailed more strongly than the monitoring process, which continues to search for contents indicating failed mental control. As a consequence, the very thoughts or sensations that one strives to ignore or suppress are amplified rather than attenuated. In terms of ironic processing theory, implementation intention participants might thus have suffered more strongly from intruding sensations of exertion once their mental capacities were limited by the straining task performance. Consistent with this interpretation, ironic effects of forming implementation intentions have been observed in other domains as well, especially when the plan negated the execution of a behavior (e.g., planning what not to eat; Adriaanse et al., 2011a). Yet, this does not imply that implementation intentions necessarily suffer from ironic effects. First, there are also studies showing that even negation implementation intentions can be effective (e.g., Verhoeven et al., 2017). Second, implementation intentions specifying to ignore undesired critical cues like intrusive thoughts and negative affect also work well (e.g., Achtziger et al., 2008; Gallo et al., 2009). Finally, a study using negation implementation intentions to regulate perceived pain during an endurance task (Thürmer et al., 2017) has successfully enhanced group performance, however without explicitly measuring perceived pain. At the bottom line, ironic processing theory provides both a compelling and a parsimonious explanation of our present results and implementation intentions seem to be susceptible to ironic effects, even though the exact circumstances of their occurrence have yet to be explored. BODY.CONCLUSION: In the present research, we set out to explore whether people can use the self-regulation strategy of making if-then plans (implementation intentions) to down-regulate RPE and thereby enhance their endurance performance. While our results demonstrate that planning to ignore sensations of strain indeed affects RPE, it did so in an unexpected and ironic way—increasing both the rate at which RPE accrued and the absolute RPE limits that people achieved before terminating the task. This observation is important given how frequently RPE is used as a correlate of physiological processes that ultimately limits performance. Moreover, the finding that implementation intentions can backfire in sport settings is of great importance as well because planning how to deal with thoughts and sensations during physical performance is presumably widespread among recreational and professional athletes, whether these plans are made consciously or not. Making plans is also recommended by the scientific community (e.g., Achtziger et al., 2008; Brick et al., 2016; McCormick et al., 2016) and part of psychological skills trainings that aim to improve sports performance (e.g., Birrer and Morgan, 2010). Our results show that athletes and coaches who use plans to ignore aversive sensations of exertion might in turn suffer from even more severe sensations of exertion, which might easily impair performance. As such, the present research contributes to the existing literature on ironic processes in sport (Janelle, 1999). BODY.ETHICS STATEMENT: This study was carried out in accordance with the recommendations of the ethics guidelines of the University of Konstanz' ethics committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethics committee of the University of Konstanz (approval #24/2016). BODY.AUTHOR CONTRIBUTIONS: MB and WW developed the research question and conducted the empirical part of the study. MB conducted the statistical analyses. MB and WW then analyzed the results, and jointly wrote the manuscript. BODY.AUTHOR CONTRIBUTIONS.CONFLICT OF INTEREST STATEMENT: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

TITLE: A Prospective, Double-Blind, Randomized, Controlled Clinical Trial in the Gingivitis Prevention with an Oligomeric Proanthocyanidin Nutritional Supplement ABSTRACT.AIM: To evaluate the effectiveness on tissue response of the new nutritional supplement made of oligomeric proanthocyanidins in induced gingivitis after 21 days of use. ABSTRACT.MATERIAL AND METHODS: A prospective, double-blind, randomized, controlled clinical trial was carried out on 20 patients; it is divided into an experimental group and a control group after fulfilling the selection criteria. Patients had to come 4 times during the study to register the Silness and Löe index, the gingival bleeding index, the plaque index, the inflammatory crevicular fluid study (IL6), and the changes in the brightness of the gingiva. No complementary hygiene methods were allowed during the 21 days. ABSTRACT.RESULTS: The Silness and Löe index was higher in the control group than in the experimental group, reaching a twofold difference between the groups (p < 0.0001). The gingival bleeding index also supports this fact, since the bleeding was lower in the experimental group (p < 0.005). However, the dental plaque on the tooth surface according to the plaque index was 33% higher in the experimental group (p < 0.006). Some differences in the IL-6 were found in the crevicular fluid (p < 0.0001). ABSTRACT.CONCLUSION: Oligomeric proanthocyanidins have an effect on the periodontal tissue's health. No effects on the accumulation of plaque on the tooth surface were found, so further studies are needed to determine the nature of the plaque. BODY.1. INTRODUCTION: Gingivitis is an inflammatory disease of dental tissue support that in the first instance affects the gingiva. Gingivitis could move on to more advanced stages, leading to the development of periodontitis, in which the inflammation and bacterial infection can produce the destruction of the supporting tissues of the teeth, gingiva, periodontal ligament, and alveolar bone [1–5]. Gingivitis is due to the long-term effects of plaque deposits on the teeth, which is composed of bacteria, located on the surfaces of the teeth and in the gingival sulcus. If plaque is not prevented or removed, it turns into a hard deposit called tartar (or calculus) that becomes trapped at the base of the tooth. Plaque and calculus irritate and produce gingival swelling. Bacteria and the toxins produce infection and more swelling gingiva [1–5]. Crevicular fluid is normal plasma exuded flowing through the gingival sulcus. A patient with gingivitis develops an inflammatory response with a large number of mediators involved, such as the cytokines IL-6 and IL-8 [1–6]. Interleukin 6 (IL-6) is a multifunctional cytokine that is produced by a range of cells, and it is involved in the B-cell differentiation, proliferation, and differentiation of T cells; in the immunoglobulin, stimulation by the B-cell secretion [7] can also induce bone reabsorption [8]. IL6 is a useful diagnostic indicator to determine the progression of the gingivitis to a periodontal disease [9]. The treatment goal of gingivitis is to reduce inflammation by cleaning the teeth, using different instruments to remove the dental plaque deposits according to the case [6]. It is important to educate the patient and prescribe special toothpaste, mouthwash, gel, and so forth which contain a wide spectrum of antiseptic and anti-inflammatory properties. However, the dentist has the responsibility to explain the correct tooth brushing technique to achieve good results [2, 3]. The oligomeric proanthocyanidins (OPCs) are one of the most abundant polyphenolic substances in the plant kingdom. These substances exhibit a range of rather surprising physical and chemical properties which, once applied to living organisms, are translated into a multitude of biological activities [10]. The OPCs have been recently investigated, and, apart from its powerful antioxidant activity, they have been shown to have anticancer, anti-inflammatory, antimicrobial, and vasodilatory properties, revealing to be a potentially valuable therapeutic tool for the treatment of many illnesses [11–15]. The available research has demonstrated that the acidic environment of the human stomach does not easily degrade the proanthocyanidins; therefore, the rates of absorption in the upper gastrointestinal tract are not high [16]. However, it seems that even the low levels observed in urine after an oral dose (generally less than 250/0 of doses/original) are sufficient to significantly increase the antioxidant capacity in plasma/serum. When OPCs reach the colon, they suffer an extensive degradation due to colonic flora [16]. The metabolites and biological properties of this process have not been investigated yet; it has been suggested that they may have protective effects on the antioxidant and systemic illness [11–16]. Regarding the other component included in the OPCs, vitamin C, we know that it is a good associative option to bilberry. Vitamin C generally provides properties that contribute to the formation of collagen, which helps to maintain functionality of teeth and gums and other properties such as reinforcement of the immune system and potent antioxidant opposition to oxidative stress [17–21]. Although in vitro studies published about the interaction between the OPCs and pathogens in the oral cavity have had very good results, there are no publications with the appropriate design to assess the clinical efficacy of OPCs in gingival disease in human, except an article published in 2015 comparing the chlorhexidine and the cranberry mouthwash [22–25]. The aim of this study is to evaluate the effectiveness of the new nutritional supplement made of oligomeric proanthocyanidins (OPCs) on tissue response to prevent gingivitis. We hypothesise a healthy state of the periodontal tissue in the study group versus gingivitis in the control group. BODY.2. MATERIAL AND METHODS.2.1. STUDY DESIGN AND POPULATION: A randomized, double-blind, placebo-controlled, clinical trial of 21 days of duration was conducted among 20 volunteer students of the School of Dentistry of the University of Seville. The duration of 21 days was chosen as that time allows the parameter study on the gingiva and was enough to study the inflammation of the gingiva without causing any irreversible problem in it. No previous studies have been found in the literature, so 20 subjects were determined for the pilot study to test our hypothesis. The Ethics Committee of the University of Seville approved the study protocol. Prior to participation, the purpose and procedures were fully explained to all healthy volunteers and all participants gave written informed consent in accordance with the Declaration of Helsinki. The study was designed, conducted, analyzed, and reported according to the guidelines for Good Clinical Practice. The study was approved by the http://ClinicalTrials.gov Protocol Registration and Results System with the number NCT02515929. The protocol can be accessed if necessary in http://ClinicalTrials.gov. The study was carried out between September 2013 and January 2014. The recruitment started in September, and the baseline stage took place in October 2013. After the 21-day follow-up data was treated obtaining the results in January 2014. The medical and dental histories were done at the prescreening visit, and we selected the 21 participants based on the inclusion and exclusion criteria. The inclusion criteria were the following: subjects older than 18 years old, male or female, good overall health, and a minimum of 20 teeth (teeth with big caries were crowned or extensively restored, and teeth that were orthodontic banded, abutments, or third molars were not included in the tooth count); the volunteers signed the written consent before the initiation of the study. The exclusion criteria were the following: periodontal disease (defined as 4 mm and/or positive bleeding when probing), pregnant or breastfeeding, subjects with fixed or removable prosthesis, tumour pathology in oral cavity, use of antibiotics during a 2-month period prior to the start of the trial, hypersensitivity to red fruits in general, xerostomy, active smoker, contagious-infectious pathologies, pathology with severe systemic repercussions, any other judgment that makes the investigator believe to endanger or risk the subject participant, subjects with phenylketonuria or allergy to aspartame, and the use of any oral hygiene product during the study. A single examiner previously calibrated determined assessment of patient eligibility for the study and enrolment of patients into trial. Patients eligible for the study were individually randomly assigned to oligomeric proanthocyanidins nutritional supplement treatment or placebo groups by an informatics programme by LACER S.A. The study was double-blind. The examiner and the patient did not know into which group they were assigned. The results were treated by another examiner who knew which patients belonged to the experimental or placebo group. The masking process was established by a number assignation to each patient in the study, and the treatment was the same both for the patient and the examiner who were not allowed to know to which group they belonged. Patients had to take the experimental or placebo treatment each night after dinner and after a rinse with water. The pill was maintained in the mouth until complete dissolution. Neither drinking nor eating was allowed during 30 minutes after taking the treatment. The experimental treatment consisted of 90 mg exocian cran 408 (equivalent to 36 mg OPCs) and 120 mg of vitamin C, while the placebo group was composed of the same organoleptic substance but free of active ingredients. Both were similar in appearance. No complementary hygiene methods were allowed during the 21 days (tooth brushing, rinses or irrigation with any product, or flossing), which could constitute a bias for the study. At the trial baseline stage, a tartar removal was carried out in each patient to regularize the initial situation; a patient diary, enough medication for the entire study, and instructions were given for its correct fulfillment. The inflammation of the crevicular fluid (IL6) and the brightness of the gingiva were registered. Two evaluation visits were performed on days 14 and 21 of the study for an oral clinical examination and to register the Silness and Löe index, the gingival bleeding index, the Turesky plaque index, the inflammatory crevicular fluid study (IL6), and changes in the brightness of the gingiva. BODY.2. MATERIAL AND METHODS.2.2. SILNESS AND LÖE INDEX [: Six teeth were examined according to Ramfjord criteria (16-21-24-36-41-44). Four surfaces of each tooth were examined, making a total of 24 measurements. These measurements were performed with a periodontal probe by the same examiner. BODY.2. MATERIAL AND METHODS.2.3. GINGIVAL BLEEDING INDEX [: A periodontal probe was used to take this index. The values established for this examination are 0—absence of inflammation; 1—mild inflammation, slight change in color, no gingival edema, and no bleeding on probing; 2—moderate inflammation, redness, edema, and gingival hypertrophy, and bleeds to probe (after 10 seconds); 3—severe inflammation, marked redness, and hypertrophy. There may be ulcerations; the gingiva tends to be spontaneous in bleeding. BODY.2. MATERIAL AND METHODS.2.4. TURESKY PLAQUE INDEX [: The buccal surfaces of the anterior teeth were examined using a mouthwash of basic fuchsine as developing agent plaque, and the following numerical scoring system from 0 to 5 was established: 0—there is no plaque; 1—independent streaks of plaque in the cervical margin of the tooth; 2—a thin continuous band of plaque (up to 1 mm) at the cervical margin; 3—a band greater than one millimeter wide, but covers less than one-third of the crown; 4—the plaque covers a third, but not more than two-thirds of the crown; 5—the plaque covers two-thirds or more of the crown. BODY.2. MATERIAL AND METHODS.2.5. INFLAMMATORY CREVICULAR FLUID STUDY (IL6) [: Crevicular fluid samples were collected from interdental areas (lingual, buccal, mesial, and distal) of six teeth distributed in the four quadrants (16, 21, 24, 36, 41, and 44) using five strips of pressed paper that were 2 cm long, especially for crevicular fluid. The impregnation time for each patient was 5 seconds, and the strips were immediately inserted into microtubes that contained 0.5 ml of Eppendorf with 50 μl saline at 4°C for preservation. The samples were then transported to the biological laboratory in a refrigerator and were stored frozen at −80°C at the laboratory until they were processed. Then, we proceeded to analyze the concentration (pg/ml) of interleukin 6 present in each sample using panels 96-well bioplex brand Luminex®. BODY.2. MATERIAL AND METHODS.2.6. BRIGHTNESS OF THE GINGIVA: The brightness of the gingiva was taken to identify possible changes in the gingival color. The reddening of the gingiva accompanies the inflammation of the tissue, which is a factor that may help to differentiate inflammatory changes at this level. The luminosity of the gingiva was registered with Micro SpectroShadeTM MHT Optic Research AG. This instrument is designed to take dental color. However, besides including color guides, the SpectroShade has the ability to measure the brightness of any color, not just shades of white. The change of color of the gingiva was observed in each patient, and the color reference was measured in the same point in each evaluation visit, which helped us compare the change in the coloration of the same point. BODY.2. MATERIAL AND METHODS.2.7. STATISTICAL ANALYSIS: The management of the data was performed using the SPSS statistical program. A similar database to Notebook Data Collection was created. There are a minimum range and a maximum range for each variable, and the name of the variable and its values were defined. The data are presented as the mean ± standard deviation of the measurements, and a p value less than 0.05 was considered statistically significant. Chi-square test was performed to qualitative variable and Student's t-test to the quantitative variable, after the Kolmogorov-Smirnov to assure the normality. The Ethical Committee approved this study, and all examinations and treatments were performed with the consent of the subjects and according to the guidelines of the Declaration of Helsinki. BODY.3. RESULTS: A statistical analysis between the two study groups, 10 patients in each group, in terms of sex, age (years), weight (kg), height (cm), tobacco use, alcohol, and use of concomitant medication, was carried out to ensure the reliability of the results. We found no significant differences between the study group and the control group, so we can say that the homogeneity between the groups was achieved (Table 1). The medication perception of both groups, as the initial flavour, the product durability, the appearance, size, and efficiency, was the same; there were no statistically significant differences between the data obtained (Table 1). No adverse events were notified during the study. Once the study groups were analysed, the data processing was conducted depending on the different views held. BODY.3. RESULTS.3.1. PERIODONTAL INDEX TO 15 DAYS FOR TREATMENT (INTERMEDIATE VISIT): The Silness and Löe gingival index (of 0–3 from least to greatest gingival inflammation) (Table 2) was higher in the control group than in the experimental group, reaching a twofold difference between the two groups (p < 0.0001). The gingival bleeding index also corroborates this fact, as the bleeding was lower in the experimental group versus the control group (p < 0.005) (Table 2). In contrast to the values obtained in the Silness and Löe and gingival bleeding indexes, the amount of dental plaque deposited on the surface of the patients according to the Turesky index plaque was slightly higher (33%) in the experimental group versus in the control group (p < 0.006) (Table 2). BODY.3. RESULTS.3.2. PERIODONTAL INDEX TO 21 DAYS FOR TREATMENT (FINAL VISIT): The results obtained 21 days after starting the treatment showed the tendency observed in the intermediate visit, increasing the difference between the study groups. Silness and Löe index was higher in the control group compared to the experimental group, with a difference of 50% between the study groups (p < 0.0001). The gingival bleeding index, which showed bleeding in the control group, was higher in the experimental group (p < 0.0001). However, the plaque deposition was higher in the experimental group than in the control group according to the Turesky plaque index having plaque deposit doubled in the patients in the experimental group (p < 0.0001) (Table 2). BODY.3. RESULTS.3.3. GINGIVAL BRIGHTNESS: The gingival brightness was taken in each patient using the Micro SpectroShade Optic Research MHT AG. This value was taken at the beginning, middle visit, and in the final visit. The data obtained from these three measurements in each patient showed no statistically significant differences (p values from 0.351 to 0.545) (Table 3). BODY.3. RESULTS.3.4. INFLAMMATORY CREVICULAR FLUID STUDY (IL6): Statistically significant differences were found at the baseline between the experimental group and the control group and in the subsequent visits. No statistically significant differences between the first visit and the second visit, and the first and third visits were found, although it was significant between the second visit and the third visit (Table 4). BODY.4. DISCUSSION: Gingivitis is caused by deposition of plaque on the tooth surface; this deposition constitutes an irritant that triggers the gingival inflammation and bleeding. Gingivitis could be reversible if the patient has a constant and good hygiene. These measures could be carried out by a proper brushing technique [1–5]. The drug used in the current study is a nutritional supplement based on blueberry and red fruit rich in oligomeric proanthocyanidins (OPCs) [7]. Proanthocyanidins have been recently investigated, as we suggested in the introduction, due to their powerful antioxidant and anticancer activities and due to their anti-inflammatory, antimicrobial, and vasodilator systemical properties. The beneficial effect of proanthocyanidins is attributed to its ability to reduce oxidative stress, lipid peroxidation, free radical generation, and LDL oxidation [11]. Mohana et al. published a study in 2015 about the effect of the OPCs in the prevention and treatment of atherosclerosis. Oxidation of low-density lipoproteins (OxLDL) has been strongly suggested as a key factor in the pathogenesis of atherosclerosis [11]. Due to the antioxidant action of the proanthocyanidins and the modulation of macrophage differentiation, Mohana et al. reported the benefit of the product as prevention and treatment of the atherosclerosis [11]. Wang et al. have also tested the anti-inflammatory mechanisms of OPCs [12]. They designed an in vivo study in mouse models to investigate the protective effect of OPCs against alcohol-induced liver steatosis and injury. The results showed that OPC significantly improved alcohol-induced dyslipidemia and alleviated liver steatosis by reducing levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total triglyceride (TG), total cholesterol (TC), low-density cholesterol (LDL-c), and liver malondialdehyde (MDA) and increasing levels of serum high-density lipoprotein (HDL-c) and liver superoxide dismutase (SOD). Further investigation indicated that OPC markedly decreased the expressions of lipid synthesis genes and inflammation genes such as sterol regulatory element binding protein-1c (Srebp-1c), protein-2 (Srebp2), interleukins IL-1b and IL-6, and TNF-α 10. These studies suggested that an effect in the inflammation genes and mediators could be modified by the OPCs, reducing the inflammation obtained [11, 12]. Our results do not show significant differences in the IL-6 between the baseline and the second visit and between the baseline and the third visit. Significant differences were found between the 14th and 21st days and between groups in each visit, including in the baseline stage, so the groups were not similar at the beginning of the study, which could be a bias in the results. That reason forces us to not consider significantly the results obtained. However, the data is lower in the experimental group in each visit and this fact suggests that an effect could be in the IL-6, but we cannot assure this like in other studies because of our difference in the baseline group. However, the clinical parameters, the Löe and Silness index and gingival bleeding index, showed us that the clinical inflammation was higher in the control group than in the experimental group, finding statistically significant differences. During the study, we observed that the deposition of plaque, Löe and Silness index, gingival bleeding index, and the brightness of the gingiva, increased sequentially, so the inflammatory factors must have also increased due to the results obtained in each parameter, although there were no differences between the study groups in the brightness of the gingiva. We attribute this fact to the mechanical irritant that the plaque constitutes itself, without taking into account its nature. Besides the anti-inflammatory effect, OPCs have shown antimicrobial effect, reported by further studies [10–16, 23, 25]. This property also justifies testing the effectiveness of the OPCs in periodontal disease. After performing the current study, we can affirm that the OPCs have an effect in the health of the periodontal tissues, as we could see through the Silness and Löe and gingival bleeding indexes, which were significantly lower in the experimental group than in the control group, although it was not corroborated by the Turesky plaque index, where the presence of plaque was paradoxically higher by 33% in the study group than in the control group (Table 1). These results lead us to suspect that this supplement certainly has antimicrobial effect, since the gingival bleeding index and Silness and Löe index were lower even in the presence of more dental plaque, so the nature of the plaque must be modified, possibly because of a change in the pH or by a different bacterial composition. In March 2015, Mahesh et al. published in the Contemporary Clinical Dentistry a randomized parallel clinical trial comparing a 0.2% chlorhexidine gluconate mouthwash versus a 0.6% cranberry mouthwash on Streptococcus mutans in 50 subjects, obtaining similar results in the colonisation of this bacteria in the plaque [25]. This study confirms the results obtained in our clinical trial, in which we hypothesised that the plaque could be different because of the minor inflammation of the periodontal tissue. Until this moment, only one study has been published about the use of cranberry mouthwash (a component of OPCs) in periodontal disease in humans [25]. However, in 2011, Löhr et al. published an in vitro study about the antimicrobial effect of the OPCs acting in the adhesion of Porphyromonas gingivalis to KB cells [23]. They postulated that the OPCs had an antiadhesive effect against P. gingivalis in the KB cells. The results of this study showed that OPCs exert antiadhesive effects against P. gingivalis, concluding that OPCs may be useful for the prevention of P. gingivalis-associated periodontal diseases [23]. Our study does not include bacterial analysis, but our results support it due to the fact that in the experimental group the parameters showed less inflammation and less cell response to the plaque, which could be attributed to its nature. Due to the previous study [23], we suspect that the antiadhesion effect modified the plaque of the subjects in the experimental group, making the inflammation lower. Lörh et al. showed that polyphenols interact with protein surface (e.g., gingipains, predominantly because of its activity against Arg-gingipain) and change the functionality and reactivity. Thus, OPCs mainly interact with the initial adhesion process of the bacteria to the cell membrane. This process could be extrapolated to our study, giving us an explication of our results. IL-6 has not shown significant differences, but a real effect in the inflammation of the gingiva took place in our patients; thus, the Löe and Silness index and gingival bleeding index were lower in the experimental group. BODY.5. CONCLUSION: The nutritional supplement made of oligomeric proanthocyanidins (cranberry and vitamin C) induces an improvement in the health of the periodontal tissues, since the Silness and Löe index and gingival bleeding index were markedly lower in the experimental group, although that supplement has not been shown to have effects on the accumulation of plaque on the tooth surface. Further studies are needed to determine the nature of the plaque.

TITLE: Calcipotriol Plus Betamethasone Dipropionate Aerosol Foam is Effective, Independent of Body Mass Index and the Extent and Severity of Psoriasis ABSTRACT.INTRODUCTION: Good treatment adherence is important in the effective management of psoriasis and is related to both the frequency of applications and the amount of product used versus the recommended dose. The efficacy and safety of fixed combination calcipotriol 50 μg/g (Cal) and betamethasone 0.5 mg/g as dipropionate (BD) in the treatment of psoriasis is well established; an aerosol foam formulation has been developed to enhance adherence. This subanalysis from the Phase III PSO-FAST study evaluates the amount of Cal/BD foam used during treatment and the association between the extent and severity of baseline disease. ABSTRACT.METHODS: Patients (≥18 years) with mild-to-severe body psoriasis were randomized 3:1 to once-daily Cal/BD foam or vehicle. The amount of Cal/BD foam and vehicle used over the 4-week study period was evaluated according to three baseline disease assessments: extent of body surface area (BSA) affected by psoriasis, physician's global assessment of disease severity (PGA) and modified psoriasis area and severity index (mPASI). Treatment success and mPASI75 rates were assessed according to body mass index (BMI) and body weight. ABSTRACT.RESULTS: 323 patients were randomized to Cal/BD foam and 103 to vehicle. At week 4, the mean total amount of Cal/BD foam used was 120.8 g (n = 293), which was similar to the amount of vehicle used (128.9 g; n = 98). The total amount of Cal/BD foam used at week 4 was greater with increasing BSA and increasing severity of baseline PGA and mPASI. Throughout the study, 93.1% of patients in the Cal/BD foam group and 99.0% of patients in the vehicle group missed ≤10% of treatment applications. Treatment success and mPASI75 rates were generally similar when stratified according to BMI and body weight. ABSTRACT.CONCLUSIONS: This subanalysis demonstrates that Cal/BD aerosol foam is used appropriately and is effective for the treatment of psoriasis, independent of BMI and the extent or severity of disease. ABSTRACT.CLINICAL TRIALS NUMBER: NCT01866163. ABSTRACT.FUNDING: LEO Pharma A/S. BODY.INTRODUCTION: Therapeutic guidelines for psoriasis recommend the topical use of corticosteroids and vitamin D3 analogs as first-line treatment, either as separate products (used together) or as a fixed combination treatment [1]. Treatment adherence has been recognized as an important issue in the management of chronic inflammatory skin diseases such as psoriasis [2]. Although many patients can manage their psoriasis with topical therapy alone [1, 3, 4], adherence remains a significant issue. Adherence is related to both the frequency of applications and the amount of product used compared with the recommended dose; a systematic literature review found that the frequency of topical treatment application in randomized controlled trials ranged from 55% to 100%, while patients applied between 35% and 72% of the recommended dose [2]. A number of reasons for poor adherence to topical therapy were identified, including a lack of efficacy, excessive time applying the medication, and poor cosmetic characteristics [2]. Adherence was shown to be greater for once-daily treatment as compared with twice daily [2]. Studies have also shown that the topical vehicle used can impact on adherence [5, 6]; patients generally prefer a vehicle that is simple and fast to apply, quickly absorbed and not greasy [7]. The efficacy and safety of the fixed combination of calcipotriol 50 μg/g (Cal) and betamethasone 0.5 mg/g as dipropionate (BD) has been confirmed in long-term trials [8–11]. The ointment and gel formulations of this fixed combination are established first-line treatments for psoriasis [12]. An aerosol foam formulation of the fixed Cal/BD combination has been developed to enhance adherence and increase the therapeutic options available to patients. Previous Cal/BD aerosol foam studies have shown greater in vitro skin penetration compared with other formulations [13], and a significantly greater anti-psoriatic effect over 4 weeks of treatment than Cal/BD ointment [14, 15], gel [16], and individual active ingredients [17], with a comparable tolerability profile [15–17]. In a large, double-blind, Phase III, 4-week study in patients with psoriasis vulgaris (PSO-FAST [NCT01866163]), treatment with Cal/BD aerosol foam led to significantly greater treatment success rates (53.3% vs. 4.8%; P < 0.001) and lower mean modified psoriasis area and severity index (mPASI; excluding head; 2.0 vs 5.5; P < 0.001) scores compared with aerosol foam vehicle [18]. This subanalysis from the PSO-FAST study evaluates the amount of Cal/BD aerosol foam used during treatment and the association between the extent and severity of baseline disease, in patients with psoriasis. BODY.METHODS.PATIENTS AND STUDY DESIGN: Patients eligible for inclusion in the PSO-FAST study were ≥18 years of age with a clinical diagnosis of mild-to-severe body psoriasis. Patients had between 2% and 30% of their body surface area (BSA; i.e., trunk and limbs) affected by the disease, with an mPASI score of ≥2. Full inclusion and exclusion criteria have been reported in Leonardi et al. [18]. PSO-FAST (Cal/BD aerosol foam in PSOriasis vulgaris, a Four-week, vehicle-controlled, efficacy And Safety Trial) was a Phase III, multicenter, double-blind study. Patients were randomized (3:1) to Cal/BD aerosol foam or aerosol foam vehicle once daily for up to 4 weeks. BODY.METHODS.ASSESSMENTS: The total amount of Cal/BD foam used by each patient during the 4-week study was determined by calculating the difference between the weight of the cans dispensed and the weight of the returned cans, multiplied by a correction factor of 0.41 to account for the propellant gases. Adherence was assessed by calculating the percentage of missed treatment applications (≤10%, >10–≤20%, >20–≤30%, >30–≤40%, >40–≤50% and >50%), as follows: the number of applications of Cal/BD foam missed for a particular visit interval was divided by the total number of days for the interval, multiplied by 100. Treatment success was defined as the proportion of patients who were clear or almost clear of disease with at least a two-step improvement at week 4, according to the physician's global assessment of disease severity (PGA). The rate of treatment success was the primary endpoint of the study and the data have been reported in Leonardi et al. [18]. BODY.METHODS.ANALYSIS: The total amount of Cal/BD foam used during the study was evaluated according to three baseline disease assessments: the extent to which a patient's BSA was affected by psoriasis vulgaris; PGA; and mPASI. The proportion of patients achieving treatment success was stratified according to body weight and body mass index (BMI). BODY.METHODS.COMPLIANCE WITH ETHICS GUIDELINES: The institutional review boards of all investigational sites approved the protocol. This study was conducted according to Good Clinical Practice guidelines and the principles of the Declaration of Helsinki 1964, as revised in 2013. All patients provided written informed consent prior to enrollment. The ClinicalTrials.gov identifier is NCT01866163. BODY.RESULTS: In total, 323 patients were randomized to Cal/BD foam and 103 to vehicle; 313 (96.9%) and 99 (96.1%) patients, respectively, completed the study. Of the 14 patients who withdrew, eight were lost to follow-up. The overall mean (±standard deviation) total amount of Cal/BD aerosol foam used after 4 weeks was 120.8 ± 85.7 g (n = 293/323), which was similar to the mean total amount of aerosol foam vehicle used (128.9 ± 92.9 g; n = 98/103). The mean amount of drug used per week was also similar between treatment groups: Cal/BD aerosol foam 29.8 g/week, vehicle 32.1 g/week. The mean amount of Cal/BD aerosol foam used was consistent from baseline to week 1 (29.9 g/week), from week 1 to week 2 (28.5 g/week), and from week 2 to week 4 (30.2 g/week). The total amount of Cal/BD aerosol foam used at week 4 was greater with increasing BSA and increasing severity of baseline PGA and mPASI (Fig. 1a–c). Most patients enrolled in the PSO-FAST study had ≤10% of their BSA involved at baseline (n = 227), and used on average between 88.3 and 117.6 g of Cal/BD aerosol foam during treatment (Fig. 1a). Throughout the 4-week treatment period, 93.1% of patients in the Cal/BD aerosol foam group and 99.0% of patients in the vehicle group missed ≤10% of treatment applications.Fig. 1Mean total amount of Cal/BD aerosol foam used at week 4 by baseline a BSA; b PGA; and c mPASI. BD betamethasone 0.5 mg/g as dipropionate, BSA body surface area, Cal calcipotriol 50 μg/g, mPASI modified psoriasis area and severity index, PGA physician's global assessment of disease severity, SD standard deviation Treatment success rates were generally similar irrespective of body weight and BMI (Fig. 2a, b). Similarly, additional evaluation of mPASI75 rates (i.e., the proportion of patients who achieved a 75% decrease in mPASI) did not demonstrate a difference in response according body weight or BMI (data not shown).Fig. 2Treatment success (defined as the proportion of patients who were clear or almost clear of disease with at least a two-step improvement at week 4, according to PGA) rates by a body weight and b BMI. BMI body mass index, PGA physician's global assessment of disease severity, SE standard error BODY.DISCUSSION: In the Phase III PSO-FAST study, most patients had mild-to-moderate disease severity (BSA <10%) and, on average, used 88–118 g of Cal/BD foam. The amount of Cal/BD aerosol foam used was comparable to vehicle and the product was used consistently throughout the 4-week study, suggesting that treatment persistence with the foam was good. In addition, patients used an appropriate amount of Cal/BD aerosol foam for the extent and severity of their psoriasis at baseline, since the mean total amount of Cal/BD aerosol foam used increased with increasing baseline BSA, PGA and mPASI. The primary analysis demonstrated that significantly more patients using Cal/BD foam achieved treatment success versus vehicle and mean mPASI score was significantly lower for patients using Cal/BD foam than vehicle at week 4 [18]. This subanalysis shows that the effectiveness of Cal/BD aerosol foam, based on treatment success rates, was not affected by body weight or BMI, so can be used in all patients irrespective of their size or extent of their BSA that is covered with plaques (as long as it is within the approved label). It has previously been suggested that BMI may be a prognostic factor for the response to treatment in psoriasis [19]; indeed, a number of studies have shown that some biologic therapies are less effective in patients with a BMI ≥25–30 compared with a BMI <25 [19–23]. One limitation of this analysis is that the regular monitoring that occurred during this clinical study of relatively short duration may reinforce patient adherence compared with that observed in the real world. Outside of the clinical trial setting, some patients are unintentionally non-adherent with treatment (e.g., because they forget to use the medication as directed), whilst others are intentionally non-adherent (e.g., altering or stopping treatment because of perceived ineffectiveness or side effects) [24]. BODY.CONCLUSION: In conclusion, this subanalysis from the PSO-FAST study demonstrates that Cal/BD aerosol foam is used appropriately and is effective for the treatment of psoriasis, independent of BMI and the extent or severity of disease.

TITLE: Early multidisciplinary assessment was associated with longer periods of sick leave: A randomized controlled trial in a primary health care centre ABSTRACT.ABSTRACT.OBJECTIVE: To study the effects on sick leave from an early multidisciplinary assessment at a primary health care centre. ABSTRACT.ABSTRACT.DESIGN: Randomized controlled trial. ABSTRACT.ABSTRACT.SETTING: Patients who saw GPs at a primary health care centre in mid-Sweden and asked for a sickness certificate for psychiatric or musculoskeletal diagnoses were invited to participate. Patients included were sick-listed for less than four weeks; 33 patients were randomized either to an assessment within a week by a physiotherapist, a psychotherapist, and an occupational therapist or to "standard care". The therapists used methods and tools they normally use in their clinical work. ABSTRACT.ABSTRACT.MAIN OUTCOME MEASURE: Proportion of patients still sick-listed three months after randomization, total and net days on sick leave, and proportion who were on part-time sick leave. ABSTRACT.ABSTRACT.RESULTS: At follow-up after three months, in contrast to the pre-trial hypothesis, there was a trend toward a higher proportion of patients still sick-listed in the intervention group (7/18) as compared with the control group (3/15). The intervention group also had significantly longer sick-listing periods (mean 58 days) than the control group (mean 36 days) (p = 0.038). The proportion of patients who were part time sick-listed was significantly higher in the intervention group (10/18) than in the control group (2/15) (p = 0.027). ABSTRACT.ABSTRACT.CONCLUSIONS: In this study an early multidisciplinary assessment was associated with longer periods on sick leave and more individuals on part-time sick leave. BODY: Multimodal/multidisciplinary rehabilitation has been regarded as "a gold standard" for shortening long periods of sick leave but the effect on sick leave has been questioned.In this RCT, early multidisciplinary assessment was associated with an increased total number of days on sick leave during the first three months.Early multidisciplinary assessment was correlated with a higher proportion of part-time sick listing.Further studies are needed to understand which patients can benefit from multidisciplinary assessment, the initiation point in the sick leave period, and what the content of the intervention should be. BODY.INTRODUCTION: In Sweden sick-leave issues have had high priority within the medical and political debate in recent years due to a rapid and large increase in the total number of granted sick-leave days in Sweden. In the period 1998–2003 the total number of sick-leave days more than doubled and the sum of sick- leave days and disability pension days divided by the number of persons insured in the national social insurance system increased from 33.6 days in 1998 to 43.2 days in 2003. The corresponding figures are now decreasing rapidly and in September 2011 the figure was 28.2 days. Approximately 20% of this figure was sick-leave days and 80% was disability pension days [1]. Disability pensioners report decreased quality of life and show increased mortality in several studies [2–4]. In Sweden the dominant causes of disability pension 2008 were psychiatric diseases (41%) and musculoskeletal disorders (25%). Most people in the general population who suffer from musculoskeletal pain are not sick-listed. In a study from the Swedish county of Dalarna of people who were not sick-listed, 49% of the men and 59% of the women reported "frequent pain in arm, back, or legs" [5]. The scientific knowledge of why some people are on sick-leave and others are not is scarce. Differences of opinion on sick-leave among patients, physicians, employers, and Social Insurance Agency staff have been demonstrated and may provide one explanation [6–9]. Even so doctors seldom refuse to sick-list their patients [10]. Symptom intensity and workplace requirements as well as individual differences in how symptoms affect perceived ability to work are other possible explanations. Multimodal rehabilitation is the gold standard method in Sweden to shorten long periods of sickness certification and is included in many recommendations from health authorities [11,12]. By definition, the term "multimodal" means that professionals from more than two health care disciplines are involved, working in an integrated team [13]. The authorities have taken initiatives and invested large sums of money (two billion SKr/year 2009–2012) in accelerating and improving the rehabilitation of sick-listed individuals [12,14,15]. When it was difficult to rehabilitate patients, GPs often said that this was because rehabilitation started too late. There are no studies showing positive effects of early interventions on return to work [16]. In a report "Sjukskrivning – orsaker, konsekvenser och praxis" the Swedish Council on Health Technology Assessment (SBU) identified a need for randomized intervention studies to investigate effects of interventions on sick-leave [17]. The aim of this study was to see if GPs, with support from an early multidisciplinary assessment carried out in a primary health care setting, could help patients to achieve faster and more appropriate rehabilitation to lower the risk of long term sick-leave. BODY.MATERIAL AND METHODS: The study took place at a county council-operated primary health care centre in mid Sweden. The health centre had a catchment area of 8500 inhabitants and had 4.5 full-time physician posts, one of which was vacant. One physiotherapist, one psychotherapist, and one occupational therapist made all assessments. All intervention patients met all three professionals. Patients eligible to participate in the study were sick-listed, either full-time or part-time, according to ICD 10-diagnoses chapter V F00-F99 (psychiatric diseases) or Chapter XIII M00-M99 (musculoskeletal diseases), and had an ongoing sick-leave period of a maximum of 28 days at randomization. The inclusion process took place from spring 2007 until winter 2008/2009. The GPs invited the patients to participate in the study after the sickness certification was issued, and gave them vocal and written information. Randomization was done by the sick-listing GP by opening randomly mixed closed envelopes. Patients randomized to intervention were given an appointment within a week to meet the assessors. Controls received "treatment as usual", which did not include this kind of early assessment. The physiotherapist performed a clinical examination of the musculoskeletal system. The psychotherapist made an assessment of the psychosocial situation at work and at home. The occupational therapist performed an assessment of the patient's general working capacity. All three therapists used the methods and tools they normally use in their clinical work (Appendix 1). For each patient, only methods judged relevant were used. The intervention did not include any treatment, but if a patient was judged to have potential to benefit from treatment, he or she was referred by the GP to standard healthcare resources. All information from the assessments was documented in the electronic patient record and usually also discussed with the doctor who had issued the medical certificate within a week. The data on duration and extent of the sick-listing periods in the study were taken from the electronic patient records and from the records of the Social Insurance Agency. Gross and net days were calculated. All patients who were included after randomization and who did not actively decline to attend were analysed (n = 33). We called this an analysis according to "intention to treat". Power calculation assumed that 30% of patients sick-listed after 14 days would still be on sick leave after three months. The aim of this study was to halve the number of patients still sick-listed at three months. With a p-value of 0.05 and a desired power of 0.8, 64 subjects were needed. Statistical analyses were performed in PASW 18 (SPSS). As the material was relatively small and not normally distributed, the tests used were non-parametric (Mann–Whitney U-test and Fisher's Exact Test). All analyses were calculated using two-sided tests. BODY.RESULTS: A total of 58 patients were invited to take part in the study. Eight GPs recruited the patients. In all, 36 patients agreed to participate in the study and were randomized, but three women (one in the control group and two in the intervention group) later withdrew from participation before assessment; 33 patients were finally committed to the study (Figure 1). Figure 1.Flow chart of eligible patients invited to participate in the study and randomized participants. The randomization resulted in groups that were similar regarding age, sex, and diagnoses on the sickness certificate (Table I). In the intervention group at randomization 15 of 18 were on full-time sick-leave and in the control group 14 of 15. Two in the intervention group and one in the control group were unemployed. Among patients who declined participation (n = 22) before randomization no significant difference in age compared with the study participants was found. In the analysis, seven out of 18 in the intervention group were still sick-listed at three months and three out of 15 in the control group (Table II). The number of days on sick-leave was significantly higher (p = 0.038) in the intervention group, with a mean value of 58 days, as compared with 36 days in the control group. The difference was slightly smaller and not significant (p = 0.070) when net days of sick-leave during the three first months were analysed. Net days of sick-leave were calculated as number of days in the period multiplied by the percentage of sickness certification. This can be explained by the fact that the proportion of individuals who were sick-listed part-time for a period during these three first months was significantly higher (p = 0.027) in the intervention group, 10 out of 18, as compared with two out of 15 in the control group. Table I. Comparison of age, sex, and diagnosis on sick note between all invited patients and those who participated in the study. n Age Women% Pain Psych. Pain+ Psych. Invited to participate 58 46 72 * * * Declined participation 22 46 82 * * * Randomized 36 46 67 27 6 3 Control group 15 48 67 11 3 1 Intervention (intention to treat) 18 44 61 13 3 2 Intervention (completed) 16 45 62 11 3 2 Table II. Sick-leave measures at three and 12 months for intervention and control groups. Intervention (n = 18) Control (n = 15) Still on sick leave after three months 7/18 3/15 p = 0.283 Total number of gross sick leave days in the first three months Mean (SD) 58 (32) 36 (33) p = 0.038 Median 65 21 IQR 69 51 Range 81 87 Total number of net sick leave days in the first three months Mean (SD) 48 (32) 32 (29) p = 0.070 Median 42 21 IQR 73 39 Range 84 87 Number of individuals who were on partial sick leave 0–3 months 10/18 2/15 p = 0.027 Still on sick leave after 12 months 4/18 1/15 p = 0.346 Total number of gross sick leave days 3–12 months, mean (SD) 91 (123) 58 (95) p = 0.727 Total number of net sick leave days 3–12 months, mean (SD) 77 (109) 37 (62) p = 0.580 Notes: (SD = standard deviation, IQR = Interquartile range). BODY.DISCUSSION: In contrast to our hypothesis, early multidisciplinary assessment was found to significantly increased days on sick-leave in the first three months. The proportion of people on part-time sick-leave was significantly higher in the intervention group, but sick-leave was longer in this group even when counted as net days. The strength of this study is the randomized design and the fact that it was carried out in a primary health care setting, where many sickness certification periods begin. The information on sick-leave days was complete, as both data from the electronic patient records and data from the Social Insurance Agency were included. Weaknesses are that data on sickness absence before inclusion are missing but randomization probably minimized any differences. The fact that this study could not randomize the planned number of individuals and that only one centre was involved adds further weakness. The relatively large number of patients who declined participation before randomization could be explained by the fact that some patients were on sick-leave for uncomplicated ailments with a good prognosis and considered the extensive assessment unnecessary. Another possible explanation could be the media debate on high sickness absence, which was very intensive when the study was being conducted. This may have been a reason for some patients eligible for inclusion to abstain, because they were concerned that an expanded assessment would question their need for sickness absence. This weakens the generalizability of this study. On the other hand the study gains generalizability from having been performed in an average Swedish, county council operated, primary health care centre as regards size, population, and access to doctors and other rehabilitation resources. Some studies carried out at pain or rehabilitation clinics have shown effects of multidisciplinary/multimodal treatment later in the sick-leave period (after four weeks) on patients sick-listed for low back pain [18]. The results from our study, indicating that early multidisciplinary assessment had the opposite effect, are not unique. In a Swedish study of an early multidisciplinary rehabilitation programme for neck and shoulder disorders lasting eight weeks, significantly more days of sick-leave in the first year were noted [16]. "Raskijobb" was a Danish study aiming to prevent sickness absenteeism but the effect was an increase of 15% more sick-leave days [19]. In a Swedish randomized evaluation of a "special resource team" for patients at risk of prolonged sick-leave, GPs, physiotherapists, behavioural therapists, and officials from the Social Insurance Agency made assessments of rehabilitation needs. The effect was a 20% increase in the number of sick-leave days as compared with a control group [20]. "SASSAM" is a method often used at the Social Insurance Agency offices in Sweden to support the patient's return to work and to assess the need for rehabilitation efforts. This method does not include any treatment. In a randomized study on early "SASSAM" there was a non-significant tendency towards longer sick-leave periods with this intervention [21]. In a Dutch study, efforts to reduce sickness absence also resulted in increased sick-leave days [22]. Two evaluations of "Rehabgarantin", where the Swedish government invested almost 1 billion SKr/year from 2009 to 2012 with the most common measure being multimodal rehabilitation for musculoskeletal pain, showed no effect on total sick-leave [23], or increased sick-leave days [24]. There are several possible explanations for the result of this study. Hanne Hollnagel, a Danish professor of Family Medicine, stresses the importance of focusing on the patient's own resources and opportunities in the consultation instead of only on symptoms and problems [25]. The extensive assessment by the physiotherapist, psychotherapist, and occupational therapist early in the sick-leave period might have the effect of focusing more on symptoms and problems. This could adversely affect "recovery expectations", "internal locus of control", "fear-avoidance", "catastrophizing", "self-perceived poor health", and "self-efficacy", all factors shown to be predictors of return to work [26–29]. Part-time sick-leave has been shown to have a potential to be a way back to work as well as posing a risk for extending the sick-leave [10,30]. In this study 3/18 in the intervention group and 1/15 in the control group were sick-listed part time at inclusion. Correcting for this difference, by excluding part-time sick-listed individuals at inclusion, did decrease the p-value for the difference in total days of sick-leave between the two groups. This is an argument against the idea that the difference in the proportion of people on part-time sick leave at inclusion could explain the results. Another possible explanation could be that the information that emerged at the multidisciplinary assessment process was not properly handled in the subsequent rehabilitation and sickness certification process that took place outside the study within ordinary healthcare resources. BODY.CONCLUSION: In this study, the total number of sick-leave days was significantly higher in the intervention group with early multidisciplinary assessment. Further randomized studies are needed to obtain better knowledge of which patients can benefit from multidisciplinary assessment in primary health care, when in the sick-leave period this should optimally be performed, and what the content of the intervention should be. BODY.ETHICAL APPROVAL: The study was approved by the ethics committee at Uppsala University registration number 2006/305.

TITLE: Effect of educational intervention on knowledge, perceived benefits, barriers and self-efficacy regarding AIDS preventive behaviors among drug addicts ABSTRACT.BACKGROUND AND OBJECTIVES:: Addicts account for approximately 68.15% of AIDS cases in Iran and injection drug users are considered as a major factor in the spread of AIDS in Iran. The purpose of this study was to determine the effect of an educational intervention on the perceived self-efficacy, benefits, and barriers concerning AIDS preventive behaviors among drug addicts in Khorramabad, Iran. ABSTRACT.METHODS:: This is a quasi-experimental study carried out in 2013 on 88 addicts kept in rehabilitations center in Khorramabad. The data collection instruments included a questionnaire on self-efficacy, perceived benefits, perceived barriers, knowledge and preventive behaviors regarding HIV. Data were analyzed by paired t-test, independent t-test, Chi-square and analysis of covariance. ABSTRACT.RESULTS:: Paired t-test showed that the mean scores for perceived benefits and barriers, knowledge and preventive behaviors significantly increased in the intervention group after the intervention than before the intervention. But the increase in self-efficacy score was not statistically significant. ABSTRACT.CONCLUSIONS:: The results of this study showed that training and education based on the health belief model led to an increase in knowledge, self-efficacy, perceived benefits, performance and reduction in perceived barriers in addicts. It is recommended that future studies should include strategies for enhancing self-efficacy and perceived benefits as well as strategies for reducing barriers to the adoption of preventive behaviors. BODY.INTRODUCTION: The HIV virus is one of the most lethal viruses discovered in the modern age and due to its high mortality rates and excessive caring costs, it is considered as a serious health and financial burden among all societies.[12] HIV has turned into an epidemic disease that threatens the global community.[3] Drug users (injecting drug users [IDUs]) represent the highest risk group for acquiring HIV and in some Eastern European and Asian countries almost all reported HIV cases are related to drug addicts. Studies and statistics show that the age of using drugs in these countries has reduced and that drug abuse is rapidly on the rise.[45] Besides, most addicts are not aware that they are infected with HIV.[6] According to estimates 3.4% of the world population or 4.7% of the world population aged 15 and older are drug addicts. In Iran, between 1.7% and 2.8% of the population aged 15 and older and 7.5% and 8% of the country's adult population are addicted to opium.[7] According to a report by the World Health Organization (WHO), the numbers of countries that report HIV infection in drug addicts are on the rising and this problem is particularly severe in developing countries.[8] According to the Ministry of Health and Medical Education to 92/7/1 in sum 27,041 persons with AIDS/HIV has been detected in the country. About 89.3% of men and 10.7% of women have been infected. 45.9% of patients at 25–34 have been that highest proportion among the age groups into account. The total of infection cases so far have been records in the country, sequencing intravenous drug abuse (68.15%), sexual intercourse (12.66%), blood and blood products (0.9%), mother to child (1.22%), (17.05%) is unknown.[9] The majority of HIV infection cases in Iran are IDUs. Injection drug use-related HIV transmission accounts for 5–10% of HIV cases in the world, 36% in the US and more than 60% of infection cases in Iran.[79] Studies have showed that IDU's are able to change their behaviors in a manner to protect themselves and others from HIV infection, and this point is very important for researchers that study in this field.[8] According to the WHO the only effective way to combat AIDS is health education and vulnerable groups should be prioritized.[10] HIV infection are highest among drug users and the problem of addiction combined with issues such as poverty, illiteracy, unemployment, and homelessness contributes to the risk of HIV infection.[10] The emerging HIV epidemic in Iran is serious and doubtlessly the rise of this epidemic will have a vast negative impact on the economic, social, and political situation in the country. Thus, efforts to contain this epidemic in Iran are a national obligation and urgent intervention strategies in the risk groups specifically injecting drug users is key to solving this problem.[11] Unawareness of or disregard for correct health behaviors is inevitable in any society. Individuals and societies require learning correct health behaviors through education in order to recognize and adopt a good lifestyle, maintain their health and prevent diseases.[12] The only way to combat this disease in the absence of treatment and effective vaccines is health education and behavior change.[13] Informing the society can be very effective in reducing HIV infections.[14] Designing and implementing an effective HIV prevention and control program based on reducing high-risk behavior has turned into a major challenge for health workers and researchers all over the world.[15] The value of educational health programs are assessed based on their effectiveness and their effectiveness is highly dependent on the correct utilization of theories and models in teaching health education. In other words, the existence of an appropriate support theory in accordance with the major health needs increases the effectiveness of educational health programs.[16] The effectiveness of the health belief model (HBM), which has been used as a theoretical framework in this study has been proved to be very useful in predicting AIDS preventive behaviors by different researchers. The HBM constructs have also proved to be effective in HIV/AIDS education programs as it helps understand behaviors that prevent AIDS and drug abuse.[1718] The health belief model is one of the oldest health behavior theories and different researchers have applied it in different behavioral fields in order to design and evaluate behavioral interventions. Plus the effectiveness of this model has been proved in the area of AIDS education in different internal and foreign researches.[192021] Based on this model, a person's decision to adopt a certain health behavior is influenced by three main categories of variables; individual perceptions, modifying factors and the likelihood of action. Individual perceptions refer to the subjective ideas regarding one's well-being, factors that affect the understanding of the disease or illness and the consequences of adopting a certain health behavior. Modifying factors are those variables, which affect an individual's appraisal of risk for disease or illness and include demographic variables, perceived threat of disease and ques to action: Which are influences external to the individual that provide a stimulus for activity.[10162223] Likelihood of action discusses the collective weighing of risks, benefits and costs in order to determine whether to take action or not. In fact, the health belief model allows us to study the probable psychological factors that influence individuals' decision-making process.[22] According to this model in order to implement HIV prevention methods, people should understand their chances of being infected by AIDS (perceived susceptibility), understand the seriousness of its symptoms in all physical, social, psychological, and economical aspects (perceived severity) and with the how-to information they receive from others or in the form of incentives (cues to action) they believe in the efficacy of the advised action, its benefits and applicability (perceived benefits) and find the deterring factors in taking the action less costly (perceived barriers), so that ultimately they take the measures. The researcher considers the HBM constructs of self-efficacy, perceived benefits and perceived barriers as important factors that influence health behaviors, specifically HIV/AIDS prevention behaviors. The constructs of self-efficacy, perceived benefits and perceived barriers are common in most health behavior theories and are three of the major components of the health belief model. Self-efficacy refers to the depth of one's trust in his/her ability.[24] The association between self-efficacy and AIDS prevention behaviors has been proved in different researches and can be effective in reducing high-risk behavior, which leads to HIV infection.[25] The shallow analysis of benefits minus barriers can happen in a situation where individual assess the benefits of taking of an action more costly, dangerous, unlikeable, saddening, and time-consuming compared with the barriers to it. Based on such an analysis, an individual adopts a certain behavior or refrains from doing so.[3] In order to change and improve health behaviors, professional health workers apply the constructs of self-efficacy, perceived benefits and perceived barriers as a theoretical basis in many of their health education programs. In fact, understanding factors that influence health behaviors can develop better health promoting strategies and success indicators can be chosen in a more logical manner.[26] A study by Mahmoud Karimi[3] and researches[2728293031] prove the predictive nature of the health belief model. This research was carried out due to the fact that there have been very few educational intervention programs as to lower high-risk behaviors in drug addicts based on this very model. The current research studies the influence of education on knowledge, perceived self-efficacy, perceived benefits and barriers as well as the performance of Khorramabad drug addicts when it comes to AIDS prevention behaviors. BODY.MATERIALS AND METHODS: A quasi-experimental and pre- to post-test research design was used to assess the impact of the HIV/AIDS educational intervention program. The selected population included 88 male addicts that were being kept in a rehabilitation center. The samples were selected from four different centers; 44 out of 2 of the centers were regarded as the intervention group and 44 of the 2 other centers as the control group. Due to limitations in the sample size, the samples were selected through the census method. And all of the addicts were being kept in the rehabilitation centers were entered to research. In the rehabilitation centers, HIV prevention and education programs are not offered. Patients are monitored 24 h, 7 days a week by trained staff. Apart from addicts that are under treatment by caregivers, no specific drugs are given to other patients. The researcher presented an official introduction letter from Isfahan University of Medical Sciences to the drug rehabilitation center in Khorramabad. In the beginning of the study, sufficient information regarding the aim of the study was given to the participants, and they were reassured that their data will remain confidential. Data gathering was undertaken by a questionnaire designed in four sections including:[1] Personal information (age, marital status, occupation, and...);[2] the second segment consisted of 10 questions to evaluate the knowledge level about HIV/AIDS pointed on a scale from 0 and 1 meaning that for a correct answer, 1 point and for a wrong answer or "I don't know no" no point was given.[3] The third segment consisted of 10 questions related to the perceived self-efficacy regarding AIDS preventive measures using a 4-point Likert scale, which ranged from "strongly agree" to "strongly disagree." Four-eight questions regarding the perceived benefits related to AIDS preventive measures with their answers scored on a 4 Likert scale that ranged from strongly agree to strongly disagree. Five-seven questions related to the perceived barriers related to AIDS preventive, which were also measured on a 4 Likert scale. Six-six yes/no questions related to the Practice, which were scored 0 and 1 therefore the maximum points acquired was 6 and the minimum. A knowledge and practice questionnaire was developed by the researcher. In order to evaluate the scientific validity of the questionnaire it was handed out to a couple of professional and experienced health education professors working at Isfahan University of Medical Sciences. As to examine the scientific reliability of the questionnaire the internal consistency test was used and the Cronbach's alpha was calculated (Cronbach's alpha = 76.3%). The self-efficacy, perceived benefits and barriers questionnaire used in this research was devised by Mahmoud Karimi under the title "Perceived benefits and perceived barriers to preventive behaviors of AIDS in addicts in Zarandieh".[26] Its validity and reliability of the questionnaire through content validity and test–retest methods was evaluated. Thus, the questionnaire was designed with the resources and scientific articles and polls research experts and use their feedback has been confirmed. Its reliability test–retest correlation coefficient Cronbach's alpha was calculated as 76% of health belief model. The information from the research departments were gathered through interviews (from the illiterate or less literate) and written responses from the samples themselves and analyzed through the SPSS 20 (IBM Corp. in Armonk, NY) descriptive analyses (means, standard deviations, and ranges) and analytical statistical methods (paired t-tests, independent t-test, Chi-square, and analysis of covariance). After extracting the necessary information from the pretest questionnaires, the results were analyzed and an educational model was designed. Pirzadeh conducted a study on students (27) in which educational interventions were implemented in 2 × 45 min sessions. These sessions were based on acquainting drug addicts with AIDS, its transmission and prevention along with improving the perceived self-efficacy, benefits of and barriers to this disease. The educational sessions was carried out among groups of 10–12 inside the center by means of speeches, face to face Q and As, group discussions and displaying educational films. Every group had 3 × 1.5 h sessions. To sustain the educational program, pamphlets, booklets, and educational films were distributed among the treatment group. The control group received no kind of education. Given that the addicts were retained in the center for an average 1 month period, to carry out the research phone contacts had to be made with the samples to complete the data gathering process. Therefore, 2 months after the educational intervention, the samples were invited to come over to complete posttest questionnaires through phone or personal visits. BODY.RESULTS: The mean age of the study participants was 33.31 years. Chi-square tests showed that there was no statistically significant difference between the two groups in terms of their marital status (P = 0.32), with married participants having the highest frequency and widowed participants, the lowest. The tests also showed that there was no significant difference between the two groups in terms of substance abuse (injectable and noninjectable drugs) (P = 0.5). The Mann-Whitney test showed that there was no significant difference between the two samples based on education level (P = 0.34) with the participants having less than a high school diploma having the highest frequency and participants with a master's degree the least [Tables 1–3]. Table 1 Marital status of participants (intervention and control groups) Table 2 distribution of participants based on their addiction status Table 3 Distribution of participants based on their educational status Paired t-test in the intervention group showed that after the intervention, the mean score for knowledge had increased; however, it was not statistically significant [Table 4]. Besides, this test showed that there was no significant difference in the mean score for perceived self-efficacy in the control group before and after the intervention. The independent t-test showed that before the intervention, the mean scores of perceived self-efficacy in the two groups had no significant differences with one another but after the intervention, the intervention group showed higher scores than the control group. Table 4 The mean score of knowledge about HIV preventive behaviors before and after intervention in the two groups The paired t-test showed that the mean score of perceived benefits had increased significantly in the intervention group after the intervention, but it was not statistically significant (P = 0.17). This test also showed that there was no significant difference in the control group's mean score of perceived self-efficacy before and after the intervention (P = 0.18). The independent t-test showed that there was no significant difference between both groups' mean score of perceived self-efficacy before the intervention (P = 0.91), but following the intervention the mean score of perceived self-efficacy in the intervention group was significantly higher than the control group (P = 0.04) [Table 5]. Table 5 Mean scores of health belief model constructs before and after intervention in the treatment group The paired t-test showed that in the intervention group, the mean score of perceived benefits had significantly increased after the intervention compared to before it (P = 0.002) but in the control group, no significant difference was seen (P = 0.16). The independent t-test showed that before the intervention, there was no significant difference between both groups' mean score of perceived benefits (P = 0.49), but after the intervention the mean score of perceived benefits in the intervention group was significantly higher than the control group (P = 0.02) [Table 5]. The paired t-test showed that after the intervention the mean score of perceived barriers had significantly decreased in both the intervention and control groups, compared to before the intervention (P < 0.001). The independent t-test showed that before the intervention, there was no significant difference between the mean scores of perceived barriers in both groups (P = 0.59), but after the intervention the mean score of perceived barriers had significantly decreased in both groups (P = 0.004) [Table 5]. The paired t-test showed that in the intervention group, the mean score of performance regarding Aids preventive behaviors increased significantly after the intervention (P = 0.03), but in the control group there was no significant difference between the mean score of performance before and after intervention (P = 0.61). The independent t-test showed that before the intervention there was no significant difference between the mean scores of performance in both groups (P = 0.59), but after the intervention the mean score of performance in the intervention group had raised significantly higher than the control group (P = 0.03) [Table 6]. Table 6 The mean score of performance about HIV preventive behaviors before and after intervention in the two groups The correlation coefficient showed that age correlated positively with performance (P = 0.01) but there was no correlation between education with knowledge, performance, self-efficacy, and perceived benefits, barriers. Knowledge positively correlated with performance (P = 0.01). Perceived self-efficacy correlated positively with performance (P = 0.009). Knowledge correlated positively with perceived benefits (P = 0.003). Knowledge correlated negatively with perceived barriers (P = 0.002). Benefits positively correlated with perceived self-efficacy (P < 0.001) [Table 7]. Table 7 Correlations BODY.DISCUSSION AND CONCLUSION: Poor health behaviors are seen in all kinds of societies whether illiterate, literate, rich, or poor. Culture imbibes into us the knowledge of good and bad behavior. People's readiness to recognize and pursue positive lifestyles in order to maintain their health and prevent diseases requires the shaping of their behaviors.[32] The results of this study showed that educating male drug addicts based on the health belief model, increases their knowledge, perceived benefits and barriers, and improves their overall performance. The knowledge levels of drug addicts in the intervention group before and after the intervention showed a significant difference. This change shows the effects of the educational intervention in increasing the awareness of addicts. The results of this study showed that implementing the health education program had a significant effect on improving the awareness and performance of the researched samples concerning the AIDS disease. These results comply with the results of studies carried out by Niknami et al. on the addicts in Kermanshah,[8] Zareban's study on the seamen of Chabahar,[12] and Clark's study on American women.[33] Informing people about the ways of HIV transmission, the preventive measures to take against it and its acceptance from people as to change dangerous and risky behavior is effective and would control and contain the disease.[32] In this study, there was a significant relationship between knowledge and perceived benefits of and barriers to adopting AIDs prevention behavior meaning that when a person's knowledge regarding the preventive behaviors against AIDS increases, the perceived benefits derived from taking preventive measures against AIDS would rise and the barriers to carry out these measures would decrease. The results of this study show that the mean score of perceived benefits after the intervention significantly increased in the intervention group (P = 0.002). The effectiveness of education on increasing the perceived benefits related to the preventive behaviors of AIDS in addicts was also seen in the studies conducted by Koopman and Voll.[20] Lollis et al., (23) and Karimi et al. (3). Various studies have shown the strong relationship between perceived benefits and taking preventive measures; an individual's understanding of the benefits of a specific measure paves the way for him/her to take it.[34] The results of this study showed that the mean score of perceived barriers before education was 65.8, which reduced significantly to 38.85 (P = 001). The effectiveness of education on decreasing the perceived barriers-related to AIDS prevention was seen in studies by Karimi et al. on drug addicts and Pirzadeh and Sharifiradon students.[27] In other studies, it has also been confirmed that educational programs have a positive and significant effect on reducing the perceived barriers, which complies with our study.[33536] A study by Adij in Ghana also demonstrated that people with low perceived barriers use condoms in their sexual intercourses almost 6 times more than other people.[37] The results of this study showed that before the intervention, there was no significant difference between the mean score of perceived self-efficacy in both groups (P = 0.91), but after the intervention, the mean score of perceived self-efficacy in the intervention group was significantly higher than the control group (P = 0.04). Studies examining the relationship between perceived self-efficacy and healthy behaviors have shown that self-efficacy has a strong effect on health behaviors and some studies suggest that self-efficacy determines >50 % variance of health behaviors.[38] The role of self-efficacy as an important basis for reducing risky behavior related to AIDS has been confirmed in studies conducted by Zamboni,[39] Smith[40] Bandora.[41] Improving self-efficacy is proposed as an intermediate goal to reduce HIV infection. According to the social cognitive theory, a person who has low self-efficacy is more probable to engage in a dangerous-risky behavior compared to a person with high self-efficacy.[42] In Adih's study in Ghana, people with high self-efficacy had used condoms 3 times more than those with low self-efficacy.[37] Kasen's study showed that students who had a low self-efficacy had engaged in sexual activity twice more than students with high self-efficacy and they had used condoms 5 time less.[43] Lin's study in Taiwan also showed that self-efficacy is the most reliable predictor in safe or unsafe sexual behavior.[28] The results of this study showed that there is a significant relationship between the perceived benefits of applying preventive AIDS behavior and perceived self-efficacy meaning that with the growth of the perceived benefits of a person regarding AIDS preventive behaviors, the self-efficacy of the person toward practicing this behavior increases. Besides, with the growth of perceived self-efficacy, practicing preventive behaviors also increases. According to the results of this study, there is a significant relationship between the perceived self-efficacy toward taking preventive measures and the actual adopting of those behaviors. Future studies should contain strategies to increase perceived self-efficacy and benefits as well as strategies to decrease perceived barrier to adopt preventive behaviors.[3] The results of this study show that there is a significant relationship between the demographic variable of age and the variable of performance in a way that the higher the age, the performance of the samples improved. Plus the demographic variable of age had no relationship with knowledge or any other studied construct and from this aspect it complies with the results attained by Momenion the students in Yasouj city[44] and Mahmoud Karimi on Zarandiyeh addicts.[3] In addition, the results showed that there was no significant relationship between the samples' education and awareness, performance and any of the other studied constructs in this study, which does not comply with studies by Karimi on the students of Yazd[45] and Niknami on self-proclaimed addicts in Kermanshah[8] in the aspect that there is a significant relationship between the education level and performance. However, it did comply with Mahmoud Karimi's study[3] in the sense that there was no significant relationship between the level of education and performance. Among the limitations of this study: There was a lack of cooperation among officials at the addicts rehabilitation centers due to their dislike of the educational subject (AIDS), the physical and psychological sickness of the addicts did not made it unlikely for them to take part in the educational sessions or complete the questionnaires, the time-consumingness of filling out the questionnaires for illiterate and less literate addicts, summoning up addicts to complete posttest questionnaires and their lack of cooperation. The results of this study show that the health education program designed based on the health belief model had a significant effect on improving the level of awareness of addicts as well as their perceived benefits, barriers and self-efficacy toward adopting preventive AIDS behavior in Khorramabad, which is in compliance with the results of[232728293031] studies. BODY.DISCUSSION AND CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: Deputy of Research and Technology of Isfahan University of Medical Sciences. BODY.DISCUSSION AND CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Comparison of clinical performance of the I-gel with LMA proseal ABSTRACT.AIM:: To compare insertion characteristics of 2 different supraglottic devices [I-gel and Proseal laryngeal mask airway (PLMA)] and to observe any associated complications. ABSTRACT.STUDY DESIGN:: This prospective, randomized study was conducted in 80 patients [Group I - I-gel insertion (n = 40) and Group P - LMA Proseal insertion (n =40)] of ASA grades I/II, of either sex in the age group 18-65 years. Both groups were compared with respect to ease of insertion, insertion attempts, fiberoptic assessment, airway sealing pressure, ease of gastric tube placement, and other complications. ABSTRACT.MATERIALS AND METHODS:: All patients were asked to fast overnight. Patients were given alprazolam 0.25 mg orally at 10 p.m. the night before surgery and again 2 hours prior to surgery with 1-2 sips of water. Glycopyrrolate 0.2 mg, metoclopramide 10 mg, and ranitidine 50 mg were administered intravenously to the patients 45 minutes prior to the surgery. Once adequate depth of anesthesia was achieved either of the 2 devices, selected using a random computerized table, was inserted by an experienced anesthesiologist. In group I, I-gel was inserted and in patients of group P, PLMA was inserted. ABSTRACT.STATISTICAL ANALYSIS:: Student t-test and Mann-Whitney test were employed to compare the means; for categorical variables, Chi-square test was used. ABSTRACT.RESULT:: Mean insertion time for the I-gel (11.12 ± 1.814 sec) was significantly lower than that of the PLMA (15.13 ± 2.91 sec) (P = 0.001). I-gel was easier to insert with a better anatomic fit. Mean airway sealing pressure in the PLMA group (29.55 ± 3.53 cm H2O) was significantly higher than in the I-gel group (26.73 ± 2.52 cm H2O; P = 0.001). Ease of gastric tube insertion was significantly higher in the I-gel group (P = 0.001). Incidence of blood staining of the device, sore throat and dysphagia were observed more in PLMA group. No other complications were observed in either of the groups. BODY.INTRODUCTION: The I-gel supraglottic airway device (Intersurgical Ltd, Wokingham, Berkshire, UK) was developed in 2007 to overcome the limitations of Proseal laryngeal mask airway (PLMA). It is made up of a thermoplastic elastomer (SEBS - styrene ethylene butadiene styrene) with a soft durometer (hardness), which has a gel-like feel.[1] It was designed to create a non-inflatable, anatomical seal of the pharyngeal, laryngeal and perilaryngeal structures while avoiding compression trauma. The shape, softness and contour accurately mirror the perilaryngeal anatomy to create the perfect fit, so that compression and displacement trauma are significantly reduced and has cheaper manufacturing costs due to the simplicity of design.[23] We compared the clinical performance of the I-gel and PLMA in terms of the efficacy and safety management in anesthetized patients on controlled ventilation, undergoing elective surgical procedures with respect to airway sealing pressure, ease of insertion, insertion attempts, fiberoptic assessment, ease of gastric tube placement, and complications. BODY.MATERIALS AND METHODS: The study was conducted after obtaining approval from the hospital ethical committee and a written informed consent from the patients. This prospective randomized study was conducted on 80 patients of American Society of Anesthesiologists physical status I/II, of either sex in the age group of 18-65 years, who were scheduled to undergo elective surgery in the supine position under general anesthesia with controlled ventilation. The exclusion criteria were patients with anticipated difficult airway, pregnancy, any pathology of the neck and upper respiratory tract or upper alimentary tract, mouth opening <2.5 cm, at risk of aspiration e.g., full stomach, hiatus hernia or gastro-esophageal reflux disease or those undergoing emergency surgery, BMI > 25 kg/m2, cervical spine disease, and head and neck surgical procedures were excluded from the study. All patients were asked to fast overnight. Patients were given alprazolam 0.25 mg orally at 10 p.m. the night before surgery and again 2 hours prior to surgery with 1-2 sips of water. Glycopyrrolate 0.2 mg, metoclopramide 10 mg, and ranitidine 50 mg were administered intravenously (IV) to the patients 45 minutes prior to the surgery. Baseline parameters [peripheral oxygen saturation (SpO2), electrocardiogram lead II, heart rate, systolic, diastolic, mean blood pressure and respiratory rate] were noted. Anesthesia was induced with fentanyl 2 μg/kg and propofol 2-2.5 mg/kg IV. Neuromuscular block was achieved with rocuronium 0.6 mg/kg IV. Both I-gel and PLMA were lubricated with water-soluble jelly. Patients were ventilated using facemask with nitrous oxide 67%, oxygen 33%, and 0.4% isoflurane for 180 seconds before attempting insertion of the chosen airway device. Once adequate depth of anesthesia was achieved either of the 2 devices, selected using a random computerized table, was inserted by an experienced anesthesiologist. In group I, I-gel of appropriate size according to the weight of the patient, was inserted as per manufacturer's instructions,[1] and in patients of group P, PLMA of appropriate size, according to the weight of the patient, was inserted as per manufacturer's introducer technique.[4] Cuff of the LMA Proseal was inflated with air to 60 cm H2O pressure and maintained at this pressure throughout anesthesia using a cuff pressure monitor. Both the devices were fixed by taping the tube over the chin and a well-lubricated gastric tube was introduced into the stomach through the gastric port. An effective airway was confirmed by bilateral symmetrical chest movements on manual ventilation, square waveform on capnography, no audible leak of gases and lack of gastric insufflation.[5] Maintenance of anesthesia was achieved with the nitrous oxide:oxygen mixture and 0.4% isoflurane and intermittent boluses of rocuronium administered IV. During anesthesia hemodynamic parameters were recorded prior to insertion of the device and then at 1, 5, 10, and 15 minutes after the insertion of device. Thereafter, monitoring was done at 15-minute intervals till the end of surgery. Insertion time was recorded by an independent observer and defined as time interval between picking up the device and securing an effective airway. However, if insertion failed at the second attempt, patient was withdrawn from the study and insertion was recorded as a failure and a cuffed endotracheal tube of appropriate size was inserted. Insertion was scored as per Table 1. Table 1 Insertion of device score If manipulation was required for achieving an effective airway, it was recorded as either 'yes' or 'no' and maneuvers required were noted. Airway sealing pressure was measured (at cuff pressure of 60 cm H2O in case of PLMA) by closing the expiratory valve of the circle system at a fixed gas flow rate of 3 L/min and recording the airway pressure at which equilibrium was reached. At this stage an audible leak at the mouth (sound of gas escaping from mouth heard by listening close to patient's mouth) and stomach (sound of gas escaping into esophagus heard by auscultation over epigastrium) was ascertained. Tidal volume loss was detected by inspiratory (set) - expiratory (outcome) volume on the ventilator display screen. Airway seal was scored as per Table 2. Table 2 Airway sealing quality score Cuff pressure of the PLMA was checked every 30 minutes till the end of surgery and was maintained at 60 cm H2O by removing air from the cuff using a syringe. Cuff pressure was checked using Portex Cuff Inflator/Pressure Gauge (SIMS Portex Limited, Hythe, UK). The amount of air removed was recorded. The anatomical position of the device was assessed by introducing a flexible fiberoptic bronchoscope into the airway tube to a position proximal to the terminal end. The scoring of fiberscope examination view was done as per Table 3. Table 3 Fiberoptic scoring system[ 6 – 8 ] Ease of insertion of the gastric tube was recorded as either: easy/difficult/failure. Its correct placement was confirmed by aspiration of the gastric contents or by injection of air and auscultation over the epigastrium. Failure was defined as inability to advance the orogastric tube into the stomach within 2 attempts. At the end of the surgical procedure anesthesia was discontinued, neuromuscular blockade was reversed with neostigmine 0.05 mg/kg and glycopyrrolate 0.01 mg/kg IV and the device was removed. Blood staining of the device, tongue, lip, and dental trauma were recorded. Regurgitation of gastric contents was also assessed. Patients were questioned after regaining full consciousness and again after 24 hours to assess pharyngolaryngeal morbidity (sore throat, dysphagia, and dysphonia). Data is presented as mean ± SD. Age, height, weight, duration of surgery, time taken to secure effective airway, number of maneuvers required to insert the device, airway sealing pressure, attempts of gastric tube insertion, and cuff characteristics were compared using the Student t-test and Mann-Whitney test. Gender, ease of insertion, manipulations required to insert the device, ease of gastric tube insertion, and fiberoptic view between the groups were compared using the Chi-square test. Complications were compared using Fisher exact test. P values of ≤ 0.05 were considered significant. BODY.RESULTS: The demographic profiles of patients in both the groups were similar [Table 4]. In all patients the supraglottic device, I-gel or PLMA, was inserted within 3 attempts. Mean insertion time for the I-gel (11.12 ± 1.814 sec) was found to be significantly lower than the mean insertion time for PLMA (15.13 ± 2.91 sec) [P = 0.001; Table 5]. A statistically significant difference (P value = 0.0004) was found between the I-gel (grade 3 = 32/40) and PLMA (grade 3 = 25/40) groups with regard to ease of insertion. Significantly, higher [P value = 0.0004; Table 5] no. of manipulations were required in the PLMA group (17/40 = 42.5% cases) as compared to the I-gel group (3/40 = 7.5% cases) to insert the device and the most frequent maneuver required was extension of the head and neck. A better anatomic fit was achieved in the I-gel group (grade 1 = 97.5% cases) as compared to the PLMA group (grade 1 = 75% cases) [P = 0.001; Table 5]. The mean airway sealing pressure in the PLMA group (29.55 ± 3.53 cm H2O) was found to be significantly higher than that observed in the I-gel group (26.73 ± 2.52 cm H2O) [P value = 0.0001; Table 6]. Table 4 Demographic data and type of surgeries performed Table 5 Insertion characteristics Table 6 Maintenance characteristics Adequate ventilation was achieved in both the groups. No patient, in either group, was observed to have a major loss i.e., > 40% of the tidal volume. Airway sealing quality as determined by percentage loss of delivered tidal volume was comparable between the 2 groups [P = 1; Table 6]. Other parameters like heart rate, blood pressure, end-tidal carbon dioxide and SpO2 were comparable between the 2 groups and within normal limits during the perioperative period. No episode of hypercapnia or desaturation was observed. There were statistically significant differences regarding postoperative adverse events between the 2 groups. Higher incidence of macroscopic blood staining of the supraglottic device, sore throat, and dysphagia were observed in PLMA group as compared to the I-gel group [P = 0.045; Table 7]. None of the patients had dysphonia or any other complications in either of the groups. The mean intra-cuff pressure of the PLMA increased to 77.65 ± 7.57 cm H2O after 30 minutes and 96.82 ± 6.82 cm H2O after 90 minutes, from the initial value of 60 cm H2O, during the course of general anesthesia. The mean amount of air removed after 30 and 60 minutes were observed to be 2.99 ± 0.72 ml and 4.01 ± 0.67 ml, respectively. Table 7 Recovery characteristics BODY.DISCUSSION: The inflatable cuff of PLMA may be the cause of various malpositions after insertion.[2] The cuff of PLMA may impede its proper placement and lack of back-plate may lead to a fold over malposition.[910] The mean insertion time in the I-gel Group I was significantly lower (11.12 ± 1.814) than mean insertion time of the PLMA group (15.13 ± 2.91) cases. A statistically significant difference was found between group I (grade 3 = 32/4) and group II (grade 3 = 25/40) with regard to ease of insertion (P = 0.0004). None of the patients, in either of the groups, required a second attempt for inserting the device. The median insertion time of 11 seconds has been reported with I-gel, with a first attempt insertion rate of 90% and the balance requiring a second attempt while none needed a third.[11] Since no cuff inflation is required in the I-gel, there is a shorter time required to achieve an effective airway, it is easier to insert and success at first attempt is more as compared to the PLMA.[12–15] In our study significantly more number of manipulations (P value = 0.0004) were required in cases in group P to insert the device. Fiberoptic scores confirmed that the I-gel has an excellent anatomic fit (Grade 1 view = 97.5%), which is significantly better than the PLMA (Grade 1 view = 75%). I-gel consistently achieves proper positioning for supraglottic ventilation and causes less hemodynamic changes as compared to other supraglottic airway devices.[16] In our study the mean airway sealing pressure in the group P (29.55 ± 3.53) patients was found to be significantly higher than that observed in group I (26.73 ± 2.52) patients. The average airway sealing pressure was reported as 25.27 cm H2O with I-gel and 29.6 cm H2O PLMA.[12] The mean leak pressure has been reported as 25.6 ± 4.9 with the use of I-gel.[17] The seal pressure appears to improve over time in a number of patients due to the thermoplastic properties of the gel cuff, which may form a more efficient seal around the larynx after warming to body temperature.[13] A gradual increase was seen in the cuff pressure of PLMA well over a 3-hour period during nitrous oxide and oxygen anesthesia.[18] We found a similar increase in values of intra-cuff pressure of PLMA at 30 and 60 minutes post-insertion. The ease of gastric tube insertion was significantly higher (P = 0.001) in group I (easy = 95%) as compared to group II (easy = 72.5%). The success rate of first time insertion of gastric tube was 100% with the I-gel (30/30) than with the PLMA (26/30). These observations are in close approximation to the results reported by others.[121419] Hemodynamic parameters were comparable between the 2 groups throughout the course of the surgical procedures. In the present study use of the PLMA is associated with a higher incidence of pharyngolaryngeal morbidity (blood staining of the device, sore throat, and dysphagia) in comparison to the I-gel. Blood staining of the device was more with the PLMA (18%) than with the I-gel (3%). There were no incidences of bronchospasm, laryngospasm, aspiration, regurgitation, and hoarseness in both the groups. Singh et al.[12] reported that the incidence of tongue, lip, and dental trauma was observed in 16.7% (5/30) patients in the PLMA group and in 3.3% (1/30) patients in the I-gel group. After removal of the I-gel a short coughing episode and a transient moderate sore throat was reported.[14] With use of PLMA reported incidence of sore throat is 23% after operation and 16% after 24 hours, with 90% of the sore throats being described as mild.[20] Devices with an inflatable mask have the potential to cause tissue distortion, venous compression, and nerve injury, which explains the increased incidence of associated postoperative morbidity.[2] Trauma on insertion, multiple insertions, and pressure exerted by cuff against the pharyngeal mucosa,[21–26] cuff volumes[27] and pressure[26] have all been incriminated for postoperative complications. The only parameter in which PLMA is better than I-gel is the airway sealing pressure. PLMA provides higher airway sealing pressures (oropharyngeal leak pressures) as compared to I-gel however the airway sealing pressure of I-gel is also within the normal limits and is reported to be effective in preventing aspiration. The present study has not examined the performance of the I-gel in patients with a full stomach. Our results reflect that the use of I-gel in such patients may not be appropriate. To conclude, I-gel is comparable to the PLMA in securing a patent airway during controlled ventilation. It is better than PLMA in terms of faster insertion and ease of insertion with a low incidence of pharyngolaryngeal morbidity. It requires less manipulation and no cuff inflation is required, therefore securing an airway is rapid in most of the patients. Although, the sample size of the present study is relatively small, it clearly elucidates that the I-gel appears to be efficacious in insertion characteristics. Our study however offers almost no conclusive evidence of unflinching safety of the device, which requires data from a considerably larger cohort in a routine practice.

TITLE: Acute Dietary Nitrate Supplementation and Exercise Performance in COPD: A Double-Blind, Placebo-Controlled, Randomised Controlled Pilot Study ABSTRACT.BACKGROUND: Dietary nitrate supplementation can enhance exercise performance in healthy people, but it is not clear if it is beneficial in COPD. We investigated the hypotheses that acute nitrate dosing would improve exercise performance and reduce the oxygen cost of submaximal exercise in people with COPD. ABSTRACT.METHODS: We performed a double-blind, placebo-controlled, cross-over single dose study. Subjects were randomised to consume either nitrate-rich beetroot juice (containing 12.9mmoles nitrate) or placebo (nitrate-depleted beetroot juice) 3 hours prior to endurance cycle ergometry, performed at 70% of maximal workload assessed by a prior incremental exercise test. After a minimum washout period of 7 days the protocol was repeated with the crossover beverage. ABSTRACT.RESULTS: 21 subjects successfully completed the study (age 68±7years; BMI 25.2±5.5kg/m2; FEV1 percentage predicted 50.1±21.6%; peak VO2 18.0±5.9ml/min/kg). Resting diastolic blood pressure fell significantly with nitrate supplementation compared to placebo (-7±8mmHg nitrate vs. -1±8mmHg placebo; p = 0.008). Median endurance time did not differ significantly; nitrate 5.65 (3.90–10.40) minutes vs. placebo 6.40 (4.01–9.67) minutes (p = 0.50). However, isotime oxygen consumption (VO2) was lower following nitrate supplementation (16.6±6.0ml/min/kg nitrate vs. 17.2±6.0ml/min/kg placebo; p = 0.043), and consequently nitrate supplementation caused a significant lowering of the amplitude of the VO2-percentage isotime curve. ABSTRACT.CONCLUSIONS: Acute administration of oral nitrate did not enhance endurance exercise performance; however the observation that beetroot juice caused reduced oxygen consumption at isotime suggests that further investigation of this treatment approach is warranted, perhaps targeting a more hypoxic phenotype. ABSTRACT.TRIAL REGISTRATION: ISRCTN Registry ISRCTN66099139 BODY.INTRODUCTION: Exercise limitation is a common symptom in patients with COPD. While the benefits of supplemental oxygen, at least in non-hypoxaemic subjects, on exercise performance remains uncertain[1], substantial skeletal muscle hypoxia is observed during exercise in both normal subjects and COPD patients whose oxygen saturation is maintained by the administration of supplemental oxygen[2]. Taken together with other data which suggest that anaerobic metabolism is frequent in patients with COPD[3, 4], a potential contributor to exercise limitation in COPD could be through the mechanism of tissue oxygen delivery, intramuscular oxygen transport or mitochondrial dysfunction. Thus approaches which reduce the oxygen cost of exercise might be of therapeutic benefit. Both nutrient and oxygen delivery, as well as mitochondrial function (and thus oxygen consumption), are under regulatory control by the physiological signalling molecule nitric oxide (NO). NO is produced endogenously by the action of the NO synthase (NOS) family of enzymes via oxidation of the amino acid L-arginine. NO may also be produced by the reduction of exogenous dietary nitrate (NO3 -), found at high levels in beetroot and green leafy vegetables[5], to nitrite (NO2 -) and then further to NO in a NOS-independent manner[6]. Ingested nitrate is enterally absorbed into the blood, with approximately 25% of the circulating amount being taken up by the salivary glands (enterosalivary circulation). Nitrate is then excreted in saliva[7], and reduced from nitrate to nitrite via the nitrate reductase activity of oral commensal facultative anaerobic bacteria[8]. Salivary nitrite is subsequently swallowed and may enter the circulation directly as nitrite[8], or be further reduced to NO and absorbed following oxidation to nitrite or nitrate. Various studies have reported benefits from dietary nitrate supplementation through the mechanism of a reduction in the oxygen cost of submaximal exercise in young, healthy individuals[9–13], and this has been associated with improved measures of exercise performance[9–12, 14]. The majority of studies have used beetroot juice as the nitrate source, the oxygen-sparing effect being dependent largely on nitrate itself since nitrate-depleted beetroot juice fails to elicit the same effect[11]. This effect has been shown to persist over at least 15 days of continual supplementation [12]. A beneficial effect of nitrate supplementation has also been demonstrated during exercise in hypoxic conditions[15], which is of interest for people with chronic respiratory disease. Two early studies suggested benefit in patients with COPD [16, 17], although both studies were limited by the placebo preparation selected, and other groups have failed to reproduce these findings using a robust placebo[18, 19]. However, in the two latter studies the dose of nitrate administered immediately prior to testing was below that which has been shown in health to induce improvement in exercise performance[6], and in one case testing was performed only one hour post dosing[19] when nitrite levels may not yet have peaked. Thus these findings need further investigation with a higher dosing regimen given at an appropriate time interval prior to exercise testing, particularly since the studies failed to show any effect on blood pressure[18, 19]. The aim of this study was to investigate the effects of a higher dosing regimen of nitrate, in the form of beetroot juice, on exercise performance in COPD with concomitant measurements of pulmonary oxygen uptake and with comparison to a robust placebo preparation. BODY.MATERIALS AND METHODS.PATIENT SELECTION: All participants provided written informed consent prior to enrolment in the study, which was approved by the Bromley Research Ethics Committee (reference 13/LO/0372) and conducted in line with the principles of the Declaration of Helsinki. COPD patients of GOLD stage II-IV[20] were considered for inclusion and recruited through outpatient clinics at the Royal Brompton Hospital and through public outreach events including World COPD Day. Recruitment started on 24th June 2013 and was completed on 28th April 2014, with follow-up completed on 3rd June 2014. This study was registered prospectively on a publicly accessible database (www.iscrn.com/ISRCTN66099139). The authors confirm that all ongoing and related trials for this intervention are registered. Exclusion criteria for the study included patients taking nitrate-based medications or phosphodiesterase V inhibitors, those with other medical conditions likely to benefit from nitrate supplementation (including ischaemic heart disease and peripheral vascular disease), and patients on long-term oxygen therapy or antibiotic therapy (whether on an acute or prophylactic basis). Those patients considered clinically unstable (within one month of pulmonary exacerbation) or suffering from hypotension or significant renal impairment (eGFR<50ml/min/1.73m2) were also excluded from the study. BODY.MATERIALS AND METHODS.STUDY DESIGN AND RANDOMISATION: The study was a double-blind, cross-over, placebo-controlled trial. Allocation of the order of beverages was performed using a computer-generated randomisation in blocks of 4, produced by an independent statistician with consecutive numbers linked to preparations of placebo or active treatment. The researcher responsible for enrolment and outcome measurements remained separate from the randomisation process and remained blinded throughout the study and during data analysis. The principal investigator who was not involved in any patient visits held the randomisation list and indicated the patient assignment order. We hypothesised that nitrate supplementation would increase time to exhaustion during symptom-limited cycle ergometry at 70% peak workload as assessed previously using an incremental, maximal, symptom-limited cardiopulmonary exercise test. Secondary outcome measures included plasma nitrate and nitrite levels, blood pressure response, area under the VO2 curve to isotime, and plasma metabolomics. BODY.MATERIALS AND METHODS.INTERVENTION: 140ml of BEET-IT Sport Stamina Shot (James White Drinks, Ipswich, UK) containing 0.8g nitrate and 140ml of a matched placebo of beetroot juice specifically depleted of nitrate were used as the active and placebo preparations, respectively. The placebo preparation was identical in appearance and taste[11], and also produced beeturia. The dose chosen was intended to provide a bolus of nitrate (12.9mmoles) exceeding that necessary to reduce oxygen consumption during submaximal exercise in young healthy subjects[6], and was selected as a convenient dosing method (2x 70ml bottles) which was readily acceptable and easily ingested. Subjects were requested to avoid foods naturally high in nitrate in the 48hour period prior to the dosing visit and were asked to match food and caffeine consumption on the two days of testing. Subjects were additionally asked to avoid use of mouthwash and chewing gum in the 48hour period prior to testing as this has previously been shown to reduce oral bacterial nitrate reductase activity[21]. Subjects were also advised to avoid heavy exercise in the 24 hours prior to each dosing visit. BODY.MATERIALS AND METHODS.STUDY CONDUCT: All data were collected at the NIHR Respiratory Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust. Following successful screening the subjects attended for measurement of baseline characteristics during which several parameters were recorded including vital signs, anthropometric and bioimpedance measurements, quality of life assessment, full pulmonary function testing, quadriceps maximal volitional contraction, physical activity monitoring, and an incremental maximal symptom-limited cycle ergometry test was performed. On the dosing visits the subjects attended on the day of testing in the morning; after a 10 minute rest period blood pressure measurements and baseline plasma samples were obtained, after which the patients were dosed. 3 hours later both blood pressure measures and blood sampling was repeated prior to endurance cycle ergometry at a constant work rate (70% peak workload achieved on prior maximal incremental exercise testing). The cycle test was conducted at the same time on each occasion. Further blood draws were taken at peak exercise. After a minimum wash-out period of 7 days the protocol was repeated with the crossover beverage. BODY.MATERIALS AND METHODS.BLOOD PRESSURE, ANTHROPOMETRICS AND FAT FEE MASS MEASUREMENTS: Blood pressure was measured using an automated blood pressure monitor (Omron M6, Omron Healthcare Europe, Hoofddorp, Netherlands). Measurements were performed in the seated position with the subject's arm supported. A mean of three measurements was recorded. Height (cm) without shoes was measured using a wall mounted measure, and weight (kg) measured using standardised scales. Fat free mass (FFM) was determined using bioelectrical impedance analysis by measuring the electrical resistance between the wrist and ankle using a Bodystat 4000 device (Bodystat, Isle of Man, UK). Subjects lay supine for 10 minutes prior to measurement. Electrodes were placed on the dominant hand and foot, at 2cm proximal to the carpometacarpal joint and the ulnar level of the wrist on the dorsal aspect of the hand, and at 2cm proximal to the carpometatarsal joint and level of the malleoli of the ankle respectively. The value obtained is dependent on body water content and based on this FFM can be calculated using a disease specific regression equation[22]. Fat Free Mass Index (FFMI) was calculated by dividing FFM by height in metres squared (kg/m2). Individuals with a FFMI below 16 kg/m2 in males and 15 kg/m2 in females were considered to have evidence of FFM depletion[22]. BODY.MATERIALS AND METHODS.PHYSICAL ACTIVITY MEASUREMENT: Patients were asked to wear a physical activity monitor (SenseWear Pro Armband, Bodymedia, Pittsburg, USA) continually for a week that incorporates a biaxial accelerometer and energy expenditure measurements[23]. The armband was placed over the body of the triceps muscle of the right arm and the subject asked to wear it continuously except when bathing. Data was analysed over a period of 5 days including 2 weekend days. BODY.MATERIALS AND METHODS.QUADRICEPS STRENGTH: Maximum isometric quadriceps force (QMVC) was measured using the technique of Edwards et al.[24]. Prior to each measurement calibration was performed using a standardised weight. Subjects were seated with the leg flexed at an angle of 90° relative to the edge of the couch. The right leg was tested unless it was not feasible to do so. An inextensible strap was placed around the subject's ankle and connected to a strain gauge. The maximum force generated and sustained for at least 1 second during at least five contractions with vigorous encouragement was measured (recorded in kg). A minimum of 30 seconds was allowed between each effort. Output from the strain gauge was processed as a digital output by a Powerlab recording unit and analysed using LabChart software (AD Instruments, Oxfordshire, UK) sampling at 10kHz. The percentage predicted QMVC was determined according to regression equations based on age, gender and FFM[24, 25]. BODY.MATERIALS AND METHODS.PULMONARY FUNCTION TESTING: Measurements were made in the lung function department of the Royal Brompton Hospital according to international guidelines and with rigorous quality assurance in place with a Jaeger master lab system (CompactLab system, Jaeger, Wurzburg, Germany). Spirometry, gas transfer and plethysmographic lung volumes were also measured in accordance with European Respiratory Society (ERS)/ American Thoracic Society (ATS) guidelines[26–28]. Standardised lung function testing reference equations were based on the European Coal and Steel Community (ECSC) reference values[29]. Capillary blood gas samples were taken at rest. BODY.MATERIALS AND METHODS.HEALTH-RELATED QUALITY OF LIFE MEASURES: Subjects completed the St George Respiratory Questionnaire for COPD patients (SGRQ-C) and the COPD Assessment Test (CAT). Each is validated in assessing health-related quality of life in COPD [30, 31]. BODY.MATERIALS AND METHODS.CYCLE ERGOMETRY TESTING: A symptom limited incremental exercise test was performed at baseline on a bicycle ergometer (Ergoselect 100, Ergoline, Bitz, Germany) with metabolic measurements collected using a mouthpiece (Masterscreen CPX metabolic cart, CareFusion, Basingstoke, UK) and analysed using JLAB software (JLAB Lab Manager version 5.32, Jaeger, CareFusion, Basingstoke, UK). Following a 2 minute rest period and 2 minute free cycling workload increased by 5 Watts every 30 seconds with subjects being asked to maintain a speed of 60–70 revolutions per minute. Measurements were taken of peak workload, VO2, VCO2, minute ventilation, respiratory rate and tidal volume. Peak workload was defined as the greatest workload that the subject was able to maintain for a 30 second period. To evaluate the effects of the intervention on cardiorespiratory fitness and skeletal muscle function endurance cycle ergometry testing was performed 3 hours following dosing at 70% peak workload achieved on incremental cycle ergometry. The endurance time was calculated as the time from the beginning of loaded cycling to the point of test cessation due to symptom limitation. Breath-by-breath data was obtained and rolling 8-breath averages were used in the isotime analysis (with isotime being defined as the duration of the shorter of the two exercise tests). BODY.MATERIALS AND METHODS.PLASMA NITRATE/NITRITE ANALYSIS: All plasma samples (baseline, 3 hours post dosing and peak exercise) were obtained by venesection in lithium heparin tubes and immediately spun for 10 minutes at 520g at 4°C. The resulting supernatant was pipetted into 2ml polypropylene cryotubes, taking care to avoid any contamination from the buffy coat containing the white cells/ platelets atop the erythrocyte pellet, and immediately frozen at -80°C. Nitrate and nitrite plasma levels were assessed using a post-column diazo coupling reaction (Griess reaction) in combination with high-performance liquid chromatography (HPLC) (Eicom NOx analyser, ENO-20, San Diego, USA)[32]. Nitrate and nitrite were first separated on the analytical column. Nitrite then reacts with the Griess reagent generating a red diazo compound, the absorbance of which was measured at 540nm using a spectrophotometric detector. Nitrate passing through the on-line reduction column was reduced to nitrite before undergoing the same diazo coupling reaction. The peak areas of absorption of solutions of know standard concentrations were then compared to those produced by the test samples to provide measures of plasma nitrate and nitrite. BODY.MATERIALS AND METHODS.PLASMA NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY ANALYSIS: Plasma samples were defrosted at room temperature and centrifuged at 16,000g for 10 minutes. An aliquot of 300μl was then mixed with 300μl of NMR buffer (double distilled water containing 8% deuterium oxide (D2O) for the magnetic lock). The resulting mixture was transferred to 5mm NMR tubes within a 96-tube rack. 1H-NMR spectra of plasma samples were obtained using a Bruker Avance III 700 MHz spectrometer (Bruker Biospin, Karlsruhe, Germany) at the operating 1H frequency 700.2 MHz with a temperature of 298K. A Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence (Bruker experiment: cpmgpr1d) was applied to better visualise low molecular weight metabolites. A spin relaxation delay of 76ms was used and a total of 32 scans were acquired. Spectra were processed and analysed as described previously[33]. An exponential window function was applied with a line broadening of 0.3 Hz prior to Fourier transformation. All chemical shifts were manually referenced to formate (δ = 8.44 ppm). BODY.MATERIALS AND METHODS.DATA ANALYSIS AND STATISTICS: We studied a convenience sample of 25, offering a good chance of identifying a clinically meaningful effect. Data are presented as mean ± standard deviation and were analysed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, California, USA). Repeated measures analysis of variance (ANOVA) was utilised to compare plasma nitrate metabolites between treatment conditions and at each specific time-point. To compare oxygen consumption between study conditions, individual cardiopulmonary exercise test data periods were expressed as percentiles of isotime (the duration of the shorter of the two tests) and the individual responses grouped to allow analysis of VO2 against percentage of isotime (plotted at the midpoint of each 10th percentile of isotime). The area under this curve was assessed for each subject and the two conditions, then compared using a paired t-test. Resting and isotime measurements between treatment conditions were compared using paired t-tests, or the appropriate non-parametric test for data that was not normally distributed. A p-value <0.05 was considered to be statistically significant. BODY.RESULTS.SUBJECTS: 25 patients were enrolled into the study, of whom 21 completed the full study protocol (Fig 1). Recruitment started in June 2013 and the trial completed in June 2014 when the 25 enrolled patients had either completed or been withdrawn. The baseline characteristics of the participants are presented in Table 1. Patients were 100% compliant with the dosing protocol with no adverse effects noted except beeturia. 10.1371/journal.pone.0144504.g001Fig 1CONSORT recruitment diagram for enrolment and study completion. 10.1371/journal.pone.0144504.t001 Table 1 Demographic and baseline clinical characteristics of the subjects (n = 21). Measurement Mean (SD) Sex (♂:♀) 16:5 Age (years) 68 (7) Systolic BP (mm Hg) 137 (19) Diastolic BP (mm Hg) 79 (7) Smoking PYH 40 (29) MRC dyspnoea score 2 (1) CAT score 14 (7) SGRQ-C total 35.37 (13.00) BMI (kg/m 2 ) 25.2 (5.5) FFM (kg) 48.9 (8.4) FFMI (kg/m 2 ) 16.9 (2.2) QMVC (kg) 37.8 (10.7) QMVC % predicted 87.8 (19.3) Average daily step count 6669 (4436) Average PAL 1.50 (0.20) FEV 1 (L) 1.33 (0.58) FEV 1 % predicted 50.1 (21.6) TLCO c % predicted 54.1 (19.3) RV % predicted 167 (53) TLC % predicted 119 (14) RV/TLC ratio (%) 52.3 (8.7) PaO 2 (kPa) 10.7 (1.2) Peak power on cycle (watts) 71.9 (30.8) Peak VO 2 (ml/min/kg) 18.0 (5.9) Data shown are mean (SD). Abbreviations: BP–blood pressure; PYH–pack year history; MRC–Medical Research Council; CAT–COPD assessment test; SQRC-C–St. George’s respiratory questionnaire for COPD; BMI–body mass index; FFMI–fat free mass index; QMVC–quadriceps maximal volitional contraction; PAL- physical activity level; FEV 1 –forced expiratory volume in 1 second; TLCO c −carbon monoxide diffusing capacity; RV–residual volume; TLC–total lung capacity; VO 2 –pulmonary oxygen uptake. BODY.RESULTS.VENOUS NITRATE AND NITRITE MEASURES: Venous plasma nitrate measurements at all timepoints are shown in Fig 2. Following dosing with nitrate-rich beetroot juice plasma nitrate levels increased substantially (37.0 ± 16.4μM baseline vs. 820.2 ± 187.7μM post dosing; p<0.0001) and remained significantly elevated at peak exercise (917.1 ± 291.6μM; p<0.0001). There were no significant changes in plasma nitrate concentrations following dosing with the placebo preparation (45.7 ± 15.8μM baseline vs. 45.3 ± 16.5μM post dosing; p>0.99). Plasma nitrite levels were below the quantifiable limit (0.2μM) in all but three patients at baseline and remained so in the placebo condition but increased following nitrate supplementation to 1.57 ± 0.98μM and remained elevated at peak exercise (1.37 ± 0.65μM). 10.1371/journal.pone.0144504.g002Fig 2Alterations in plasma nitrate following dosing.Plasma nitrate concentrations prior to ('baseline') and following consumption of a nitrate-rich or placebo beverage. 'Post dosing' indicates a time point 3 hours following consumption which was immediately prior to cardiopulmonary exercise testing. 'Peak' was a time point at the point of exhaustion during endurance cycle ergometry testing. Repeated measures analysis of variance (ANOVA) was used to compare plasma nitrate metabolites between treatment conditions and at each specific time-point; *significantly different from baseline, p<0.0001; †significantly different from placebo group, p<0.0001. BODY.RESULTS.ENDURANCE EXERCISE TIME: The median endurance time during cycle ergometry performed at 70% maximum workload was not significantly different between the treatment groups (5.65 (3.90–10.40) nitrate vs. 6.40 (4.01–9.67) minutes placebo; p = 0.50). The individual responses are depicted in Fig 3. We also tested for an order effect, but none was present (the change in endurance time if nitrate supplementation was applied first was -2.03±4.64 minutes, compared to 1.92±4.16 minutes if placebo was administered first; p = 0.13). 10.1371/journal.pone.0144504.g003Fig 3Endurance time during cycle ergometry at 70% maximal workload measured in the placebo and nitrate-rich beetroot juice dosing conditions.A Wilcoxon test was used to compare median values in the two treatment groups; no significant difference was found. BODY.RESULTS.BLOOD PRESSURE RESPONSES: The changes in resting blood pressure parameters following ingestion of nitrate-rich beetroot juice and placebo are shown in Fig 4. The reduction in systolic blood pressure after dosing between treatment conditions was not significantly different, however, the reduction in diastolic blood pressure was greater in the nitrate supplemented group (7±8 nitrate vs. 1±8mmHg placebo; p = 0.008). There was a trend towards greater lowering of the mean arterial pressure following nitrate supplementation, although this was not statistically significant (7±8 nitrate vs. 3±8mmHg placebo; p = 0.07). 10.1371/journal.pone.0144504.g004Fig 4Blood pressure parameters following dosing.Alterations in blood pressure parameters (systolic blood pressure sBP, diastolic blood pressure dBP and mean arterial pressure MAP) relative to presupplemented baseline 3 hours following dosing with nitrate-rich beetroot juice or placebo preparation. Paired t-tests were used to compare blood pressure parameters between treatment groups; *significantly different from placebo, p<0.01. BODY.RESULTS.VO: At rest there was no significant difference in minute ventilation or VO2 following dosing with either nitrate-rich beetroot juice or placebo (Table 2). At isotime (i.e. the longest time of equivalent exercise completed under either study condition) both pulmonary VO2 and minute ventilation were lower in the nitrate supplemented condition than placebo (Table 2). There was a numerical improvement in the power/VO2 relationship (P/O ratio) in the nitrate supplemented condition, although this was not statistically significant. 10.1371/journal.pone.0144504.t002 Table 2 Rest and isotime analysis of the cardiopulmonary exercise test parameters. Parameter Timepoint Placebo Nitrate-rich beetroot juice Difference p value HR (bpm) Rest 85 (11) 87 (11) 2 (7) 0.063 HR (bpm) Isotime 122 (17) 121 (20) -1 (10) 0.30 BF (breaths/min) Rest 16 (4) 17 (4) 1 (3) 0.095 BF (breaths/min) Isotime 32 (8) 32 (5) 0 (7) 0.48 VT (L) Rest 0.93 (0.43) 0.87 (0.25) -0.06 (0.26) 0.33 VT (L) Isotime 1.44 (0.39) 1.40 (0.47) -0.04 (0.18) 0.12 VE (L/min) Rest 13.27 (3.90) 13.37 (3.95) 0.10 (2.29) 0.34 VE (L/min) Isotime 43.85 (13.94) 41.88 (14.85) -1.97 (4.52) 0.029 * VO 2 (ml/min/kg) Rest 4.5 (1.2) 4.4 (1.3) -0.1 (0.9) 0.32 VO2 (ml/min/kg) Isotime 17.2 (6.0) 16.6 (6.0) -0.6 (1.48) 0.043 * VCO 2 (ml/min/kg) Rest 4.2 (1.2) 4.2 (1.3) 0.0 (0.82) 0.49 VCO2 (ml/min/kg) Isotime 17.1 (6.0) 16.7 (6.3) -0.4 (1.74) 0.14 Power/VO 2 ratio (W/ml/min/kg) Isotime 2.92 (0.73) 3.03 (0.73) 0.11 (0.29) 0.053 SpO 2 (%) Rest 95 (2) 96 (2) 0 (2) 0.26 SpO 2 (%) Isotime 92 (4) 93 (4) 1 (2) 0.15 Oxygen pulse (ml/beat) Rest 3.8 (1.0) 3.6 (0.9) -0.2 (0.69) 0.12 Oxygen pulse (ml/beat) Isotime 10.0 (2.6) 9.7 (2.3) -0.3 (1.2) 0.09 Relative oxygen pulse (ml/beat/kg) Rest 0.053 (0.0138) 0.051 (0.014) -0.002 (0.010) 0.15 Relative oxygen pulse (ml/beat/kg) Isotime 0.143 (0.039) 0.138 (0.036) -0.005 (0.014) 0.043 * Data are shown as mean (SD) *p<0.05. Abbreviations: HR—heart rate; BF–breathing frequency; VT–tidal volume; VE–minute ventilation; VO 2 –pulmonary oxygen uptake; VCO 2 —exhaled carbon dioxide production; SpO 2 –peripheral arterial oxygen saturation. VO2 curves to isotime revealed a significant separation of the curves between the two treatment conditions (Fig 5). The area under the curve to VO2 isotime was also calculated for each subject to enable a comparison of the two treatment conditions (Fig 6). 10.1371/journal.pone.0144504.g005Fig 5Isotime VO2 analysis.The graph represents the VO2 at each 10th percentile of isotime with 0% representing the initiation of loaded cycling at 70% maximal workload achieved on incremental cycle ergometry. The area under the curves to isotime was compared using a paired t-test showing significant separation of the curves representing the two treatment conditions (p = 0.027). 10.1371/journal.pone.0144504.g006Fig 6Individual responses for area under the curve (AUC) to VO2 isotime.There was a significant difference between the two treatment conditions with a reduction in the area of the curve to VO2 isotime in the nitrate supplemented condition. BODY.RESULTS.PLASMA NMR SPECTROSCOPY ANALYSIS: Exercise related plasma metabolites showed no difference when comparing pre and peak exercise values under the two treatment conditions (Fig 7). 10.1371/journal.pone.0144504.g007Fig 7Alteration in plasma metabolomics during endurance cycle ergometry.Values are expressed as a ratio of peak exercise:pre exercise, with a value of 1 indicative of no change. Paired t-tests were utilised to compare metabolite levels between treatment conditions, with no significant difference seen for any metabolite analysed. BODY.DISCUSSION: Our trial, in patients with COPD, failed to demonstrate an improvement in endurance exercise performance following acute nitrate dosing in the form of beetroot juice. However, nitrate supplementation was biologically active, causing reductions in resting diastolic blood pressure and oxygen consumption at isotime during exercise, which may warrant further investigation. BODY.DISCUSSION.CRITIQUE OF THE METHOD: Adequate nitrate supplementation was confirmed by the observation of a 22-fold rise in plasma nitrate within 3 hours. Baseline plasma nitrate levels were similar to those of young recreationally active individuals[6, 34] and, whilst we were concerned that possible age related changes in digestion, enterosalivary circulation and oral commensal bacteria might impair nitrate delivery, the plasma nitrate levels achieved exceeded even those achieved by administration of a higher dose of nitrate in healthy young individuals[6]. Secondly, we observed a statistically significant reduction in diastolic blood pressure as previously noted in healthy individuals[35, 36], seniors with peripheral vascular disease[37], and COPD patients[16, 17]. One previous study has failed to show a reduction in blood pressure parameters following acute nitrate dosing in COPD[18]. However that study, which also failed to show an effect on exercise performance, used a lower treatment dose of nitrate and achieved plasma nitrate levels that were 4-fold lower than our dosing regimen (215±84μM vs. 820±288μM). Previous work has established that both reduction in blood pressure and post-dose plasma nitrate levels are dose-dependent[6]. Thus an important conclusion of the current study is that a single higher dose of nitrate is both safe and biologically active in COPD. The plasma nitrite levels fell below the limit of quantifiability in the majority of baseline samples. Part of the explanation for this, besides a small interfering peak apparently originating from the beetroot juice, is that samples were not pre-treated with a thiol-blocking agent prior to centrifugation; such treatment ensures there is no flux of nitrite across blood cell membranes, which may lower plasma concentrations. However, high levels of plasma nitrite were readily measurable following nitrate dosing. We failed to observe an improvement in exercise performance following nitrate dosing, despite a reduction in the oxygen cost of exercise. There are several possible explanations for this. Firstly, this could be due to statistical power and variation in endurance time. The variability in endurance time at 70% maximal workload on endurance cycle ergometry was beyond what we have observed in previous studies in a similar population[38]. In part this may be explained by day-to-day symptom variability in COPD[39], and might thus be an expected feature of studies in this condition using this outcome measure as has been reported by other groups studying nitrate supplementation in COPD[16, 17, 19]. A trial run might have reduced this variability, although it is important to note no order effect was identified. Studies using cycle ergometry methodology and agents of proven efficacy (for example bronchodilators) have tended to have larger sample sizes than ours[38, 40, 41]. Certainly, significant caution is needed when interpreting the results of non-blinded studies in this field, and emphasises the importance of using nitrate-depleted beetroot juice (which also causes beeturia) as a placebo[16, 17]. Secondly, it may simply be the case that this small improvement in the oxygen cost of exercise does not impact on the rate limiting parameters in exercise in ventilatory limited patients. Thirdly, we may have studied the wrong patient group. By design we did not include patients on oxygen supplementation but it may be the case that it is this specific patient phenotype where a reduction in the oxygen cost of exercise produces the greatest benefit. BODY.DISCUSSION.SIGNIFICANCE OF THE FINDINGS: While it is possible that a larger sample size might have given a positive result this seems relatively unlikely given exercise time was also numerically shorter in the treatment group. However, the observation (confirmed by the fall in blood pressure) that nitrate supplementation is biologically active and associated with a measurable reduction in oxygen consumption is of interest. Cardiovascular comorbidities are common in patients with COPD, so an intervention that lowers blood pressure may produce clinical benefit outside of a direct effect on exercise performance. Some prior data are available regarding nitrate supplementation with beetroot juice in COPD. An improvement in endurance cycle ergometry exercise time has been previously noted in a randomised cross-over study[16]. However, we believe that caution is needed in interpreting this result, partly because a matching placebo beverage (i.e. one which caused beeturia) was not used. This study as noted by the authors, also noted inter-individual variability in endurance time despite the use of a familiarisation constant work rate exercise test, and appropriate adjustment if the endurance time was outside of the 4–10 minute range. In fact, some individual responses were of a greater magnitude than could be explained by a plausible biological mechanism, thus skewing the overall data for endurance time. Despite this, in line with our own study, a similar reduction in pulmonary oxygen uptake at isotime was noted (of the order of 0.7ml/min/kg), although this failed to reach statistical significance (p = 0.099) most likely due to under-powering[16]. This lends support to our own findings regarding the influence of nitrate supplementation on the oxygen cost of exercise. Two further studies in COPD (both reported after we started our study) used a robust placebo but either failed to show a positive effect on oxygen consumption[18] or did not measure this variable[19], and both showed no improvement in exercise performance[18, 19]. Effects on oxygen consumption have previously been shown to be dose-dependent[6], and thus these negative findings may be due to the use of an insufficient dose of nitrate. Consideration should also be given to the fact that there are recognised 'responders' and 'non-responders' to nitrate supplementation when studying athletic populations[42, 43]. Whilst the lack of response in elite athletes may in part be explained by intrinsic high levels of nitrate metabolites through both exercise induced upregulation of NOS activity[44] and the selection of a diet rich in nitrate, the fact that within that group there is variability in response indicates that other unrecognised factors are likely at play[42, 43]. Our own study is too small to enable effective subanalysis but it should remain a focus of future studies to consider factors that may affect responsivity to nitrate loading, allowing those subjects most likely to benefit from nitrate supplementation to be both recognised and targeted. The reduction of VO2 at absolute isotime in our study is of a similar magnitude (3–4%) to that recorded in healthy individuals[6]. The cause of this observation is unclear, but of interest. Several potential modes of action of nitrate have been proposed including alterations to skeletal muscle blood flow[45], improved efficiency of the mitochondrial oxidative phosphorylation[46], effects on energy requiring processes in skeletal muscle[47, 48], alterations to mitochondrial biogenesis[49], blood viscosity[50] and NOS substrate availability[51]. No significant changes were noted in any of the metabolites identified in the NMR spectroscopy array, and although lactate rose appropriately following the exercise challenge this was of equal magnitude in both conditions, suggesting, as in health, that nitrate does not promote anaerobic metabolism[9]. Use of phosphorus-31 NMR spectroscopy have suggested that nitrate supplementation acts, at least in part, via reduced ATP cost of muscle force production[9]. Separately we have recently demonstrated a trend towards longer ADP recovery times in COPD patients preselected for quadriceps dysfunction[3]. The generation of ATP is dependent on the generation and maintenance of the electrochemical proton gradient. However, not all of this membrane potential is eventually coupled to ATP production due to proton leakage across the inner mitochondrial membrane, through uncoupling proteins and the adenine nucleotide translocase. Larsen et al.[46] demonstrated ex vivo that following 3 days of nitrate supplementation harvested human skeletal muscle mitochondria increased oxidative phosphorylation efficiency, correlating with a 3% reduction in whole-body oxygen consumption during submaximal workload exercise[46]. Future studies are clearly required to inform the mechanistic action of nitrate to allow the exploration of pharmacological therapies that may exploit the relevant pathways. Although our data do not support a role for general use of nitrate supplementation using beetroot juice in this setting we believe, because of the observation of reduced oxygen consumption, that targeting specific phenotypes in COPD may lead to the identification of therapeutic benefits. Thus, for example, we are now conducting a trial to investigate nitrate supplementation as an adjunct to pulmonary rehabilitation (www.controlled-trials.com/ISRCTN27860457), powered to detect an improvement in incremental shuttle walking distance. Other possible feasibility studies could be those in patients stratified for lactataemia (indicating anaerobic metabolism) during exercise, or those with either systemic or marked local hypoxia (for example detected using near-infrared spectroscopy[2]). In conclusion, acute administration of 0.8g nitrate in the form of beetroot juice failed to improve endurance exercise cycle capacity compared with placebo, but the reduction in oxygen consumption during exercise in COPD subjects and the reduction observed in resting diastolic blood pressure confirm a biological action at this dose. Several studies have now confirmed the blood-pressuring lowering properties of nitrate administered as beetroot juice in COPD, and as comorbidities are common in this population this may be an important effect. Since the intervention is safe and inexpensive further studies are warranted to establish whether it has a therapeutic role in specific COPD phenotypes. BODY.SUPPORTING INFORMATION: S1 CONSORT Checklist(DOC)Click here for additional data file. S1 Trial Dataset(XLS)Click here for additional data file. S1 Trial Protocol(DOCX)Click here for additional data file.

TITLE: A natural mineral supplement provides relief from knee osteoarthritis symptoms: a randomized controlled pilot trial ABSTRACT.BACKGROUND: This small, pilot study evaluated the impact of treatment with a natural multi-mineral supplement from seaweed (Aquamin) on walking distance, pain and joint mobility in subjects with moderate to severe osteoarthritis of the knee. ABSTRACT.METHODS: Subjects (n = 70) with moderate to severe osteoarthritis of the knee were randomized to four double-blinded treatments for 12 weeks: (a) Glucosamine sulfate (1500 mg/d); (b) Aquamin (2400 mg/d); (c) Combined treatment composed of Glucosamine sulfate (1500 mg/d) plus Aquamin (2400 mg/d) and (d) Placebo. Primary outcome measures were WOMAC scores and 6 Minute Walking Distances (6 MWD). Laboratory based blood tests were used as safety measures. ABSTRACT.RESULTS: Fifty subjects completed the study and analysis of the data showed significant differences between the groups for changes in WOMAC pain scores over time (p = 0.009 ANCOVA); however, these data must be reviewed with caution since significant differences were found between the groups at baseline for WOMAC pain and stiffness scores (p = 0.0039 and p = 0.013, respectively, ANOVA). Only the Aquamin and Glucosamine groups demonstrated significant improvements in symptoms over the course of the study. The combination group (like the placebo group) did not show any significant improvements in OA symptoms in this trial. Within group analysis demonstrated significant improvements over time on treatment for the WOMAC pain, activity, composite and stiffness (Aquamin only) scores as well as the 6 minute walking distances for subjects in the Aquamin and Glucosamine treatment groups. The Aquamin and Glucosamine groups walked 101 feet (+7%) and 56 feet (+3.5%) extra respectively. All treatments were well tolerated and the adverse events profiles were not significantly different between the groups. ABSTRACT.CONCLUSION: This small preliminary study suggested that a multi mineral supplement (Aquamin) may reduce the pain and stiffness of osteoarthritis of the knee over 12 weeks of treatment and warrants further study. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov number: NCT00452101. BODY.INTRODUCTION: Osteoarthritis (OA), also called degenerative joint disease, is a slow destructive process of the joint affecting millions of people worldwide. Although the exact biochemical cause of OA remains unknown, the process usually begins when the joint structures are abnormal or the stress placed on the joint surfaces is unusually high. The secondary inflammation due to progressive articular destruction appears to be localized to the particular joint being affected. Current anti-inflammatory treatments for OA while providing some relief from symptoms are suboptimal and the side effects associated with these treatments; in particular the COX-2 specific NSAID's are becoming increasingly recognized [1,2]. As a result of this, use of alternative treatments and complementary medicines are gaining popularity in the United States among OA sufferers. Glucosamine, a structural component of cartilage, is recognized as a nutritional supplement by the US FDA but as a pharmaceutical product in most European and Scandanavian countries as well as some Asian and Latin American Countries. Glucosamine has been the subject of many trials [3,4] and is used worldwide as an "alternative" treatment for OA although the extent to which it may provide relief to the symptoms of OA is still unclear. The recent NIH funded Glucosamine/Chondroitin Arthritis Intervention Trial (GAIT) tested the efficacy of glucosamine in providing relief for subjects with symptomatic knee osteoarthritis [5]. In this multi-centre study, glucosamine hydrochloride was tested either alone or in combination with chondroitin sulfate, a gycosaminoglycan that is also a structural component of cartilage and a popular alternative therapy for OA. In this study, the overall group of subjects failed to demonstrate an improvement in symptoms for both the individual and combined treatments possibly as a result of the large (60%) placebo effect observed. Some benefit was observed in a subset of subjects with moderate to severe knee osteoarthritis suggesting that the benefits of these nutraceuticals may be limited to this group. In addition to glucosamine and chondroitin, other nutraceutical products have been reported to provide relief from OA [6-9]. Cat's claw extract has recently been combined with a mineral based treatment (Sierrasil®) to provide symptomatic relief for a group of mild to moderate OA sufferers. While initially demonstrating some benefit with the cats claw/mineral supplement, Miller and co-workers observed that this benefit was at best temporary for a 1–2 week period [10]. Even though the positive effects were short lived in this subset of OA subjects, growing evidence suggests that minerals may play a role in joint health. Naturally occurring minerals such as magnesium, copper, manganese, selenium and zinc have shown anti-inflammatory effects in both animal and human studies. In a rat model of osteoarthritis, a deficiency of dietary magnesium was demonstrated to enhance the amount of cartilage damage [11]. Furthermore, increased magnesium in the diet may influence inflammation through reducing the serum level of the pro-inflammatory protein C-reactive protein [12]. The trace element copper is an essential cofactor in enzymes such as the collagen cross-linker lysyl oxidase and the anti-oxidant enzyme super oxide dismutase (SOD) that also requires zinc and manganese as cofactors. Recent evidence has suggested a role for oxidative stress in the pathogenesis of OA whereby an excess of reactive oxygen species arising from an imbalance in the antioxidant status of the joint (such as reduced levels of SOD) may result in cartilage degradation and joint remodeling [13]. Selenium is also an essential co-factor for glutathione peroxidase may have a role in reducing the incidence of osteoarthritic lesion [14,15] Positive roles have also been suggested for trace minerals such as boron and manganese in reducing the symptoms and slowing the pathogenesis of OA [16]. The present study was designed to evaluate the potential for a seaweed-derived multi-mineral supplement to alleviate OA symptoms. The mineral supplement (Aquamin) is derived from the red algae Lithothamnion corallioides which is rich in calcium and magnesium and has a variety of trace minerals (Table 1). The goal of this pilot trial was to gain preliminary data regarding the impact of Aquamin, Glucosamine Sulfate, the combination of Aquamin and Glucosamine Sulfate, or Placebo on symptoms and functional abilities of subjects with OA during 12 weeks of treatment. Table 1 Typical Mineral Composition of Aquamin Mineral Dry Salt Weight Calcium Carbonate 85% (34% calcium) Magnesium Carbonate 8.5% (2.4% magnesium) Salt (as chloride) 1.5% Moisture 3.0% Trace Minerals** 2.0% Sulphur 0.7% Potassium 0.6% Phosphorus 0.05% Sodium 0.25% Manganese 100 ppm. Zinc 20 ppm Iron 800 ppm Iodine 30 ppm Boron 17 ppm. Copper 8 ppm. Cobalt 0.1 ppm. Selenium 1.0 ppm. **Aquamin contains a wide spectrum of trace minerals assimilated from sea water of which the minerals outlined in the remainder of the table are a selection. Aquamin is a natural ingredient and trace mineral levels may vary over time BODY.MATERIALS AND METHODS.STUDY DESIGN: This study was a randomized, double blind, placebo controlled clinical trial with four parallel treatment groups: Aquamin, Glucosamine Sulfate, Aquamin plus Glucosamine Sulfate and Placebo. This trial was performed in compliance with all applicable regulations and guidelines (e.g. International Conference on Harmonization Good Clinical Practices, ICH-GCP, the Declaration of Helsinki, 21CFR50-Protection of Human Subjects, and 21CRF56-Institutional Review Boards) and was approved and continuously reviewed by the Quorum Institutional Review Board (Seattle, WA). BODY.MATERIALS AND METHODS.SAMPLE SIZE: Since pretrial information about Aquamin was entirely anecdotal, traditional effect sizes and sample size estimates were not possible. We used our experience from a previous study with similar endpoints to estimate the number of subjects that might be needed for this pilot trial. In our earlier trial, we found that 6 weeks of treatment with glucosamine sulfate and/or hyper-immune milk had a significant impact on WOMAC pain, stiffness and activity scores among 35 OA subjects with 10–13 subjects in each of the 3 treatment arms [6]. Based on this information, we enrolled 15 subjects in each of the four treatment arms (Aquamin, Glucosamine Sulfate, Aquamin plus Glucosamine Sulfate and Placebo) for a total of 70 subjects enrolled in this small pilot trial. BODY.MATERIALS AND METHODS.SUBJECTS: This was a single centre study conducted at the Minnesota Applied Research Centre and subjects were recruited by advertising in the Minneapolis, Minnesota area. Subjects of either gender were included if they voluntarily gave informed consent, were ambulatory, 25–75 years old, with normal digestion and absorption, diagnosed with moderate to severe OA of the knee according to their previous medical history and the modified clinical criteria of the American College of Rheumatology [17] and had a Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index [18] score ≤ 75 in the target knee. The target knee was chosen by physical examination to identify the most severely effected knee for each subject and the cut off point for the WOMAC score was enforced as a means of standardizing the extent of pain and immobility in the small number of subjects recruited for this trial. In order to establish a standardized calcium intake across all treatments, subjects were asked to consume a diet with ~600 mg calcium (e.g. two dairy servings) which was estimated to be 40–60% of their RDI (depending on age) per day. Exclusion criteria were rheumatoid arthritis, gout, pseudogout, Paget's disease, seizure disorder, insulin dependent diabetes mellitus, uncontrolled hypertension, unstable cardiovascular disease, active hepatic or renal disease, active cancer and/or HIV infection or if they required prescription drugs to control pain; had other clinical trial or experimental treatments in the past 3 months; were pregnant, lactating, or at risk of becoming pregnant; or if they received NSAIDS within 48 hours; intramuscular/systemic corticosteroid injection within 4 weeks; intra-articular corticosteroid injection within 2 months; or inter-articular hyaluronic acid injection within 4 months prior to enrollment. Each subject received one bottle of 350 two-piece hard shell test article capsules each month. Each bottle (and the capsules inside) appeared identical. Subjects were randomized in blocks of 4 using sequential treatment assignments prepared by the independent consulting statistician. The clinical investigator, statistician, clinic staff and subjects remained blinded throughout the trial to avoid bias. The sequence of the study began with a two week period when subjects were asked to discontinue any prescription or over-the-counter or alternative therapy treatments for osteoarthritis. At the baseline visit, vital signs were assessed and laboratory tests were performed. Subjects were assessed for WOMAC parameters and a 6 minute walking test was performed. After each month of treatment (at 4, 8 and 12 weeks) the subject's diaries, WOMAC questionnaires, and unused pills were collected, medications/supplements were reviewed, adverse events investigated, vital signs measured, blood was drawn and 6 MWD and WOMAC were measured. Active treatment was completed at week 12 when laboratory tests were repeated. Each subject returned to the clinic at 16, 20 and 24 weeks after their treatment began for monitoring of blood chemistry only. Although there was no reason to expect any beneficial carry over effect on the OA symptoms after removal of the treatments, subjects returned to the clinic every 4 weeks for 12 weeks after termination for blood chemistry readings in order to ensure that there were no adverse consequences on their blood metabolites including their blood calcium levels. BODY.MATERIALS AND METHODS.TREATMENTS: The duration of treatment was 12 weeks, administered as three capsules taken with a glass of water, three times per day. The capsules contained Aquamin (267 mg Aquamin F + 167 mg maltodextrin – FCC, USP & NF specifications for 10 Dextrose Equivalent Maltodextrin which was assumed inert in the capsules) designated A in the results section; Glucosamine sulfate (167 mg D-glucosamine sulfate Potassium salt, Pharmachem Labs NJ, USA + 267 mg maltodextrin) designated GS, Aquamin and Glucosamine (267 mg Aquamin F + 167 mg glucosamine sulfate) designated G+A, or Placebo (434 mg maltodextran) designated PBO in the results section. The rescue medication was acetaminophen, 325 mg, 1–2 tablets every 4–6 hours as needed for intractable pain. BODY.MATERIALS AND METHODS.STUDY MEASUREMENTS AND STATISTICAL ANALYSIS: Joint symptoms were assessed using the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index, a validated questionnaire including scores for pain, stiffness and activities as well as a composite (total) score. The WOMAC scores were transformed according to the standard orthopedic formula: Transformed Score = 100 - (Actual Raw Score × 100/Possible Raw Score) [18] The values represent "percentage of normal," such that increasing scores reflect improvement and decreasing scores reflect worsening of symptoms. The six minute walking distance (6 MWD) was conducted by marking off a 100-foot distance in an interior hallway and asking subjects to walk as far as they can as quickly as they can over 6 minutes. The total distance was measured and recorded. Adverse effects were assessed by a questionnaire and vital signs/laboratory measurements respectively. This study was conducted, monitored and audited in compliance with ICH-GCP guidelines and according to the Minnesota Applied Research Center Standard Operating Procedures (SOP's) and the SOP's of certified vendors (e.g. for WOMAC scoring). Subject compliance was assessed at each visit by pill count, interview, and review of the medication diary. Subject data was kept confidential and study records were stored in a locked and secure storage area. An independent statistician used ANOVA for between group comparisons at baseline. ANCOVA (with baseline score as co-variate) were used to assess between-group differences in change over time. Matched pair T-tests were used for within group comparisons of change over time. Data was analyzed under Intent To Treat Last Observation Carried Forward (ITT-LOCF) case condition and statistical significance was set at p < 0.05. BODY.RESULTS.BASELINE CHARACTERISTICS: Table 2 shows that all four groups were comparable (on average) for number of subjects (N = 15–20), gender (6–11 male; 7–11 female), BMI (30.5 – 32.5), age (58.5 to 60.3 years), WOMAC activity (49.4–63.0) and composite (48.8–63.4) scores and 6 MWD (1323–1427 feet) indicating that the randomization was effective for those parameters. Significant baseline differences were observed between the four groups for WOMAC pain (50.0–67.2) and stiffness scores (40.4–57.8) (p = 0.039 and 0.013 respectively). (Table 3) The finding of baseline differences for the pain and stiffness sub-scores limits the analysis of these data. Table 2 Baseline characteristics (ITT analysis). Characteristic N started (completed) Gender M/F Mean age (S.D.) BMI (calc) PBO 16(9) 6/10 58.9(7.4) 32.4 A 20(15) 11/9 58.5(12.1) 32.5 GS 19(14) 8/11 59.2(8.3) 32.1 G+A 15(12) 8/7 60.3 (9.8) 30.5 p (ANOVA*) NS NS NS NS * = ANCOVA comparison between groups Table 3 Changes in WOMAC Scores at baseline and at the end of the trial Between and Within Groups (ITT-LOCF) Variable Pain Baseline Pain End Pain baseline – pain end Stiffness Baseline Stiffness End Stiffness baseline – stiffness end Activity Baseline Activity End Activity baseline – activity end Composite Baseline Composite End Composite baseline – composite end PBO 50.0 52.9 -2.9 40.4 46.3 -5.9 49.4 56.4 -7.0 48.8 54.8 -6.1 SD 22.9 21.4 19.9 17.4 25.3 18.3 23.1 24 18.4 21.8 22.7 17.6 Sig.* NS NS NS NS A 56.8 74.3 -17.5 44.4 65 -20.6 58.8 72.4 -13.5 57.3 72.2 -14.9 SD 15.8 17.6 22.7 17 17.5 26.1 16.2 16.8 21.3 15.3 16.6 21.4 Sig.* .0.003 0.002 0.01 0.006 GS 60.6 72.9 -12.6 51.3 61.8 -10.5 60.1 70.7 -10.6 59.4 70.2 -10.9 SD 14.8 17.6 16.3 13.8 19.8 24.0 13.9 18.4 15.4 13.2 17.6 15.6 Sig.* 0.003 NS 0.008 0.007 G+A 67.2 69.1 -1.9 57.8 64.1 -6.3 63 68.6 -5.6 63.4 68.3 -4.9 SD 13.3 16.9 14.0 15.7 17.6 17.7 8.7 13.1 11.0 8.1 12.9 101 Sig.* NS NS NS NS p(ANCOVA) 0.039# 0.009 0.013# NS NS# NS NS# NS *Sig. = significance by within group paired t-test; 2-tailed; ANCOVA comparison between groups with baseline measure as covariate. # = ANCOVA comparison between groups showing any differences at baseline for these parameters. BODY.RESULTS.SUBJECT ATTRITION AFTER RELEASE OF THE TEST ARTICLE: Twenty (20) of the 70 subjects given test article withdrew from the trial prior to completion (29% attrition) and a total of 50 subjects completed the trial (Figure 1). The reasons for subject attrition were spread evenly across the test article administration groups and no pattern was obvious that might suggest withdrawal due to a problem specific to any of the test articles in this trial. Figure 1Trial flow chart. BODY.RESULTS.WOMAC: Using an ITT-LOCF analysis, only the improvements in WOMAC pain score differed significantly between the groups over the course of the study (p = 0.009 ANCOVA). All four groups displayed numerical improvements from baseline to end of treatment for WOMAC scores (Table 3 and Figure 2); however, no significant improvements were demonstrated within groups over time for the placebo or for the combined treatment groups. Within group analysis over time showed that the pain score was significantly improved by 17.5 (P = 0.003 ANOVA) for A and 12.6 (P = 0.003 ANOVA) for GS compared to non significant changes of 2.9 for PBO and 1.9 for G+A (higher score indicates less pain). Figure 2Percent change in WOMAC scores from baseline over 12 weeks of treatment. Within group analysis over time showed that the activity score was significantly improved by 13.6 (P = 0.010 ANOVA) for A and 10.6 (P = 0.008 ANOVA) for GS compared to non significant changes of 7.0 for PBO and 5.6 for G+A. Within group analysis over time showed that the composite (total) WOMAC score was significantly improved by 14.9 (P = 0.006 ANOVA) for A and 10.8 (P = 0.007 ANOVA) for GS compared to non significant changes of 6.1 for PBO and 4.9 for G+A. Of interest, the Aquamin group also displayed a significant improvement over time for stiffness score (20.6, p = 0.002) compared to non significant changes of 10.5 for GS, 5.9 for PBO and 6.3 for G+A. BODY.RESULTS.SUBJECT CONSUMPTION OF RESCUE MEDICATION: No significant differences were found between the groups in the amount of rescue medication consumed over the course of the experiment (Table 4). Table 4 Consumption of rescue medication. Group Month 1 Month 2 Month 3 Total Placebo 39(38)* 27(27) 30(33) 89(88) A 45(62) 19(22) 23(31) 87(96) GS 25(20) 29(38) 31(50) 84(103) G+A 26(55) 26(34) 27(46) 63(112) *Mean (SD). Group comparisons of rescue medication were non-significant (Kruskal-Wallis one-way non-parametric analysis of variance) at all time periods and for total medications BODY.RESULTS.SIX MINUTE WALKING DISTANCE (6 MWD): The distance covered during a 6 minute timed walk was significantly improved over time on treatment within the Aquamin group (+101 feet, p = 0.001, Table 5). The glucosamine group also demonstrated a significant improvement in 6 MWD over time on treatment (+56 feet, p = 0.030). No significant improvements were demonstrated within groups over time for the placebo group and surprisingly for the combined treatment group. Table 5 Changes in 6 MWD Between and Within Groups Over 12 weeks of treatment. Variable Baseline 6 MWD End 6 MWD Baseline-End 6 MWD PBO 1323.4 1331.9 -8.4 SD 226.1 250.2 109.3 Sig.* NS A 1427.5 1528.8 -101.3 SD 225.6 252.3 121.5 Sig.* 0.001 GS 1410.1 1456.8 -55.7 SD 246.1 256.1 103 Sig.* 0.03 G+A 1363.1 1378.1 -15 SD 253.7 253 126.6 Sig.* NS p (ANCOVA) NS *Sig. = significance by within group paired t-test; 2-tailed; ANCOVA comparison between groups with baseline measure as covariate BODY.RESULTS.ADVERSE EFFECTS: All treatments were well tolerated. Table 6 shows that a total of 51 of the 70 subjects given test product (TA) reported eighty-eight (88) adverse effects (AE) but only 7 of the 88 AE (8%) were considered at least possibly related to the TA treatment. These AE were distributed somewhat evenly across the groups: 1 on PBO, 3 on A, 2 on GS and 1 on G+A and none were considered definitely related to the TA (Table 6). Most of the adverse effects (31/88; 35%) were related to musculoskeletal complaints and these were mainly reports of increased knee pain (n = 19/88; 22%). All AE completely resolved or returned to baseline. Table 6 Adverse effects Item Total GS A PBO G+A Number of Subjects with AE 51 12 12 14 13 HEENT 25 6 8 6 5 Respiratory/Pneumonia 1 0 0 0 1 Cardiovasular/Hypertension 1 0 0 0 1 Gastrointestinal 17 5 3 6 3 GU/Reproduction 3 1 1 0 1 Neurological 5 1 3 1 0 Dermatological (hives, cat bite) 2 1 0 0 1 Musculoskeletal 31 4 9 10 8 Increased Knee Pain 19 4 5 4 6 Other 6 1 1 0 4 TOTAL 88 18 25 23 22 BODY.DISCUSSION: This trial was designed as a preliminary pilot trial to investigate the potential of a marine derived multi-mineral supplement to reduce symptoms of moderate to severe knee osteoarthritis. The dose of the mineral supplement Aquamin was determined based on previous anecdotal experience and a rigorous Intent to Treat – Last Observation Carried Forward statistical analysis was used to compare the four treatment groups: Aquamin, Glucosamine Sulfate, Combination of Aquamin and Glucosamine Sulfate, or placebo. These results were confounded because the WOMAC pain scores were significantly different between the groups at baseline and, therefore, these improvements need to be viewed with caution because further study is warranted. In general, significant differences were found between groups for pain scores after 12 weeks of treatment. Within groups over time, the Aquamin treatment group showed significantly improved WOMAC pain, stiffness, activity and composite scores over the course of the 12-week treatment. The glucosamine sulfate treatment group also showed significant improvements over time on treatment for the pain, activities and composite scores (but not for the stiffness scores); however, no significant improvements were found over time on treatment for subjects in the Placebo group or, surprisingly for subjects in the Combination treatment group. No baseline differences were observed among the four groups for the 6 minute walking distances. Over time on treatment, the Aquamin and glucosamine groups demonstrated significant improvements in 6 minute walking distances (101 feet, p = 0.001 and 56 feet, p = 0.030, respectively). This was an improvement of 7% and 3.5% respectively over their baseline walking distances. No significant differences were found for the walking distances measured for the placebo group and surprisingly for the combined treatment group. Although, these distances appear to be small, our subjects with severe OA indicated that the ability to walk even a little bit further was important to them. The main limitations of this study were its short duration (12 weeks), lack of assessment for remnant effects after treatment stopped and limited sample size (15 subjects per treatment arm). Glucosamine sulfate has been shown to provide a benefit over a longer course of treatment [4] and its efficacy may have been under demonstrated within this 12 week study period. Additional study of longer treatments in a greater numbers of subjects would be helpful to verify the treatment effect for Aquamin and to explore the lack of any treatment effect for the combination of Aquamin and Glucosamine Sulfate in this small pilot trial. Although these products are unlikely to have reacted in the tablet form it is interesting to speculate about a possible dietary interaction, possibly related to the very basic nature of Aquamin (pH 10) compared to the acidity of the Glucosamine Sulphate (pH of 3.5 to 5), and the requirement for this to ionize in the stomach to be effective. Aquamin is composed of multiple minerals and the 'active ingredient' for the complex is difficult to determine. A number of the minerals in Aquamin may have anti-inflammatory and anti-oxidant properties which might directly and/or indirectly influence the efficacy of this unique complex [13,14,16]. While the prominent mineral present in Aquamin is calcium (dosage = 80% Ca U.S RDA), its role in joint health is unclear. Magnesium however, was given at the daily dosage providing 14% (male) to 18% (female) U.S. RDA [12] and over the course of this study, this increased consumption of magnesium may have influenced OA symptoms by affecting the utilization of calcium or by potentially reducing inflammation around the affected joint. Both manganese and selenium were given at the daily dosage providing up to 16% and 4% of their RDA respectfully. Both of these trace minerals have been reported to reduce the appearance of osteoarthritic lesions and reduce the severity of symptoms in OA [14,16]. These pilot trial results suggest a potential treatment effect for Aquamin among subjects with moderate to severe OA and this preliminary finding warrants further study. BODY.COMPETING INTERESTS: Marigot Ltd. provided funding for this clinical trial and the article processing charges to publish this work. Melanie Walsh is a paid employee of Marigot Ltd., the sponsor of this work and provided only medical writing support for this manuscript. The other authors declare that they have no other competing interests. Marigot Ltd approved the protocol and reviewed the manuscript before submission for publication and can be reached at: Strand Farm, Currabinny, Carrigaline, Co. Cork, IRELAND; Phone: 353-21-437-8727; Fax: 353-21-437-8588. Marigot Ltd did not participate in any of the data collection or statistical analyses reported herein. BODY.AUTHORS' CONTRIBUTIONS: JLF and MW co-authored the manuscript. JLF co-authored the protocol and directed the research team at MARC during the conduct of this trial. JLZ provided critical review of the manuscript, co-authored the protocol and provided medical monitoring services during the trial. MAK provided critical review of the manuscript and the protocol and provided statistical services for the design, execution and analysis of the data in this trial. All authors have read and approved the final manuscript.

TITLE: Comparison of the Effects of Magnesium Sulfate and Remifentanil on Hemodynamic Responses During Tracheal Extubation After Laparotomy: A Randomized Double-blinded Trial ABSTRACT.BACKGROUND:: Because blood pressure and heart rate (HR) elevations during tracheal extubation are common, different medications have been studied to prevent such complications. ABSTRACT.OBJECTIVES:: To compare magnesium sulfate, remifentanil, and placebo regarding mean arterial pressure (MAP) and HR changes during/after tracheal extubation, in patients who underwent laparotomy. ABSTRACT.MATERIALS AND METHODS:: In this randomized double-blinded trial, 120 patients undergoing laparotomy were evenly divided into three groups, including remifentanil (1 mcg/kg), magnesium sulfate (50 mg/kg), or normal saline, as placebo. Hemodynamic responses (MAP and HR) were documented at different times (before operation, during medication administration, immediately before extubation, immediately after extubation, and also 3, 5, and 10 minutes after extubation). The double burst time (DBT) was determined using neuromuscular monitoring, as time interval, between administration of reverse medication and DBT of 100%. ABSTRACT.RESULTS:: The HR was significantly lower, immediately after extubation and 3, 5, and 10 minutes after extubation, in both magnesium and remifentanil groups, compared to normal saline (P < 0.001). The MAP was also lower in magnesium and remifentanil groups, immediately after extubation and 3 minutes after extubation, in comparison to the normal saline group (P < 0.001). Mean (± SD) DBT 100% was significantly higher in magnesium group (30.2 ± 15.3) vs. remifenatnil (13.6 ± 6.8) and normal saline (13.5 ± 8.2) groups (P < 0.001). ABSTRACT.CONCLUSIONS:: Both remifentanil and magnesium had favorable outcomes in preventing HR and MAP elevation after tracheal extubation. However, remifentanil was associated with more rapid regaining of consciousness and reversal of muscular relaxation. BODY.1. BACKGROUND: Tracheal extubation is an important part of general anesthesia, which can be associated with complications (1). Both cardiac and respiratory complications, associated with tracheal extubation, may lead to considerable morbidity and mortality (2). Cardiopulmonary adverse events resulted from tracheal extubation (12.6%) are three times more common when compared to those of endotracheal intubation and induction of general anesthesia (4.6%) (3). The major adverse events associated with tracheal extubation include mechanical complications (such as trauma to the larynx) (4), cardiovascular complications [such as 10 - 30% increase in blood pressure (BP) and heart rate (HR) increase for 5 - 10 minutes] (5), respiratory complications (atelectasis, coughing, hypoxia and etc.) (6). Documented beneficial effects of magnesium, as a selective blocker for calcium channels and N-methyl-d-aspartate (NMDA) receptor antagonist, have been the basis for multiple studies that have reported protective effects of this medication regarding hemodynamic responses, in patients undergoing general anesthesia (7, 8). Studies have noted that magnesium has an important role as vasodilator in arterioles, whereas this effect is minimal on venules, which result in improvement in cardiac output (9). This function is regulated mainly by blocking sympathetic system via inhibiting the release of catecholamines from the adrenal, as well as peripheral nerve endings (10). In addition, magnesium has beneficial effects on maintaining diastolic function and regulating sinus rhythm, during arrhythmias (11). Since magnesium is a vasodilator, it has been used in general anesthesia to decrease BP and prevent its elevation. Remifentanil, as an anilidopiperidine opioid, has similar pharmacodynamic characteristics to other opioids, however with distinct pharmacokinetic properties (12). This is an effective medication for analgesia, considering its analgesic properties. Since it has no inhibitory effect on the local nervous system, it can be used during extubation (13). In comparison to fentanyl and alfentanil, remifentanil administration is associated with better control of hemodynamic responses during surgery and lower rate of respiratory depression. However, hypotension and bradycardia have been reported, when remifentanil is administered during surgery, and its effects, after extubation, are not completely understood (14). As mentioned earlier, hemodynamic changes during extubation are common. Hence, medications have been used to prevent these unfavorable changes. Magnesium sulfate and remifentanil are two of the medications that may be used, although studies to determine their efficacy in this regard, as well as any other side effects resulted by using these agents are necessary. BODY.2. OBJECTIVES: Here, we intended to compare the effects of remifentanil and magnesium on BP, as well as heart rate changes during tracheal extubation, in patients undergoing laparotomy under general anesthesia. BODY.3. MATERIALS AND METHODS: This was a randomized double-blinded clinical trial. The study population consisted of patients who were candidates for laparotomy at our university hospital. Inclusion criteria consisted of age range between 18 and 55 years, ASA (American society of anesthesiologists) class I or II, laparotomy duration of less than 2 hours, no background medical conditions, including cardiovascular diseases, neuromuscular diseases, acute or chronic renal diseases, no dependence to narcotic drugs, and lack of history of taking anti-hypertensive or anti-arrhythmic medications. Exclusion criteria were prolongation of general anesthesia (more than 2 hours), a significant hemodynamic instability, which necessitates medical intervention, or development of arrhythmias, which required medical intervention. The study protocol was approved by the ethics committee of our university. The study steps were described for the patients and written informed consent was obtained from them, prior to participation. All data were kept confidential and the patients were not charged for the study purposes. The study protocol was in conformity with the ethical guidelines of the 1975 Declaration of Helsinki (15). A total of 120 patients were included. The patients were randomly divided (using random number table, computer-based) into three groups: remifentanil (40 cases), magnesium sulfate (40 cases), and normal saline (as placebo) (40 cases). Upon admission to the operation room, HR, systolic BP (SBP), diastolic BP (DBP) and mean arterial pressure (MAP) were documented and were used as baseline hemodynamic data of the subjects. Then, all patients underwent noninvasive intraoperative blood pressure (NIBP), bispectral index (BIS), ECG, and pulse oximetry monitoring. Then, standard general anesthesia, which was used for all patients, was initiated, as follow: first, fentanyl (2 mcg/kg) and midazolam (0.03 mg/kg) were injected intravenously. Then, for anesthesia induction, thiopental sodium (5 mg/kg) was injected and for facilitation of endotracheal intubation, atracurium (0.5 mg/kg) was injected. Three minutes after administering atracurium, tracheal intubation was done via direct laryngoscopy. In order to continue the general anesthesia, isoflurane gas at 0.5 - 1.5 MAC (minimum alveolar concentration) was used to keep MAP at about 10% of the baseline value. Capnography monitoring was attached for all patients and end-tidal CO2 was kept at 35 - 45 mmHg, with control of the respiratory rate. During the operation, atracurium (0.15 mg/kg) and fentanyl (0.7 mcg/kg) were injected every 30 minutes. The SBP, DBP and MAP were recorded by automatic oscillometric BP devices. The HR was recorded by ECG. The BIS range was kept at 40 - 50. The volume of intravenous (IV) fluids, necessary for patients, was calculated according to the weight, maintenance fluid, deficit fluid, and volume of bleeding and was replaced by lactated Ringer's solution or normal saline. The last stitch on the skin placed by the surgeon was considered as the last painful stimulus of the surgery (time 0). At this time, magnesium sulfate (50 mg/kg), or with the same volume remifentanil (1 mcg/kg), or the same volume of normal saline were administered for the three groups of the study. The injected medications had similar volumes and were prepared in similar syringes. The syringes were numbered and the technician who injected the medications was unaware of the medications. The patients remained on ventilator with 100% oxygen. When, based on muscular monitoring, train-of-four (TOF) became 2 of 4 (50%), atracurium effect was reversed, using neostigmine (0.04 mg/kg) and atropine (0.02 mg/kg). When the patient opened his/her eye spontaneously or showed purposeful movements, extubation was done. The variables recorded were gender, age, weight, operation duration, fentanyl dosage used, the time of the last dose of fentanyl, the time interval between medication administration and extubation and DBT, using neuromuscular monitoring, as the time interval between administration of reverse medication and DBT of 100%. The hemodynamic variables of interest, including SBP, DBP, and HR were recorded at time 0, immediately before tracheal extubation, immediately after extubation, and 3, 5, and 10 minutes after extubation. Coughing at 1 minute before and 1 minute after extubation was documented and its severity was categorized as mild (1 - 2 coughs), moderate (3 - 5 coughs), and severe (> 5 coughs). At 5 and 15 minutes after extubation, consciousness level was recorded based on the sedation score scale, using the following indices: Alert and responsiveDrowsy, although responsive to verbal commandUnresponsive to verbal command To report continuous data, mean ± standard deviation (SD) was used. Categorical data were reported by frequency and percentage. To compare categorical variables between the three groups, the Chi-squared test or the Fisher's exact test were applied. Continuous variables were compared between the groups via analysis of variance (ANOVA) or Kruskal-Wallis test, depending on the normal distribution of the data, determined by the Kolmogorov-Smirnov test. To investigate the changes of the studied variables, at different time points, repeated measure ANOVA was used. The significance level was set at 0.05. All analyses were performed with the SPSS software for Windows (ver. 20.0) (IBM, New York, NY, USA). BODY.4. RESULTS: Table 1 presents the baseline characteristics (age, gender, weight) of the patients. As shown, no significant differences were found, regarding baseline characteristics, between the three groups. Table 1. Comparison of Age, Gender, and Weight in Laparotomy Patients who Received Remifentanil, Magnesium Sulfate, or Normal Saline a Remifentanil Magnesium Sulfate Normal Saline P Value Gender, Male 52.5% 45% 47.5% 0.79 Age, y 43 (± 11.7) 42.7 (± 10.5) 38.7 (± 12.7) 0.18 Weight, kg 66.4 (± 10.8) 68.4 (± 8.9) 70.6 (± 10.2) 0.17 a For each group: (n = 40). Table 2 presents anesthesia-related variables. As shown, no significant differences were found regarding mean general anesthesia duration, total fentanyl dosage administered, time interval passed between fentanyl administration and studied medication administration, or time interval between studied medication administration and tracheal extubation, between the three groups. Table 2. Comparison of General Anesthesia-Related Variables in Laparotomy Patients who Received Remifentanil, Magnesium Sulfate, or Normal Saline a Remifentanil Magnesium Sulfate Normal Saline P Value Anesthesia Duration, min 108.4 (± 18.6) 108.9 (± 11.8) 106.7 (± 10.5) 0.77 Total Fentanyl Dosage, mcg 275 (± 45.3) 278 (± 51.7) 261.2 (± 57.1) 0.28 Time Interval Between Fentanyl and Studied Medication Administration, min 25.7 (± 16.9) 33.9 (± 15.4) 31 (± 19.70) 0.1 Time Interval Between Studied Medication Administration and Tracheal Extubation, min 16.4 (± 8) 14.45 (± 3.1) 14.9 (± 4.9) 0.26 a For each group: (n = 40). Regarding HR changes, recorded before starting the operation until 10 minutes after extubation, there was no significant difference between the three groups, until the time point recorded immediately before extubation. However, immediately after extubation and 3, 5, and 10 minutes after extubation, mean HR showed a significant decrease, in comparison to normal saline group (Table 3). However, analysis of the HR changes pattern, at different time points, showed that there was no significant difference between the groups, with respect to heart rate during follow-up period (P = 0.11). Table 3. Comparison of Mean (± SD) Heart Rate at Different Time Points in Patients who Underwent Laparotomy and Received Remifentanil, Magnesium Sulfate, or Normal Saline a Remifentanil Magnesium Sulfate Normal Saline P Value Before Operation 93.4 (± 17.6) 87.6 (± 15.6) 90.2 (± 14.3) 0.26 Time 0 b 79.2 (± 10.9) 81.2 (± 15.8) 78.7 (± 14.7) 0.7 Immediately Before Extubation 93.8 (± 17.3) 98.2 (± 14.9) 94.9 (± 14.9) 0.74 Immediately After Extubation 95.4 (± 10.6) 105.2 (± 16.4) 111.03 (± 14.9) < 0.001 Minute 3 Post-Extubation 85.2 (± 12.3) 85.9 (±17.8) 98.05 (± 13) < 0.001 Minute 5 Post-Extubation 85.06 (± 12.8) 85.2 (± 15.2) 92.8 (± 11.7) < 0.001 Minute 10 Post-Extubation 84.7 (± 12.8) 85.7 (± 13.9) 91.9 (± 12.1) < 0.001 a For each group: (n = 40). b Time 0 = after putting the last stitch on the skin. A similar pattern to HR changes was observed with regard to MAP. At follow-up time point before extubation, no significant difference was seen between the two groups. However, MAP was significantly lower in remifentanil and magnesium groups, compared to normal saline group, immediately after extubation, as well as at minute 3 after extubation. At minutes 5 and 10 post-extubation, again no difference was present between the groups. Two groups of remifentanil and magnesium were comparable regarding MAP changes (Table 4). Analysis of MAP changes pattern, at different time points, showed that there was no significant difference between the groups during follow-up period (P = 0.58). Table 4. Comparison of Mean (± SD) Mean Arterial Pressure (MAP) at Different Time Points in Patients Underwent Laparotomy Who Received Remifentanil, Magnesium Sulfate, or Normal Saline a Remifentanil Magnesium Sulfate Normal Saline P Value Before Operation 102.6 (± 16.7) 100.2 (± 17.08) 102.7 (± 10.6) 0.69 Time 0 b 94.3 (± 17.2) 98.8 (± 11.4) 92.7 (± 10.8) 0.11 Immediately Before Extubation 101.08 (± 14.6) 103.1 (± 19.9) 97.05 (± 13.7) 0.23 Immediately After Extubation 104.9 (± 18.6) 107.1 (± 9.9) 119.1 (± 11.02) < 0.001 Minute 3 Post-Extubation 105 (± 11.2) 101.4 (± 8.5) 111.08 (± 15.1) 0.002 Minute 5 Post-Extubation 102.2 (± 9.6) 100.1 (± 9.1) 100.7 (± 11.2) 0.63 Minute 10 Post-Extubation 99.8 (± 8.54) 98.9 (± 9.05) 98.9 (± 11.01) 0.88 a For each group: (n = 40). b Time 0 = after putting the last stitch on the skin. Remifentanil and magnesium, both resulted in less severe coughing episodes, compared to normal saline. The frequencies of mild and moderate coughing were 82.5% and 10% in remifentanil group, 80% and 20% in magnesium group, and 40% and 52.5% in normal saline group, respectively. Severe cough was not reported in magnesium group, although it was reported in 7.5% of patients of normal saline and remifentanil groups (P < 0.001). However, no difference was detected between magnesium and remifentanil groups. At 5 minutes after extubation, 60% of normal saline cases were alert and responsive; however, this percentage was 47.5% in remifentanil group and 25% in magnesium group (P = 0.004). Similar findings were noted at 15 minutes post-extubation time. Alertness and responsiveness was reported in 95% of normal saline patients, 72.5% of remifenatnil cases and in 60% of magnesium patients (P = 0.007). Mean ± SD DBT 100% was significantly higher in magnesium group (30.2 ± 15.3) vs. remifenatnil (13.6 ± 6.8) and normal saline (13.5 ± 8.2) groups (P < 0.001). BODY.5. DISCUSSION: This study aimed to compare magnesium and remifentanil regarding their effects on hemodynamic responses during tracheal extubation, in patients undergoing laparotomy. The obtained findings showed that mean HR recorded a significant decrease, in both magnesium and remifentanil group, immediately after tracheal extubation. This trend continued until 10 minutes post-extubation. Likewise, MAP was significantly lower in magnesium and remifentanil groups, compared to normal saline group, after extubation. This continued just up to 3 minutes post-extubation, and then the three groups were similar regarding MAP. When the two groups of magnesium and remifentanil are compared, we did not detect differences between them in terms of HR or MAP. Administering magnesium and remifentanil, both resulted in less severe coughing episodes, in comparison to normal saline, although neither magnesium nor remifentanil had superiority to each other in decreasing coughing severity. However, regarding consciousness level, the patients who received just normal saline had higher rate of alertness and responsiveness at 5 and 15 minutes post-extubation than patients of remifentanil or magnesium groups. The above core findings suggest that both magnesium and remifentanil have comparable effects on hemodynamic responses during and shortly after tracheal extubation. However, patients of remifentanil group had a better profile in regaining consciousness than magnesium group. In a similar clinical trial, the effects of remifentanil (1 mcg/kg) or magnesium sulfate (30 mg/kg) were compared versus placebo, regarding SBP, DBP, HR and oxygen saturation in electroconvulsive therapy (7). The authors studied 20 patients for whom anesthesia was induced by thiopental (4 mg/kg). They reported that remifentanil and magnesium administration were associated with a significant attenuation of the increase in SBP. However, only remifentanil attenuated HR, not magnesium. It was concluded that, since magnesium had no effect on HR, this effect may be considered as an advantage, compared to remifentanil, in patients who needed electroconvulsive therapy. These findings are similar to ours, in terms of BP changes, as we observed that both remifentanil and magnesium prevented increases in MAP. Plus, we observed a similar effect on HR, which was not reported by the mentioned article in magnesium group. The advantage of magnesium is attributed to the fact that it may result in bradycardia, and similarly, we did not see bradycardia in our patients who received magnesium or remifentanil. In a study to determine the hemodynamic responses after endotracheal intubation, magnesium sulfate and remifentanil were compared. Lim et al. (16) studied 80 patients who underwent general anesthesia and divided them to receive normal saline, magnesium sulfate (50 mg/kg), remifentanil (1 mcg/kg), or a combination of 25 mg/kg magnesium sulfate and 0.5 mcg/kg remifentanil, which were administered half a minute before induction of anesthesia, with propofol and succinylcholine. The results showed that SBP changes of patients, who received magnesium, remifentanil, or combination of these two agents, were lower, immediately after intubation than those of the normal saline group. The SBP in magnesium and magnesium/remifentanil groups showed an increase in relation to baseline recording, although SBP alone, in remifentanil group, decreased from that at the baseline, immediately after intubation. In contrast to our results, where we observed a decrease in HR in both remifentanil and magnesium groups, Lim et al. found an increase in HR of magnesium group, with a decrease of heart rate in remifentanil group. They concluded that magnesium was associated with increase in SBP and tachycardia after intubation, whereas remifentanil resulted in SBP drop and bradycardia (16). Kim et al. (17) studied remifentanil infusion effects on hemodynamic responses and coughing during extubation in postoperative (major abdominal surgery) intensive care unit patients, in a randomized clinical trial. They reported that MAP, HR and coughing severity did not differ between the two groups during extubation. The only significant difference was related to the time from discontinuation of propofol infusion to extubation, which was significantly longer in the remifentanil group, compared to control group. Wu et al. (18) investigated the efficacy of different dosages of remifentanil (0.05, 0.1, and 0.2 mcg/kg) for attenuating cardiovascular response to tracheal extubation, after general anesthesia, in 164 surgical (upper abdominal surgery) patients. They concluded that remifentanil at 0.1 mcg/kg was the ideal dose to prevent hemodynamic response to tracheal extubation. As the prevention of MAP and HR elevation, after tracheal extubation, is considered in anesthesiology, both magnesium and remifentanil had comparable effects in prevention of adverse hemodynamic responses. Regarding the faster regaining of consciousness and reversal of muscular relaxation, remifentanil was superior to magnesium. Future studies can focus on different dosages of magnesium and remifentanil, as well as using longer post-operation follow-ups.

TITLE: Comparison of saddle, lumbar epidural and caudal blocks on anal sphincter tone: A prospective, randomized study ABSTRACT.OBJECTIVE: To compare the effects of saddle, lumbar epidural and caudal blocks on anal sphincter tone using anorectal manometry. ABSTRACT.METHODS: Patients undergoing elective anorectal surgery with regional anaesthesia were divided randomly into three groups and received a saddle (SD), lumbar epidural (LE), or caudal (CD) block. Anorectal manometry was performed before and 30 min after each regional block. The degree of motor blockade of the anal sphincter was compared using the maximal resting pressure (MRP) and the maximal squeezing pressure (MSP). ABSTRACT.RESULTS: The study analysis population consisted of 49 patients (SD group, n = 18; LE group, n = 16; CD group, n = 15). No significant differences were observed in the percentage inhibition of the MRP among the three regional anaesthetic groups. However, percentage inhibition of the MSP was significantly greater in the SD group (83.6 ± 13.7%) compared with the LE group (58.4 ± 19.8%) and the CD group (47.8 ± 16.9%). In all groups, MSP was reduced significantly more than MRP after each regional block. ABSTRACT.CONCLUSIONS: Saddle block was more effective than lumbar epidural or caudal block for depressing anal sphincter tone. No differences were detected between lumbar epidural and caudal blocks. BODY.INTRODUCTION: Surgical procedures for haemorrhoids and other minor anorectal disorders account for a large proportion of elective ambulatory surgery.1 Anorectal surgery requires deep anaesthesia because the region is innervated by multiple nerves and it is a reflexogenic zone.2 Although each method has its advantages and drawbacks, saddle, lumbar epidural, and caudal blocks are regional anaesthetic techniques considered well tolerated and reliable for anorectal surgery.3–5 Several methods have been introduced in an attempt to quantify motor blockade but they have limited practicability and are unfeasible in some clinical situations6 such as evaluating anal muscle tone.3 The degree of motor block associated with regional anaesthesia has traditionally been assessed using the Bromage scale or modified Bromage scale.7,8 However, the information obtained by observing the movements of the muscles included in this scale (i.e., hip, knee, and foot) does not accurately reflect the motor blockade that affects the anal muscle. Often in clinical practice, the anaesthesiologist or surgeon, test anorectal motor block by palpating the patient's anus but it is difficult to quantify the degree of motor blockade using this subjective method.5 Epidural local anaesthesia does not produce anaesthetic effects uniformly across the spinal segments.9 One of the factors that can affect the blockade is the size of the nerve root. For example, the large size of the L5 and S1 nerve roots may cause resistance to the local anaesthetic effect.10 Interestingly, it has been reported that the sensory blockade of S3 is easier than that of S1 and as rapid as that of L2 in a lumbar epidural block.11 Moreover, the same study concluded that the efficacy of a lumbar epidural block is comparable with a caudal block.11 However, the authors evaluated motor blockade using the Bromage scale and did not quantify the anal motor block. To our knowledge, no objective investigation has compared saddle, lumbar epidural and caudal blocks in a prospective, randomized study. The lack of a comparative study and adequate information on the potency of paralysing the anal sphincter with these regional anaesthetic techniques prompted us to examine if significant differences in anal motor block existed using the three different methods. Anorectal manometry, with the use of a continuous water perfusion system, was used to assess the tone of the anal sphincter muscles. BODY.PATIENTS AND METHODS.STUDY DESIGN AND PATIENT POPULATION: The prospective, randomized, comparative study took place in the Department of Anaesthesiology and Pain Medicine, Kangwon National University Hospital, Gangwon-Do, Republic of Korea between 05 December 2013 and 27 June 2014. Adult patients, aged 16–65 years, scheduled for elective anal surgery with regional anaesthesia and American Society of Anaesthesiologists physical status I or II were eligible for the study.12 Exclusion criteria were as follows: (i) a known allergy to local anaesthetics; (ii) the presence of a neuromuscular disorder; (iii) contraindications to regional anaesthesia; (iv) morbid obesity (i.e., body mass index > 30 kg/m2). A computer-generated random number table was used to assign patients to one of the following three regional anaesthetic groups: saddle (SD), lumbar epidural (LE), or caudal (CD). The study was approved by the Institutional Review Board of Kangwon National University Hospital (Ref: Imsang 07–05) and all patients provided written informed consent prior to enrolment. BODY.PATIENTS AND METHODS.ANAESTHETIC PROCEDURES: Patients did not receive any premedication. Electrocardiograms, heart rate and oxygen saturation were monitored before the block and thereafter continuously throughout the study. In the SD group, the subarachnoid space was entered using a 25-gauge Quinke spinal needle (Insung, Seoul, Korea) at the L3–4 interspace with the patient in the sitting position. After confirming the free flow of cerebrospinal fluid, 1 ml of 0.5% bupivacaine (5 mg) in 8% glucose solution (Marcain; AstraZeneca, Södertälje, Sweden) was administered. The patient remained in the sitting position for 5 min after the local anaesthetic injection. Subsequently, the patient was moved to the supine position. For the LE and CD groups, the anaesthetic solution was lidocaine-epinephrine-bicarbonate (20 ml of lidocaine 2% plus epinephrine [1:200 000] plus 2 ml of sodium bicarbonate 8.4%, which was added immediately before administration). In the LE group, the epidural space was identified using an 18-gauge Tuohy needle (Perifix®; B Braun, Melsungen, Germany) with its bevel facing caudally via the midline approach at the L4–5 interspace using the loss-of-resistance-to-air technique. Subsequently, a 22-gauge multi-orifice catheter was placed 3 cm into the epidural space in the lateral position. After taping the catheter and filter, 3 ml of 2% lidocaine with epinephrine (1: 200 000) were administered via the epidural catheter as a test dose. If no evidence of intravascular or subarachnoid injection appeared, an additional 12 ml of the anaesthetic solution was injected through the catheter over 1 min. In the CD group, the patients were placed in the lateral position and an 18-gauge Tuohy needle was inserted into the caudal space through the sacrococcygeal ligament. If no blood or cerebrospinal fluid was aspirated, a caudal injection of 15 ml of the anaesthetic solution was given through the needle. All patients in the LE and CD groups were moved into supine position following the anaesthetic injection and remained in the supine position for 20 min. To avoid inter-operator variability, one staff anaesthesiologist (S.S.K.) performed all of the blocks. In all patients, the level of sensory analgesia was evaluated on the torso and lower extremity at 5-min intervals for 30 min after injecting the local anaesthetic. The anatomical landmarks and correlated dermatome levels were standardized for testing as follows: the umbilicus (T10), inguinal crease (L1), anterior thigh (L2), medial knee (L3), medial malleolus (L4), the dorsum web between first and second toe (L5), the lateral heel (S1), and the medial popliteal fossa (S2). The assessment was based on the loss of cold sensation to a cotton wool swab soaked in absolute alcohol by an independent assessor blinded to the anaesthetic treatment group. BODY.PATIENTS AND METHODS.ANAL INTRALUMINAL PRESSURE MEASUREMENTS: Anal intraluminal pressure was evaluated using water perfused anorectal manometry pre-block and post-block. The pre-block assessment was performed 1–2 h before regional anaesthesia and the post-block assessment was taken 30 min after regional anaesthesia.13 The patients had no bowel preparation before manometry. The anal sphincter pressures were measured using a 3-lumen, water-perfused catheter and pneumohydraulic capillary infusion system (R3B & PIP4-4; Mui Scientific, Mississauga, Ontario, Canada) connected to a computerized software program (MMS Database; Medical Measurement Systems, Enschede, The Netherlands). The catheter had three ports spaced 0.5 cm apart for the two proximal holes and each lumen was perfused with sterile degassed water at 0.1 ml/min. The sphincter was identified using the slow station pull-through technique.14 For the pre-block measurement, the location of the catheter was verified using the pressure increase observed during pull-through and during a voluntary squeeze and the same position was used for the post-block measurement. After the patient was comfortable and the manometric trace had stabilized, the recording was started. The patient was then instructed to contract the anal sphincter maximally for 15 s on three separate occasions 1 min apart and the average value of 3 consecutive measurements was taken for the analysis. While relaxing and squeezing, the maximal resting (MRP) and squeezing (MSP) pressures of the anus were observed. The amount of inhibition caused by anaesthesia (i.e., the pressure drop in the anal canal) was expressed as the percentage of the resting and squeezing pressures. The percentage inhibition for MRP or MSP was calculated as follows: %inhibition={preanaesthetic pressure-postanaesthetic pressure}preanaesthetic pressure×100 All anorectal manometry measurements were performed by a fully trained independent physician blinded to the anaesthetic procedures. To ensure study blindness, the area from the lumbar vertebrae to the sacrococcygeal ligament was covered with the cotton gauze. All procedures and measurements were performed in the same manometric study room. After the manometric measurements were taken, all patients were moved to operating room and anal surgery was undertaken with the patient in the prone position. During the operation, the patients were sedated with intravenous administration of midazolam (1–3 mg). BODY.PATIENTS AND METHODS.STATISTICAL ANALYSES: The sample size was based on estimates from our pilot study in which the mean percentage inhibition of the MRP was 21.2, 31.3, and 29.2% for saddle, lumbar epidural, and caudal blocks, respectively. The standard deviation was 8.6%. Therefore, approximately 45 patients (15 in each group) were required to achieve 80% power to detect a difference of 10% inhibition of MRP among the means using one-way analysis of variance (ANOVA) at the 0.05 significance level. For the same variables, an expected difference of 8% inhibition of MSP generated a similar sample size. Patients with inadequate blockade after the administration of local anaesthetic were recorded as failures and excluded from the post-anaesthetic evaluation. To allow for drop-outs, a sample size of at least 18 patients per group was chosen. All statistical analyses were performed using the SPSS® statistical package, version 19.0 (SPSS Inc., Chicago, IL, USA) for Windows®. Differences among the groups were analysed using one-way ANOVA or nonparametric Kruskal–Wallis test, as appropriate. Manometric data among the groups were compared using ANOVA and if significant results were obtained the post hoc Scheffé test was used.15 Within-group comparisons were made using the paired t-test. P-values < 0.05 were considered statistically significant. BODY.RESULTS: Of the 62 patients who were assessed for eligibility, 54 were randomized to one of the three regional anaesthesia groups. Reasons for exclusion are shown in Figure 1. In the LE group, the lumbar epidural technique was successful in all but two patients (failure to identify epidural space and catheter insertion failure) and in the CD group three patients (failed block in two patients and incomplete block in one patient) had to be excluded from the analysis. Therefore, the study analysis population consisted of 49 patients (SD group, n = 18; LE group, n = 16; CD group, n = 15). No statistically significant differences were detected among the three groups in terms of age, height, weight or sex distribution (Table 1). The anaesthesia level in the LE group was statistically significantly higher than in the SD and CD groups (P < 0.01 for both comparisons) (Table 1). All the operations were performed successfully without any need for additional intervention or analgesics. Figure 1.Flow diagram showing patient numbers at various stages in the prospective, randomized, comparative study of the effects of saddle (SD), lumbar epidural (LE) and caudal (CD) blocks on anal sphincter pressure. Table 1.Clinical and demographic characteristics of patients (n = 49) who participated in this study to compare the effects of saddle, lumbar epidural and caudal blocks on anal sphincter tone.Regional anaesthesia groupSaddle block (n = 18)Lumbar epidural block (n = 16)Caudal block (n = 15)Age, years35.8 ± 18.737.9 ± 18.139.8 ± 13.4Height, cm164.1 ± 9.5161.7 ± 8.8163.0 ± 7.3Weight, kg62.1 ± 10.164.5 ± 8.263.3 ± 9.0Male/female10/89/77/8Diagnosis† Haemorrhoids987 Anal fissure655 Anal fistula464Anaesthesia levelL1 (T10–L2)T11 (T8–L1)*L2 (T12–L3)Data are presented as mean ± SD or n of patients. Anaesthesia level is shown as median (range).†Some patients had two or more preoperative anal diseases.*P < 0.01 compared with groups SD and CD; nonparametric Kruskal–Wallis test. No statistically significant differences were observed in the pre-block MRP and MSP values among the three regional anaesthetic groups (Tables 2 and 3). However, within each group, the values for MRP and MSP after regional block had decreased significantly compared with the pre-block values (P < 0.05 for all comparisons). Table 2.Maximal resting anal pressure measured by anorectal manometry pre- and post-block with regional anaesthesia.Maximal resting pressureSaddle block (n = 18)Lumbar epidural block (n = 16)Caudal block (n = 15)Pre-block, mmHg79.7 ± 14.377.2 ± 20.374.7 ± 14.3Post-block, mmHg61.5 ± 17.7*50.7 ± 19.6*54.1 ± 21.3*Mean difference, mmHg19.2 (8.7–29.8)22.3 (6.9–37.8)20.9 (5.9–36.0)Values are the mean ± SEM. Mean differences are the mean (95% confidence interval).*P < 0.05 compared with pre-block in each group; paired t-test. Table 3.Maximal squeezing anal pressure measured by anorectal manometry pre- and post-block with regional anaesthesia.Maximal squeezing pressureSaddle block (n = 18)Lumbar epidural block (n = 16)Caudal block (n = 15)Pre-block, mmHg85.1 ± 29.893.2 ± 38.086.7 ± 33.7Post-block, mmHg16.6 ± 11.7*40.8 ± 19.6*45.1 ± 18.4*Mean difference, mmHg70.4 (42.2–98.6)54.4 (32.8–75.9)40.1 (17.4–57.5)Values are the mean ± SEM. Mean differences are the mean (95% confidence interval).*P < 0.05 compared with pre-block in each group; paired t-test. The mean ± SEM percentage inhibitions of MRP with regional block were 21.2 ± 10.5%, 31.4 ± 14.1%, and 29.2 ± 16.4% in the SD, LE, and CD groups, respectively (Figure 2). Although there were no statistically significant differences between groups in the percentage inhibition of MRP, the percentage inhibition of MSP was significantly greater in the SD group (83.6 ± 13.7%) compared with the LE group (58.4 ± 19.8%) and the CD group (47.8 ± 16.9%) (P < 0.05 for both comparisons) (Figure 2). No difference was observed between the LE and CD groups. For all groups, the percentage inhibition in MSP after each regional block was significantly greater than for the MRP (P < 0.05 for all comparisons) (Figure 2). Figure 2.Percentage inhibition of maximum resting pressure (MRP) and maximum squeezing pressure (MSP) measured by anorectal manometry following regional anaesthetic block. The amount of inhibition caused by the regional anaesthesia (i.e., the pressure drop in the anal canal) was expressed as the percentage of the resting and squeezing pressures. Values are mean ± SEM. *P < 0.05 compared with the SD group, †P < 0.05 compared with MRP. SD, saddle block; LE, lumbar epidural block; CD, caudal block. BODY.DISCUSSION: Anorectal manometry is a simple, non-invasive and reproducible method of measuring luminal pressures within the anal canal.16 The technique is now widely used as a surrogate measure of anal sphincter tone.16,17 The MRP in the anal canal is at its highest level when the patient is at rest. The resting tone of the internal anal sphincter contributes 85% to the MRP and the resting tone of the external anal sphincter 15%.11 The MSP in the anal canal is at its highest pressure during squeezing and starts from a baseline represented by the maximal resting tone. The MSP is related to the activity of the striated sphincters (i.e., the external sphincter and the puborectalis muscle). The principal finding of this study was that saddle block was the most effective technique for decreasing MSP. In addition, the reductions in MSP following lumbar epidural or caudal block were similar. Also, the decrease in MRP was similar for all types of anaesthetic technique. These current findings support the results of a study that found surgeons who were blinded to the anaesthetic method rated spinal anaesthesia better than epidural anaesthesia for minor non-obstetric surgery.18 The complex innervation of the anal zone (i.e., autonomic versus spinal) may explain the differences that this present study found between the regional anaesthetics in the anorectal manometry results. The internal anal sphincter is under autonomic control via sympathetic innervation from the hypogastric plexus, which initiates tonic contraction, and via sacral parasympathetic fibres that mediate anorectal muscle relaxation. The external sphincter, which is supplied by the inferior rectal branch of the internal pudendal nerve and the perineal branch of the fourth sacral nerve, is under voluntary control.19 The decrease in the MRP after each block is probably due to sympathetic blockade. Therefore, the difference that this current study observed between the reductions in MSP and MRP may have been the result of partial, incomplete sympathetic blockade. In this present study, the level of analgesia ranged from T10 to L2 (SD group), T8 to L1 (LE group) and T12 to L3 (CD group). Considering the innervation of this region and the sensory level that was observed with each block, partial sympathetic blockade of the hypogastric plexus was a probable cause for the differences that were found between the regional anaesthetics in this study. With a spinal block, sympathetic blockade is present two or three levels above the level of the sensory blockade, whereas with an epidural block the level of sympathetic blockade is the same as, or lower than the sensory block.9 The hypogastric plexus contracts the internal anal sphincter and receives its sympathetic innervation from preganglionic fibres, which have their cell bodies in the lower thoracic and upper lumbar segments.20 Another possible explanation for the differences that were found with the three regional anaesthetics in the anorectal manometry results in the present study is incomplete sympathetic blockade that might happen in the dermatomal segments when analgesia is present. For example, one study showed that despite a blockade sufficient for surgical anaesthesia, it was difficult to achieve a complete sympathetic blockade with a spinal or epidural block using clinical doses of local anaesthetics.21 Although caudal block is widely used for anorectal surgery, its success rate has been reported to be only 70–80%.22–24 Despite a highly experienced staff anaesthesiologist performing the procedure in the present study, three of the 18 caudal blocks were unsuccessful. Caudal block failure is often related to anatomic variations of the bony sacrum and the soft tissues over the bone, which include displacement of the hiatus, narrowing of the sacral canal, and ossification of the sacrococcygeal membrane that can occur especially in the elderly.25 In addition, following caudal entry to the epidural space, the cephalad spread of the drug may be limited by minor bony obstructions that will result in the blockade of limited segments.25 In our institute, saddle block is the most commonly performed regional anaesthetic technique for anal surgery, since it provides rapid-onset, dense block and usually takes a short time to perform.26 Caudal block is used as an alternative to saddle block in cases where dural puncture is to be avoided (e.g., patients with a history of post-dural puncture headache, increased intracranial pressure). However, as mentioned previously, caudal block in adults is often difficult because of anatomical variability. Also, we noted that the sacral spread of anaesthetic following the lumbar epidural block was sufficient to block sacral segments for anal surgery. Therefore, lumbar epidural block rather than caudal block may be a better choice in cases where saddle block cannot be used. The present study had several limitations. First, the exact level of sympathetic blockade was not checked and although measuring the level would have provided more detail, it was not the primary objective of the study. Moreover, there is some debate concerning the evaluative methods for sympathetic blockade.27–30 Secondly, the direction of the epidural catheter was not confirmed radiologically. One study showed that the initial injection of a local anaesthetic solution through a caudally oriented epidural catheter results in a large spread of anaesthesia through the sacral area.31 Although the bevel of the epidural needle was placed caudally, it did not ensure caudal advance. However, another study showed that the injection of a local anaesthetic solution through a lumbar epidural catheter oriented caudally resulted in a faster onset and superior quality of anaesthesia in comparison with a cephalad-oriented catheter in ankle surgery (operating site innervated mainly by S1), but not in haemorrhoidectomy (by S3–5).32 This interesting difference between the two aforementioned studies implies that although more anaesthetic pooled in the sacral area with the caudally oriented lumbar epidural catheter, sensory analgesia with a lumbar epidural block for the area innervated by the sacral nerve (excluding S1, the blockade of which is often delayed due to the larger size of this root8) is not affected by the direction of spread of the local anaesthetic.31,32 In this present study, it remains unclear as to whether the direction of the epidural catheter affected manometric changes within the LE group. Theoretically, we would suggest that more caudal spread of the local anaesthetic with a caudally directed catheter should result in a difference in the degree of motor blockade in the lower sacral area. Finally, different local anaesthetics were used in the three treatment groups; hyperbaric bupivacaine was used for spinal anaesthesia and lidocaine for the LE and CD groups. A significantly higher motor blockade has been reported with lidocaine than with bupivacaine.30 However, the focus of this present study was to make a clinically oriented comparison of the three anaesthetic techniques in a normal clinical practice setting. In addition, several case reports and animal studies have suggested that spinal lidocaine may result in a greater incidence of neurological complications compared with other local anaesthetics.33,34 In conclusion, this prospective, randomized, comparative study used anorectal manometry to demonstrate that saddle block was more effective than lumbar epidural or caudal block for depressing anal sphincter tone. No differences were detected between the lumbar epidural and caudal blocks.

TITLE: The Brain Effects of Laser Acupuncture in Healthy Individuals: An fMRI Investigation ABSTRACT.BACKGROUND: As laser acupuncture is being increasingly used to treat mental disorders, we sought to determine whether it has a biologically plausible effect by using functional magnetic resonance imaging (fMRI) to investigate the cerebral activation patterns from laser stimulation of relevant acupoints. ABSTRACT.METHODOLOGY/PRINCIPAL FINDINGS: Ten healthy subjects were randomly stimulated with a fibreoptic infrared laser on 4 acupoints (LR14, CV14, LR8 and HT7) used for depression following the principles of Traditional Chinese Medicine (TCM), and 1 control non-acupoint (sham point) in a blocked design (alternating verum laser and placebo laser/rest blocks), while the blood oxygenation level-dependent (BOLD) fMRI response was recorded from the whole brain on a 3T scanner. Many of the acupoint laser stimulation conditions resulted in different patterns of neural activity. Regions with significantly increased activation included the limbic cortex (cingulate) and the frontal lobe (middle and superior frontal gyrus). Laser acupuncture tended to be associated with ipsilateral brain activation and contralateral deactivation that therefore cannot be simply attributed to somatosensory stimulation. ABSTRACT.CONCLUSIONS/SIGNIFICANCE: We found that laser stimulation of acupoints lead to activation of frontal-limbic-striatal brain regions, with the pattern of neural activity somewhat different for each acupuncture point. This is the first study to investigate laser acupuncture on a group of acupoints useful in the management of depression. Differing activity patterns depending on the acupoint site were demonstrated, suggesting that neurological effects vary with the site of stimulation. The mechanisms of activation and deactivation and their effects on depression warrant further investigation. BODY.INTRODUCTION: Despite the remarkable developments in Western Medicine in modern times, public interest in Traditional, Complementary and Alternative Medicine (TCAM), such as acupuncture, remains high [1], [2]. This may be because TCAM is perceived as holistic and relatively free of adverse effects. However, these treatments sit uncomfortably alongside scientific medicine because of strikingly different explanatory systems and the empirical tests applied by each discipline. In order to bridge the gulf between high public acceptability and the lack of empirical evidence for many of these treatments, it is important to reconcile them with modern scientific concepts. Our focus here is on laser acupuncture, and we address the question whether laser acupuncture produces brain effects that are biologically plausible. There have been many studies [3]–[20] some of which have involved functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) of the brain effects of needle acupuncture. Some neuroimaging and neuroendocrine studies have suggested that needle acupuncture affects hypothalamic as well as extrahypothalamic functions and modulates mood [3], [5]–[9]. Needling of the leg acupoint ST36 and the hand acupoint LI4 was shown to activate the hypothalamus and nucleus accumbens and deactivate the rostral anterior cingulate cortex (rACC) [3]. Superficial needling (i.e. needling that did not produce the classical de qi sensation - tingling, numbness or other sensations that occur after an acupuncture needle has been properly placed in the body) and non-acupoints (i.e. points on the skin that do not lie on recognized meridians in Traditional Chinese Medicine [TCM]) did not activate the hypothalamus [7]. Stimulation of the acupoint PC6, located above the wrist, recommended for a wide range of conditions from nausea to stress management, resulted in activation of the cerebellum, dorsomedial nucleus of the thalamus, anterior cingulate gyrus and left superior frontal gyrus [8]. All of these studies have used needle acupuncture which, although more traditional, is invasive. While laser acupuncture has become an increasingly common clinical method, particularly in primary care, its empirical basis has been less well studied to date. Whereas needle acupuncture studies have shown activation and deactivation of the somatosensory cortex [3], [4], [6], [7], [11]–[14], superficial needling and laser intervention appear to stimulate cortical and subcortical structures other than the somatosensory cortex [11], [19], [20]. This is consistent with the observation that low intensity laser stimulation does not produce a skin sensation. For example, laser acupuncture of a foot acupoint, classically used for treating visual problems, was demonstrated to cause activation of the occipital cortex [19]. This study has used laser delivered at low intensity as used in primary care. Other studies have reported high intensity lasers can produce de qi sensation [21]. High intensity laser is not commonly used in primary care situations and therefore was not used in the current study. Further, as low intensity laser does not result in sensory sensation it is ideal for double blind randomized controlled studies where the subjects could not differentiate between placebo (laser off) and verum laser (laser on). The evidence suggests that cortical activation does occur with acupuncture and this activation may be specific to certain brain regions in relation to the site and type of stimulation [11]–[14]. In practice, acupoint efficacy is not specific, and one acupoint can be used for several different conditions, just as one medical condition can be managed with several acupoint locations. For instance, the antidepressant effect of laser acupuncture [22] has been attributed to a group of acupoints - CV14, LR14, LR8 and HT7 (see fig.1 for anatomical location), however there are other acupoint combinations that are also applicable for the management of depression. The neurological effects of stimulation of these acupoints CV14, LR14, HT7 and LR8 in combination have yet to be investigated. In this study, we examined the blood oxygen level dependant (BOLD) functional magnetic resonance imaging (fMRI) response to laser simulation on the above mentioned acupoints CV14, LR14, LR8 and HT7. We chose laser acupuncture as it permits blinding of application because of the lack of a local sensation at low intensity, together with the previously mentioned increases in practical usage and limited understanding of its mechanisms. We reasoned that if laser acupuncture is altering a person's mental state a neurological effect should be observable. Further, if the effect differs dependent on the site of stimulation, then the neural locus of the activity should also differ. BODY.MATERIALS AND METHODS.ETHICS STATEMENT: The study was approved by the human research ethics committee of the South Eastern Sydney & Illawarra Area Health Service and participants provided written informed consent before participation. BODY.MATERIALS AND METHODS.PARTICIPANTS: The participants (n = 10) (7 women, 3 men) were healthy volunteers aged 18–50 years (mean age = 39.8 years) who were recruited by advertisement from the staff and students of the University of New South Wales and Prince of Wales Hospital, Sydney, Australia. All participants were right-handed and had no past history of depression or other psychiatric disorder, a Beck Depression Inventory [21] score <10, no history of drug or alcohol abuse, no medication intake within 3 months of the study, and no neurological or systemic disorders. Eight were acupuncture naïve and two had had needle acupuncture more than 3 months previously and did not know what to expect from laser intervention. Any contra-indications to MRI (pacemaker, ferromagnetic implants or foreign body, claustrophobia) were exclusionary. BODY.MATERIALS AND METHODS.CHOICE OF ACUPOINTS AND CONTROL POINT: The acupoints were selected based on results from our previous study [22] and the TCM for mood disorders [23], [24]. These acupoints lay on the classically named liver (LR), heart (HT) and conception vessel (CV) meridians. The selected points, labelled LR8, LR14, HT7 and CV14 in TCM, are shown in Figure 1. LR8 is in the medial knee region, between the insertions of the sartorius and semitendinosus muscles. LR14 is in the vicinity of the 6th intercostal space on the mid clavicular line. HT7 is at the wrist crease, in the vicinity of the radial side of the flexor carpi ulnaris. CV14 is in the anterior midline, approximately 5 cm below the xiphisternum. A control non-acupoint was selected on the abdominal surface, midway between SP15 (four cun from the umbilicus) and ST25 (two cun from the umbilicus) away from the abdominal meridians. 10.1371/journal.pone.0012619.g001Figure 1Selected acupoints relevant to mood and depression.In Traditional Chinese Medicine, the Liver (LR) meridian is used to treat depression. Two LR points were used: LR14 and LR 8. Two other acupoints considered important in depression were also used (CV 14 and HT 7). The control point (sham point or non acupoint) was located midway between SP15 and ST 25 acupoints. BODY.MATERIALS AND METHODS.FMRI DESIGN: A block design was used, with each block of 20 seconds duration during which the subject received either laser stimulation (switched 'on') or placebo stimulation (switched 'off') at one acupoint. The infra red laser was held with light touch on the skin surface by the acupuncturist. Since the laser produces no sensation, the subject was able to be kept blind to the phase of stimulation. The on-off cycle was repeated 4 times for each acupoint (LR14, LR8, CV14, HT7), with the 4 acupoints being stimulated twice in random order. The control point near ST25 was stimulated once per subject. The block design accomodated for the placebo (laser off) condition during its rest phases. In total there were nine runs per subject. The subject was told to relax and keep his/her eyes closed during the entire time in the scanner. BODY.MATERIALS AND METHODS.LASER STIMULATION: A MoxlaR prototype fiberoptic infra-red light laser (808 nm) with 25 mW capacity and a fiber optic arm was developed for usage in the scanning room. The laser parameters are similar to the one used in the clinical study we have based our investigation upon [22]. The acupoints were marked with a skin marking pencil prior to entry into the scanning room. A stably held laser was applied to the skin by the acupuncturist (IQ-S) who moved it from point to point according to the time signal. The switching on and off was achieved with a computer signal time-locked to the MRI acquisition. BODY.MATERIALS AND METHODS.FMRI: Imaging was performed on a 3T Philips Intera MRI scanner (Philips Medical Systems, Best, Netherlands) for both T1-weighted 3D structural and BOLD contrast functional MRI. The 3D structural MRI was acquired in sagittal orientation using a T1-weighted TFE sequence (TR/TE = 6.39/2.9 ms; flip angle = 8; matrix size = 256×256; FOV = 256×256 mm; slice thickness 1.0 mm), yielding sagittal slices of 1.0 mm thick and an in-plane spatial resolution of 1.0×1.0 mm, producing an isotropic voxel of 1.0×1.0×1.0 mm. A gradient echo-planar imaging (EPI) technique (TR/TE = 2000/40 ms; matrix size = 128×128; FOV = 250×250 mm; in plane pixel size 1.953×1.953 mm) was used to acquire T2-weighted BOLD contrast fMRI in axial orientation. The whole brain was covered using 21 slices at 5.0 mm slice thickness and 0.5 mm gap for each volume. Each session of 96 volumes were collected with the rate of 2s/volume. BODY.MATERIALS AND METHODS.IMAGE PREPROCESSING AND STATISTICAL ANALYSIS: Imaging data were analyzed using statistical parametric mapping (SPM2, Wellcome Department of Cognitive Neurology, London, UK) implemented in Matlab version 6 (The Mathworks Inc., USA). All volumes were realigned spatially to the first volume and the time-series for voxels within each slice realigned temporally to acquisition of the middle slice. Resulting volumes were normalized to a standard EPI template based on the Montreal Neurological Institute (MNI). The normalized images were smoothed with an isotropic 8 mm full-width half-maximal Gaussian kernel. The time-series in each voxel were highpass-filtered to 1/120 Hz to remove low-frequency noise. Statistical analysis was performed in two stages, assuming a random effects design. Each stimulation site was compared to the placebo (laser off) condition for first level analysis. The BOLD response to the laser acupuncture stimulation was modeled by a canonical hemodynamic response function (HRF). The second level analysis (ANOVA) used each individual subject's contrast images, which were effectively the statistical parametric maps of the t-statistics for each voxel. The data had a threshold of p <0.001 with a spatial extent of 15 contiguous voxels. BODY.MATERIALS AND METHODS.POST-IMAGING ASSESSMENT: After the scanning session, subjects rated selected items on the Spielberger State Anxiety Inventory [25] to describe their mental state during the period of the scanning. The ratings were: 1 (not at all), 2 (somewhat), 3 (moderately so) and 4 (very much so). BODY.RESULTS.GROUP ANALYSIS: At the group level, there were significant increases (activation) in BOLD levels in some brain regions for acupoints LR14, CV14, LR8 and the control point compared to all the other points (verum laser per point > all others, p<0.001; see Table 1). Further, there were significant decreases (deactivation) in BOLD levels for acupoints LR14, LR8 and the control point compared to all the other points (all others > verum laser per point, p<0.001; see Table 1) in other brain regions. 10.1371/journal.pone.0012619.t001 Table 1 Significant brain activation patterns from laser acupuncture to LR14, CV14, LR8 and control point. ACUPOINT x y z p vox No of Voxels Brain Region Activation LR14 −28 −42 68 0 169 Left Postcentral Gyrus −26 −6 62 0 85 Left Superior Frontal Gyrus −44 34 26 0 20 Left Middle Frontal Gyrus CV14 −10 −50 6 0 84 Left Posterior Cingulate LR8 −6 0 42 0 48 Left Cingulate Gyrus Control Point 50 −26 48 0 40 Right Postcentral Gyrus Deactivation LR14 −26 −42 −32 0 54 Left Cerebellar Tonsil −8 −66 18 0 69 Left Occipital Lobe, Precuneus LR8 36 6 52 0 96 Right Middle Frontal Gyrus −26 12 62 0 83 Left Middle Frontal Gyrus 20 14 54 0 151 Right Superior Frontal Gyrus 42 −60 22 0 131 Right Middle Temporal Gyrus 10 8 8 0 35 Right Caudate Control Point −26 −56 −4 0 182 Left Parahippocampal Gyrus With LR8, activation of ipsilateral limbic cortex (cingulate gyrus) and deactivation of bilateral frontal cortices (middle frontal gyrus), (contralateral superior frontal gyrus), contralateral temporal cortex (middle temporal gyrus) and contralateral caudate occurred. Stimulation at the LR14 acupoint resulted in activation of contralateral frontal cortex (superior and middle frontal gyrii), contralateral parietal cortex (postcentral gyrus) and deactivation of contralateral cerebellum (cerebellar tonsil) and contralateral occipital cortex (precuneus). Acupoint CV14 produced activation of the left limbic cortex in the posterior cingulate and there was no significant deactivation in the grey matter. HT7 had no significant activation or deactivation. The control point (non acupoint or sham point), activated the contralateral parietal cortex (postcentral gyrus). It also deactivated the contralateral limbic cortex (parahippocampal gyrus). BODY.RESULTS.SOMATOSENSORY CORTEX AND LATERALITY OF CEREBRAL ACTIVATION AND DEACTIVATION: Our study involved randomized stimulation of the 4 acupoints and a control point. Although there was activation of contralateral postcentral gyrus (primary somatosensory cortex or SSI) with LR14 and control point, none of the other acupoints showed any activation of the somatosensory cortex. The cortical and subcortical structures activated with stimulation of the limb acupoints tended to be largely ipsilateral to the side of stimulation. All the acupoints and control point did not have deactivation at the somatosensory cortex. However they all had contralateral deactivations with the exception of LR8 that had bilateral middle frontal gyrus deactivation. BODY.RESULTS.BEHAVIORAL OBSERVATIONS: Participants did not describe anxiety or discomfort during scanning, except for one who found the headphones uncomfortable. The mean Spielberger scale ratings were on select items were: feeling calm (3.3), secure (3.3), relaxed (3.3), nervous (1.4), jittery (1.1), worried (1.1) or overexcited and rattled (1.1). BODY.DISCUSSION: This is the first fMRI study to examine the effects of laser stimulation of a suite of acupoints found to be efficacious in a clinical condition (depression). A salient feature of this study was that four acupoints and a control non-acupoint (sham point) were stimulated in a random design. The subjects were unaware of the relative significance of different acupoints. The use of low level laser acupuncture, which does not produce a skin sensation, permitted the blinding of subjects to verum or placebo stimulation, something difficult to achieve with needle acupuncture. The main finding of our study was that each acupoint or control point resulted in a different pattern of brain activity when contrasted against all the other acupoints or control point. The acupoints we investigated in this study were those that have been used in our previous treatment study for depression [22]. This finding suggests that although these acupoints are all used in the treatment of depression, the neural locus of this effect differs depending upon the site stimulated. The efficacy of these acupoints in the treatment of depression may vary greatly between patients and site stimulated, and our findings may shed some light on these effects [26]. The neuroanatomical basis of depression is not completely understood, however a number of studies have implicated abnormalities in certain brain regions, in particular the medial and dorsolateral prefrontal cortex, the cingulate gyrus and the so-called limbic brain regions (hippocampus, parahippocampal gyrus, amygdala, septal nuclei, insula, thalamus) and paralimbic regions (orbitofrontal cortex, anterior temporal lobe) [26]–[29]. There is converging evidence from drug treatment, cognitive-behavior therapy and brain stimulation techniques that antidepressant treatments work by modulating frontal-subcortical neuronal circuits. The most consistently reported finding is that antidepressant treatments lead to a normalization of activity in the dorsolateral prefrontal cortex, with additional changes in the subgenual cingulate region, the posterior cingulate, parahippocampal gyrus and insula [29]. Whether the change in prefrontal cortex is a primary event or secondary to changes in subcortical nuclei is unclear, but the relationship of treatment response to this suggests that it is biologically plausible that laser acupuncture could be an effective antidepressant treatment through its effects on the above brain regions. The results show this combination of acupoints activating frontal cortex, limbic cortex and subcortical caudate. The trend is for ipsilateral activation suggestive of neurological circuitry outside the dorsal spinal columns and more likely to be autonomically driven [30]–[32]. Most of the deactivations were contralateral. Also LR14 and control point activations included primary somatosensory cortical activations (SSI). None of the deactivations involved SSI, however they did include the regions as described earlier that could collectively be called the affective cortex (the frontal, limbic and temporal cortices as well as the subcortical caudate). This combination of ipsilateral and contralateral activations and deactivations may perhaps be representative of the combined actions of both the spinal and autonomic nervous systems during laser acupuncture. In classical acupuncture, there are primary and secondary acupoints for the treatment of any disorder. The approach to acupoint selection can be variable, with primary acupoints being considered essential and secondary acupoints additive for some patients. In our study, we cannot predict from these results whether any acupoint should be preferred over others for clinical use, even though LR8 deactivated more brain regions (middle and superior frontal gyrus, middle temporal gyrus and the subcortical caudate) than all the other points. These are results from a sample of healthy subjects. The question of whether the results would be different in a sample of clinically depressed subjects, needs to still be answered. Further studies are required to explore the relative value of different acupoints, the final test for which naturally lies in a clinical trial. It also cannot be stated from our study whether the treatment response can be achieved with stimulation at one point alone, or if multiple points are necessary. There is conflicting evidence regarding acupoint specificity and whether that specificity is relative rather than absolute for any particular disorder [33]–[38]. Furthermore, it is debatable whether the clinical effects of acupuncture are restricted to stimulation on points that lie on the classical meridians in TCM. Our finding that laser stimulation of a non-acupoint produced some brain activation suggest that there is unlikely to be a completely neutral control non-acupoint, and this should prompt a re-examination of the use of sham points (in needle acupuncture studies) as control hence minimizing the true statistical effects of any acupoint [36]–[39]. It is also interesting that laser acupuncture in this study appeared to preferentially activate the limbic cortex ipsilaterally and deactivate the limbic cortex contralaterally. It has been suggested that laser stimulation preferentially activates unmyelinated afferent fibers that project ipsilaterally to the insula [40]–[42], which might also explain the differences from needle acupuncture. This laser acupuncture fMRI study demonstrated the central effects of stimulation of a suite of acupoints found to be efficacious in treating depression in a primary care setting. The multiple acupoints each activated different groupings of frontal-limbic-striatal brain regions, suggesting some acupoint specificity but also a commonality in the regions affected. There was a trend for the limb acupoints to cause ipsilateral activation and contralateral de-activation. The results of the study suggest that laser acupuncture is a biologically plausible anti-depressant treatment. Its efficacy and the relative merits of the different proposed acupoints must be empirically examined.

TITLE: Lithium as add-on to quetiapine XR in adult patients with acute mania: a 6-week, multicenter, double-blind, randomized, placebo-controlled study ABSTRACT: Quetiapine extended release (XR) and lithium are treatments with proven efficacy in acute mania. This randomized study evaluated the efficacy and safety of lithium or placebo as add-on to quetiapine XR in adult patients with manic or mixed symptoms of bipolar I disorder. In this 6-week, double-blind study (Trial D144AC00003), adult patients with DSM-IV-TR-diagnosed bipolar I disorder (current episode manic or mixed), a Young Mania Rating Scale (YMRS) total score ≥20, and score ≥4 on two of four core YMRS items were administered quetiapine XR (400 to 800 mg/day) and randomly assigned to receive add-on lithium (600 to 1,800 mg/day) or placebo. The primary efficacy end point was change in the YMRS total score from baseline to day 43, analyzed using a mixed-model for repeated measures (MMRM) approach. Secondary efficacy and safety end points were also measured. Rating scales were administered by trained staff. Three hundred fifty-six patients treated with quetiapine XR were randomized to add-on lithium (n = 173) or placebo (n = 183). Two hundred ninety-one patients (81.7%) completed the study. At day 43, least squares mean change in YMRS total score was −22.8 for add-on lithium and −20.1 for add-on placebo, a statistically significant treatment group difference of −2.69 (p < 0.001). On secondary measures, add-on lithium was associated with significant improvements in response, remission, illness severity, and overall illness versus add-on placebo (p < 0.05). The number needed to treat was 9.1 for response and 7.9 for remission for add-on lithium compared with add-on placebo. Lithium in combination with quetiapine XR was generally well tolerated, with a similar profile to quetiapine XR in combination with placebo. The addition of lithium to quetiapine XR therapy was associated with significantly greater efficacy than placebo as add-on and was generally well tolerated in patients with acute bipolar I mania. This study was registered under Clinicaltrials.gov Identifier NCT00931723. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40345-014-0014-9) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Bipolar disorder is a complex, debilitating illness that typically follows a chronic and recurrent course. Manic, depressed, or mixed symptoms range in severity and rate of onset and in their most extreme forms require hospitalization (Sanchez-Moreno et al. 2009). Given the severe impact and rapid onset of manic symptoms in many patients, prompt and effective control of symptoms is a primary treatment goal (Garlow 2008; Oral 2005). Guidelines typically recommend lithium, divalproex, or an atypical antipsychotic in the treatment of acute mania, frequently as monotherapy initially, with combination therapy in cases of inadequate response (Connolly and Thase 2011; Goodwin 2009; Grunze et al. 2009; Nivoli et al. 2011; Yatham et al. 2009). Guidelines also recommend combination therapy as first-line treatment for severe manic symptoms (Goodwin 2009; Grunze et al. 2009; Nivoli et al. 2011). However, there is a lack of evidence to support the efficacy and safety of many combination therapies, and not all agents demonstrating antimanic efficacy as monotherapy offer additional efficacy and acceptable tolerability when used in combination (Brooks et al. 2011; Geoffroy et al. 2012; Sachs and Gardner-Schuster 2007; Smith et al. 2007). Quetiapine extended release (quetiapine XR) demonstrates efficacy as monotherapy in acute mania (Cutler et al. 2011) and bipolar depression (Suppes et al. 2010). Yatham et al. (2004) previously reported that quetiapine immediate release (IR) added to lithium or divalproex provided superior efficacy in treating mania when compared with lithium or divalproex alone. The potential benefits of adding lithium to quetiapine XR have not previously been investigated. The current study compared the efficacy and safety of lithium versus placebo as add-on to quetiapine XR in adults with bipolar I disorder with a current or recent episode of severe manic or mixed symptoms. BODY.METHODS.STUDY DESIGN: This was a 6-week, multicenter, double-blind, randomized, parallel-group placebo-controlled study (Trial D144AC00003; Clinicaltrials.gov ID: NCT00931723), conducted at 38 study centers in eight countries (Belgium, Bulgaria, Germany, India, Poland, Russia, South Africa, and Ukraine) between June 24, 2009 and November 22, 2010. The study included an enrollment period (with medication washout for 7 to 28 days, according to medication) and a 6-week treatment period, when quetiapine XR was administered as flexible dose to all patients. Patients were randomly assigned in a 1:1 ratio to receive, in addition, either flexible-dose lithium or placebo at the start of treatment. Following mandatory hospitalization during randomization, patients were permitted to continue study treatment as outpatients. The study was performed in agreement with the ethical principles of the Declaration of Helsinki and was consistent with the International Conference on Harmonization (ICH)/Good Clinical Practice (GCP). Written informed consent was provided by all patients. BODY.METHODS.PATIENT POPULATION: Males or females aged 18 to 65 years with a Diagnostic and Statistical Manual for Metal Disorders, Fourth Edition, Text Revision (DSM-IV-TR) diagnosis of bipolar I disorder (most recent episode manic or mixed) (American Psychiatric Association 2000), confirmed by the Structured Clinical Interview for DSM-IV (First et al. 2007), were eligible for inclusion. Patients were required to have a Young Mania Rating Scale (YMRS) (Young et al. 1978) total score ≥20 and ≥4 on two of four core items (irritability, speech, content, disruptive/aggressive behavior), a Clinical Global Impressions for Bipolar Disorder (CGI-BP) (Spearing et al. 1997) score ≥4 (i.e., moderately ill), and to have experienced ≥1 manic or mixed episode (other than the current episode) in the past 5 years. Key exclusion criteria included current DSM-IV-TR Axis I disorders other than bipolar mania; >8 mood episodes within the previous 12 months; mania-like syndrome associated with a medical condition or treatment, substance abuse, or withdrawal; continuous hospitalization for an acute episode of bipolar mania for >3 weeks before randomization; current substance dependence or abuse; current serious suicidal or homicidal risk; or history of nonresponse to study treatments. The use of an antipsychotic other than quetiapine, mood stabilizer, antidepressant, anxiolytic, hypnotic, other psychoactive drug, or inducer/inhibitor of cytochrome P 3A4 enzymes was prohibited within 7 to 28 days before randomization. Nonpsychoactive and anticholinergic medications were allowed during the study, as were zolpidem tartrate (maximum 10 mg/day), zaleplon (20 mg/day), zopiclone (7.5 mg/day), chloral hydrate (1 g/day), and lorazepam (2 mg/day). BODY.METHODS.STUDY TREATMENTS: Open-label quetiapine XR was administered once-daily in the evening, at a starting dose of 300 mg/day on day 1, increasing to 600 mg/day on day 2, and adjusted between 400 and 800 mg/day from day 3 onward depending on efficacy and tolerability, in agreement with prescribing guidelines (Seroquel XR (quetiapine fumarate) 2013). For patients who received quetiapine prior to the study, the existing dose was administered on day 1 and increased to 600 mg at day 2 if the previous dose was lower. Patients were treated with lithium or placebo twice daily. Lithium (immediate release) was administered at a starting dose of 600 mg on days 1 and 2, increasing to 900 mg/day on days 3 to 8. Lithium (or placebo) dosing was adjusted (range, 600 to 1,800 mg/day) from day 9 at the discretion of the investigator to minimize side effects or achieve target trough concentrations of 0.8 to 1.2 mEq/L. On days 8, 15, 22, 29, and 43, lithium was administered after serum lithium sampling. On each occasion that a lithium dose recommendation was sent for a patient randomized to lithium, a matching sham recommendation was sent to non-lithium patients in relation to the dose of placebo capsule. The lithium and placebo capsules were identical in appearance, smell, and taste to maintain blinding. In addition, blood samples were drawn from all patients (whether treated with lithium or placebo) on days 8, 15, 22, 29, and 43 for determination of trough serum lithium concentrations. BODY.METHODS.EFFICACY ASSESSMENTS: The primary outcome measure was change in YMRS total score from baseline to day 43. Secondary outcome measures included changes to day 43 in scores for individual YMRS items, Clinical Global Impressions for Bipolar Disorder-Severity of Illness (CGI-BP-S), CGI-BP-Change (CGI-BP-C) (Spearing et al. 1997), Positive and Negative Syndrome Scale (PANSS) total and activation and positive subscales (Kay et al. 1987), and Montgomery-Åsberg Depression Rating Scale (MADRS) (Montgomery and Asberg 1979). Response (i.e., ≥50% reduction in YMRS total score to day 43), remission (YMRS total score ≤12 at day 43), and improvement in overall bipolar illness (CGI-BP-C score of 'much' or 'very much' improved overall bipolar illness at day 43) were also assessed, using conventional cutoff criteria (Bourin and Thibaut 2013). Rating scales were administered by staff who were trained in their use and who were blinded to study treatments. BODY.METHODS.SAFETY AND TOLERABILITY ASSESSMENTS: Safety and tolerability assessments included treatment-emergent adverse events (TEAEs; incidence and severity), adverse event (AE)-related withdrawals, and changes from baseline in vital signs, laboratory parameters, and body weight. Extrapyramidal symptoms (EPS) were assessed by TEAEs, changes in EPS rating scales (Simpson-Angus Scale [SAS] (Simpson and Angus 1970), Abnormal Involuntary Movement Scale [AIMS] (Guy 1976), and Barnes Akathisia Rating Scale (BARS) (Barnes 1989)), worsening of SAS, AIMS, or BARS score category, and initiation of anticholinergic medication for EPS. Suicidality was assessed by TEAEs and prospective use of the Columbia-Suicide Severity Rating Scale (C-SSRS), with mapping of C-SSRS responses also to the Columbia-Classification Algorithm for Suicide Assessment (C-CASA) (Posner et al. 2007, 2011). Other TEAEs of special interest, besides EPS and suicidality, included somnolence, diabetes mellitus, QTc prolongation, neutropenia/agranulocytosis, and depression/depressed mood. BODY.METHODS.STATISTICAL ANALYSIS: It was estimated that 166 evaluable patients were required in each treatment group to reject the null hypothesis of no difference with a power of 80% when using a two-sided t-test at an overall type I error rate of 5%, assuming a true effect of four points on YMRS between the two groups and a standard deviation (SD) of 9. Efficacy analyses were performed on the modified intent-to-treat analysis set (ITT), which included all randomized patients who received ≥1 dose of quetiapine XR and lithium or placebo and had a baseline YMRS score and ≥1 post-baseline score assessment. Group differences in change in efficacy rating scale score were analyzed using a mixed-model for repeated measures (MMRM) approach; while group differences in response, remission, and overall bipolar illness were tested using generalized estimating equations. The robustness of the primary study analysis was tested using the per-protocol (PP) set (i.e., all ITT patients without major protocol violations or deviations affecting efficacy) and in the ITT set using robust variance estimates, as well as by analysis of covariance (ANCOVA) modeling using last observation carried forward (LOCF) methodology, including baseline YMRS total score as covariate, treatment group as fixed effect, and centers as random effect. Safety variables were evaluated on the safety analysis set (all randomized patients who received at least one dose of study medication) and are presented using descriptive statistics. All statistical comparisons were based on a two-sided test using a significance level of 5%. Analyses were performed with SAS® software, Version 9.2 or higher (SAS Institute, Cary, NC, USA). BODY.RESULTS: Overall, 441 patients were enrolled and screened, of whom 356 received quetiapine XR and were randomized to add-on treatment with lithium (n = 173) or placebo (n = 182) (Figure 1). The most common reason for nonrandomization was incorrect enrollment due to patients not meeting the inclusion criteria (n = 57). The majority of patients (91.0%) received study treatment as outpatients. Forty-four of the patients (12.4%) were diagnosed with severe mania and psychotic features at baseline.Figure 1 Patient disposition. a One randomized patient did not receive study treatment. The ITT and safety analysis sets comprised 349 and 356 patients, respectively. The PP set included 143 patients (n = 45, add-on lithium; n = 98, add-on placebo). Failure to achieve lithium concentrations within the target range (n = 112 patients) was the most common protocol deviation. Baseline demographics and disease characteristics were generally similar between the treatment groups (Table 1). Mean YMRS total scores at baseline were 29.9 and 30.0 in the add-on lithium and add-on placebo groups, respectively, indicating a severely ill population.Table 1 Baseline demographic and clinical characteristics (safety set) Demographic and clinical characteristics Quetiapine XR + Lithium ( n = 173) Quetiapine XR + Placebo ( n = 183) Total ( n = 356) Age (years) Mean (SD)37.9 (12.7)38.8 (12.1)38.3 (12.4) Median37.038.037.0 Range (min, max)18, 6418, 6518, 65Gender, n (%) Male101 (58.4)121 (66.1)222 (62.4) Female72 (41.6)62 (33.9)134 (37.6)Race, n (%) White98 (56.6)102 (55.7)200 (56.2) Asian73 (42.2)78 (42.6)151 (42.4) Other2 (1.2)3 (1.6)5 (1.4)Weight (kg) Mean (SD)69.1 (15.9)71.0 (17.1)70.1 (16.5) Median68.069.068.0 Range (min, max)37, 12037, 11537, 120BMI (kg/m2) Mean (SD)24.3 (4.3)24.9 (5.2)24.6 (4.8) Median23.824.123.9 Range (min, max)15.8, 37.114.7, 43.814.7, 43.8Psychiatric diagnosis, n (%) Most recent episode manic159 (91.8)173 (94.5)332 (93.3) Most recent episode mixed14 (8.1)10 (5.5)24 (6.7)Rapid cycling, n (%) Yes3 (1.7)5 (2.7)8 (2.2) No170 (98.3)178 (97.3)348 (97.8)Time since first diagnosis of acute mania, years Mean (SD)7.1 (6.6)7.8 (7.0)7.5 (6.8)Time since first mania/mixed episode, years Mean (SD)7.2 (6.6)8.1 (7.0)7.7 (6.8)Total number of mania/mixed episodes in the past year, n (%) 056 (32.4)56 (30.6)112 (31.5) 188 (50.9)88 (48.1)176 (49.4) ≥ 229 (16.8)39 (21.3)68 (19.1)Previous medication use, n (%) Lithium4 (2.3)2 (1.1)6 (1.7) Quetiapine8 (4.6)17 (9.3)25 (7.0) Open-label quetiapine XR was administered at a mean modal dose of 623.1 mg/day (range, 200 to 800 mg/day) in the add-on lithium group and 669.9 mg/day (range, 300 to 900 mg/day) in the add-on placebo group. The mean modal lithium dose was 1,085.5 mg/day (range, 300 to 1,800 mg/day). The mean serum lithium level was 0.72 mEq/L (range, 0.00 to 1.43 mEq/L) at day 43; mean lithium levels did not differ notably during the study (range of means, 0.64 to 0.77 mEq/L). Mean durations of exposure to quetiapine XR were 38.5 days (range, 2 to 48) in the add-on lithium group and 36.9 days (range, 1 to 48) in the add-on placebo group; mean durations of exposure to add-on lithium or placebo were 38.6 days (range, 3 to 48) and 37.2 days (range, 2 to 48), respectively. The use of concomitant medications was similar between the lithium (24.9%) and placebo (23.0%) add-on groups, with anilides (acetaminophen) representing the most common concomitant medication in both groups (>3% each) (Additional file 1: Table S1). A total of 291 (81.7%) patients completed the study, while 65 (18.3%) patients discontinued the study prematurely (n = 26 (15.0%), add-on lithium; n = 39 (21.3%), add-on placebo group), most commonly because of voluntary discontinuation (Figure 1). The majority of patients were compliant with quetiapine XR (98.5%, add-on lithium; 94.4%, add-on placebo group) and with lithium (99.2%) or placebo (96.1%). BODY.RESULTS.EFFICACY ASSESSMENTS.PRIMARY EFFICACY END POINT: Mean YMRS total scores decreased from baseline to day 43 in both treatment groups (Figure 2). Least squares (LS) mean (SE) reductions in YMRS total score were −22.8 (0.71) in the add-on lithium and −20.1 (0.71) in the add-on placebo group, a significant between-group difference of −2.69 (95% CI, −4.09 to −1.29; p < 0.001) (Table 2).Figure 2 Mean change in YMRS total score (with 95% CIs) from baseline (observed case data, ITT set). Table 2 Mean changes from baseline to day 43 in primary and secondary efficacy measures (ITT set) Measure Quetiapine XR + Lithium ( n = 173) Quetiapine XR + Placebo ( n = 176) Young Mania Rating Scale (YMRS) total score LS Mean change (SE) (MMRM)−22.8 (0.71)−20.1 (0.71) Mean difference (95% CI)−2.69 (−4.09, −1.29) p-value <0.001 Mean change (SD) (OC)−22.6 (7.24)−20.4 (7.64) n at day 43149147 Clinical Global Impressions Bipolar–Severity (CGI-BP-S) LS Mean change (SE) (MMRM)−2.5 (0.08)−2.2 (0.08) Mean difference (95% CI)−0.24 (−0.43, −0.04) p-value 0.017 Mean change (SD) (OC)−2.5 (0.99)−2.3 (1.00) n at day 43149147 Clinical Global Impressions Bipolar-Change (CGI-BP-C) LS Mean (SE) (MMRM)1.7 (0.09)1.9 (0.09) Mean difference (95% CI)−0.20 (−0.37, −0.03) p-value 0.020 Mean (SD) (OC)1.7 (0.69)1.9 (0.85) n at day 43149147 Positive and Negative Syndrome Scale (PANSS) total score LS Mean change (SE) (MMRM)−19.2 (0.91)−15.6 (0.91) Mean difference (95% CI)−3.7 (−5.74, −1.63) p-value <0.001 Mean change (SD) (OC)−19.8 (12.34)−16.1 (10.82) n at Day 43148147 PANSS positive subscale LS Mean change (SE) (MMRM)−8.1 (0.32)−7.0 (0.32) Mean difference (95% CI)−1.1 (−1.85, −0.39) p-value 0.003 Mean change (SD) (OC)−8.1 (4.47)−7.1 (4.54) n at day 43148147 PANSS activation subscale LS Mean change (SE) (MMRM)−7.1 (0.28)−5.9 (0.28) Mean difference (95% CI)−1.2 (−1.77, −0.61) p-value <0.001 Mean change (SD) (OC)−7.3 (3.60)−5.9 (3.77) n at day 43148147 Montgomery-Åsberg Depression Rating Scale (MADRS) total score LS Mean change (SE) (MMRM)−4.8 (0.37)−4.1 (0.37) Mean difference (95% CI)−0.6 (−1.36, 0.07) p-value 0.077 Mean change (SD) (OC)−5.0 (4.84)−4.5 (3.56) n at day 43149147ITT, intent to treat; LS, least squares; MMRM, mixed-model repeated measures; OC, observed cases. A negative change in score indicates improvement, with the exception of Clinical Global Impressions Bipolar-Change, where a positive change in score indicates improvement. Significant p-values shown in bold. The robustness of the primary efficacy analysis was confirmed by supportive analyses, where significant between-group differences at day 43 were observed in MMRM analysis of the PP set (p = 0.005), MMRM modeling of the ITT set with robust variance estimates (p < 0.001), and ANCOVA modeling with LOCF methodology (p = 0.001). In post hoc analysis of patients categorized by serum lithium level, LS mean (SE) change in YMRS total score was −23.6 (0.79) in the ≥6 mEq/L and −21.6 (1.04) in the <6 mEq/L group (p < 0.001 and 0.171, respectively, vs. placebo). BODY.RESULTS.EFFICACY ASSESSMENTS.SECONDARY EFFICACY END POINTS: Response (≥50% reduction in YMRS score at day 43) occurred in 79.2% and 68.2% of the lithium and placebo add-on groups, respectively, and remission (YMRS total score ≤12 at day 43) was reported in 72.3% and 59.7%, respectively. Between-group differences in response and remission rates were significant in favor of add-on lithium (p = 0.005, both). The number needed to treat was 9.1 for response and 7.9 for remission for add-on lithium compared with add-on placebo. The majority of individual YMRS item scores were also significantly improved in the lithium versus the placebo add-on group (Figure 3).Figure 3 Mean changes in YMRS item scores from baseline (ITT set, MMRM analysis). In post hoc analyses of patients categorized by serum lithium level, response rates were 85.1% and 72.7%, respectively, in the ≥6 and <6 mEq/L groups (p = 0.006 and 0.164, respectively, vs. placebo). Remission rates in the respective lithium groups were 78.1% and 65.5% (p = 0.004 and 0.170, respectively, vs. placebo). Add-on lithium was associated with significantly greater LS mean reductions in CGI-BP-S (mean difference, −0.24, p = 0.017) and CGI-BP-C (mean difference, −0.20; p =0.020) than add-on placebo. Proportions of patients 'much improved' or 'very much improved' at day 43 were similar in the two groups (77.5% vs. 71.0%; p = 0.177). Psychotic features and agitation and aggression, assessed by PANSS total and positive and activation subscales, were improved in both treatment groups, with significantly greater improvements in the add-on lithium than add-on placebo group (p < 0.001, 0.003, and < 0.001, for respective scales; Table 2). Severity of depressive symptoms, measured by MADRS total score, improved in both groups, without significant between-group difference (Table 2). BODY.RESULTS.SAFETY AND TOLERABILITY ASSESSMENTS: The incidence of TEAEs was 63.0% in the add-on lithium and 48.1% in the add-on placebo group. Tremor, somnolence, dizziness, diarrhea, and vomiting occurred at higher rates in the add-on lithium than add-on placebo group (Table 3). Treatment-emergent adverse events in both groups were mostly mild to moderate in intensity.Table 3 Incidence of treatment-emergent adverse events (≥2% in any group; safety set) Adverse event, n (%) Quetiapine XR + Lithium ( n = 173) Quetiapine XR + Placebo ( n = 183) Tremor27 (15.6)9 (4.9)Somnolence22 (12.7)10 (5.5)Constipation16 (9.2)16 (8.7)Dry mouth14 (8.1)14 (7.7)Dizziness11 (6.4)8 (4.4)Insomnia11 (6.4)12 (6.6)Headache9 (5.2)11 (6.0)Pyrexia10 (5.8)9 (4.9)Diarrhea8 (4.6)2 (1.1)Vomiting8 (4.6)0Sedation5 (2.9)3 (1.6)Dysarthria4 (2.3)2 (1.1)Nausea5 (2.9)3 (1.6)Weight increased2 (1.2)5 (2.7)Increased appetite4 (2.3)1 (0.5)Patients with multiple events falling under the same category are counted only once in that category. Adverse events led to discontinuation in a greater proportion of patients in the add-on placebo than add-on lithium group (n = 13 (7.1%) and n = 6 (3.5%), respectively). Psychiatric disorders, including mania, were the most common AE leading to discontinuation, reported in two patients in the add-on lithium and seven patients in the placebo add-on group. Serious adverse events (SAEs) included mania (n = 3 patients) and gastroenteritis (n = 1) in the add-on lithium group and mania (n = 6), aggression (n = 1), hostility (n = 1), and no therapeutic response (n = 1) in the add-on placebo group. The SAEs of emergent mania led to study discontinuation in two patients in the add-on lithium and six patients in the add-on placebo group. Incidences of TEAEs potentially related to EPS were 16.8% in the add-on lithium and 6.6% in the add-on placebo group, including tremor in 15.6% and 4.9% of patients, respectively. All these TEAEs were mild to moderate in intensity. Two TEAEs (tremor and dystonia) resulted in study discontinuation in the add-on placebo group. The majority of patients showed either improvement or no change in EPS severity during the study, assessed by SAS, AIMS, and BARS scores. For example, SAS score was improved in 11.6% and 8.2% of the add-on lithium and placebo-add groups, respectively, unchanged in 67.1% and 75.4% and worsened in 15.6% and 9.8%, respectively, at day 43. No patients initiated anticholinergic medication for new-onset EPS. No TEAEs potentially related to suicidality were reported during the study. Columbia-Suicide Severity Rating Scale assessment identified two patients with suicidal behavior and one with suicidal ideation in the add-on lithium group. Columbia-Suicide Severity Rating Scale and C-CASA assessments at end of treatment indicated that no patients had any type of suicidal ideation in either group. Rates of emergent depression were low in both add-on lithium (1.2%) and add-on placebo (0.5%) groups. Among other TEAEs of special interest, there were no reported events potentially related to diabetes mellitus, neutropenia, or QTc prolongation. No clinically meaningful between-group differences were reported in incidences of physical examination, vital signs, hematology, clinical chemistry, or urinalysis findings (Additional file 1: Table S2). There were no notable between-group differences in mean changes in liver function parameters, electrolytes, or lipids. The mean change in prolactin concentration was greater in the add-on lithium than add-on placebo group (mean (SD) –210.8 (488.1) mU/L vs. −116.9 (602.4) mU/L). A greater proportion of patients in the add-on lithium group had weight gain ≥7% at end of treatment compared with add-on placebo (8.0% vs. 4.7%). BODY.DISCUSSION: In this 6-week study, lithium as an add-on therapy to quetiapine XR was significantly more effective than add-on placebo for improving acute severe symptoms of bipolar mania, measured by change in YMRS total score at day 43. Reductions in YMRS score of 22.8 and 20.1, respectively, occurred in the add-on lithium and add-on placebo groups, with a clinically relevant between-group difference of 2.69 in favor of add-on lithium (p < 0.001). Consistent with this primary efficacy measure, the secondary efficacy measures showed that add-on lithium significantly improved a range of manic symptoms and the overall severity of illness compared with add-on placebo. The combination of quetiapine XR with lithium was generally well tolerated, and reported AEs were consistent with the known safety profiles of these medications in bipolar disorder (Bowden 2000; Cutler et al. 2011; Suppes et al. 2010). In agreement with studies of quetiapine IR (Emsley et al. 2005), worsening of glycemic control was not observed during quetiapine XR treatment combined with lithium or placebo. Incidences of potentially EPS-related TEAEs were also consistent with studies of quetiapine IR combined with lithium or divalproex (Suppes et al. 2009; Yatham et al. 2004). Tremor, a known side effect of lithium (Bowden 2000), occurred at higher rates in the add-on lithium than add-on placebo group. More patients in the add-on lithium than add-on placebo group also had weight gain ≥7% at the end of treatment. Add-on lithium was not associated with an altered rate of switch to depressive symptoms, which occurred at low incidences in both groups. The current study is unique in comparing quetiapine XR combined with lithium against quetiapine XR combined with placebo. Direct comparison with previous studies - which compared a mood stabilizer combined with quetiapine IR against a mood stabilizer combined with placebo - is not possible (Sachs et al. 2004; Suppes et al. 2009; Vieta et al. 2008a; Yatham et al. 2004), although all these studies indicate the superior efficacy of combination therapy versus monotherapy. In the earlier studies, quetiapine IR combined with a mood stabilizer was significantly more effective than mood stabilizer alone in acute mania (Sachs et al. 2004; Yatham et al. 2004) and in the prevention of recurrent mood disorder (Suppes et al. 2009; Vieta et al. 2008a). A post hoc analysis of the two maintenance studies (Suppes et al. 2009; Vieta et al. 2008a) demonstrated that quetiapine IR combined with lithium was associated with a 68% reduction in risk of recurrence of a mood event (manic, depressive, or mixed) compared with placebo and lithium (p < 0.001), while quetiapine combined with divalproex reduced recurrence risk by 72% compared with placebo and divalproex (p < 0.001) (Suppes et al. 2013). A second post hoc analysis of the maintenance studies showed that quetiapine combined with mood stabilizer (vs. mood stabilizer alone) significantly reduced the risk of recurrence of a mood event, whether the index episode was manic, depressive, or mixed (Vieta et al. 2012) (p ≤ 0.001, all). Quetiapine (IR or XR) in combination with lithium or divalproex is also associated with quality-adjusted life expectancy and cost-effectiveness benefits relative to maintenance treatment with mood stabilizers alone (Plosker 2012). Together, these findings may provide a rationale for initiating treatment with combined quetiapine XR and lithium in patients with severe acute manic symptoms, rather than escalating the dose of monotherapy up to the highest dose approved, and hence also for subsequent use of this combination as maintenance treatment to prevent recurrence. Notable features of this study include the initiation of medications (quetiapine XR and lithium or placebo) concurrently at the start of the study, which reflects management of severe acute mania in practice. To demonstrate efficacy for combination therapy in this setting is challenging, when compared with study of patients who show partial response to monotherapy. This study also required an inpatient status at randomization, which provides reassurance on the reliability of YMRS and other scoring at baseline. Direct comparison of the quetiapine XR and lithium combination versus other combination therapies in acute mania is hampered by the limited availability of other studies and differences in study design, duration, and patient population. Most studies report superior efficacy for combinations versus monotherapy (measured as symptom control, speed of onset, or relapse prevention), but also increased rates of AEs and related discontinuations (Geoffroy et al. 2012). For example, when compared with a mood stabilizer alone, combinations including a mood stabilizer and olanzapine or asenapine are associated with weight gain, combinations including aripiprazole lead to greater risk of akathisia, and combinations including quetiapine are associated with increased somnolence (Geoffroy et al. 2012; Sachs et al. 2004; Szegedi et al. 2012; Tohen et al. 2002; Vieta et al. 2008b; Yatham et al. 2004). While the quetiapine XR and lithium combination in the current study was associated with elevated rates of tremor and somnolence when compared with quetiapine alone, the combination was generally well tolerated and rates of treatment-related discontinuations were below add-on placebo-group levels. This study raises a number of questions that would require further investigation. Add-on lithium showed significant benefits despite a high proportion of patients (approximately 65%) having serum lithium levels outside the target range. The target lithium range selected (0.8 to 1.2 mEq/L) was based on evidence for the efficacy of lithium as monotherapy in bipolar disorder (Goodwin and Jamison 2007). Post hoc analyses in the current study reported greater efficacy (assessed as YMRS score change, response rate, and remission rate) for add-on lithium versus add-on placebo at a lithium level ≥6 mEq/L, but not <6 mEq/L. These data are consistent with an earlier study that reported significant efficacy against manic symptoms for lithium monotherapy versus placebo at higher (0.72 and 0.5 mEq/kg/day) but not lower (0.24 mEq/kg/day) lithium doses (Stokes et al. 1976). It may also be conjectured that the use of lower quetiapine doses in the add-on lithium than add-on placebo group (mean modal 623.1 mg/day vs. 669.9 mg/day) contributed to reduce the between-group difference in YMRS score. Investigators may have judged that there was no requirement to increase the quetiapine dose in a substantial proportion of patients due to the effective control of manic symptoms achieved by the addition of blinded lithium. Furthermore, the lower doses of quetiapine used in the add-on lithium group may have contributed to the beneficial side effect profile of combination therapy observed in this study. The study design excluded patients with a known lack of response to lithium or quetiapine, although only 1.7% and 7.0%, respectively, of the study population were treated with lithium and quetiapine previously. The benefits of combination therapy in lithium nonresponders are therefore unknown from this study, although other studies (Hardoy et al. 2005; Yatham et al. 2004) have suggested that lithium nonresponders may benefit from combination therapy with quetiapine and lithium. The pharmacological rationale for combining quetiapine and lithium also remains to be confirmed, although it may be conjectured that the mechanisms of action of quetiapine (Nemeroff et al. 2002; Nord et al. 2011) and lithium (Quiroz et al. 2010; Shim et al. 2012) are complementary and additive as combination therapy. BODY.CONCLUSIONS: Add-on lithium enhanced the efficacy of quetiapine XR in patients with acute bipolar I mania, without compromising safety and tolerability.

TITLE: Pioglitazone Attenuates Drug-Eluting Stent-Induced Proinflammatory State in Patients by Blocking Ubiquitination of PPAR ABSTRACT: The inflammatory response after polymer-based drug-eluting stent (DES) placement has recently emerged as a major concern. The biologic roles of peroxisome proliferator-activated receptor-γ (PPAR-γ) activators thiazolidinedione (TZD) remain controversial in cardiovascular disease. Herein, we investigated the antiinflammatory effects of pioglitazone (PIO) on circulating peripheral blood mononuclear cells (MNCs) in patients after coronary DES implantation. Methods and Results. Twenty-eight patients with coronary artery disease and who underwent DES implantations were randomly assigned to pioglitazone (30 mg/d; PIO) or placebo (control; Con) treatment in addition to optimal standard therapy. After 12 weeks of treatment, plasma concentrations of high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase-9 (MMP-9) were significantly decreased in PIO group compared to the Con group (P = 0.035, 0.011, 0.008, and 0.012, resp.). DES-induced mRNA expressions of IL-6, TNF-α, and MMP-9 in circulating MNC were significantly blocked by PIO (P = 0.031, 0.012, and 0.007, resp.). In addition, PIO markedly inhibited DES-enhanced NF-κB function and DES-blocked PPAR-γ activity. Mechanically, DES induced PPAR-γ ubiquitination and degradation in protein level, which can be totally reversed by PIO. Conclusion. PIO treatment attenuated DES-induced PPAR loss, NF-κB activation, and proinflammation, indicating that PIO may have a novel direct protective role in modulating proinflammation in DES era. BODY.1. INTRODUCTION: Polymer-based drug-eluting (everolimus, zotarolimus, sirolimus, paclitaxel, etc.) stents (DES) have been known as a standard treatment for coronary artery diseases (CAD) undergoing percutaneous coronary angioplasties. Although DES has demonstrated efficacy and safety in clinical studies, human pathological data have raised concerns about the long-term healing and the potential for local inflammatory reactions [1, 2]. Also, it has been reported that DES induced hypersensitivity reactions with interacting lymphocytes, macrophages, multinucleated giant cells, and eosinophils and pervasive inflammation throughout the stented arterial segment by autopsy [3]. DES-induced inflammatory reaction has been found as early as 30 days after implantation but progressed in frequency and severity through 90 to 180 days in porcine model [1]. Furthermore, our previous studies found that DES implantation induced specific systematic inflammatory state, as evidenced by the enhanced NF-κB activity, suppressed PPAR-γ activity, and elevated plasma inflammatory factors, compared with no-stent implantation or bare metal stent (BMS) implantation [4, 5]. However, how to refine DES-induced proinflammation remains unknown. In vascular wall and atherosclerosis, PPAR-γ is expressed in macrophages, T cells, endothelial cells, and vascular smooth muscle cells [6, 7]. Recent data have shown that synthetic antidiabetic thiazolidinediones (TZDs), which are known as PPAR-γ activators, inhibit inflammatory cytokine production by cells of monocyte/macrophage lineage [8]. These activators inhibit gene expression in part by antagonizing the activities of transcription factors such as activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) [9, 10]. Several animal studies have demonstrated the antiatherogenic effects of TZDs [11, 12]. Moreover, pioglitazone (PIO) significantly decreased the occurrence of all-cause mortality, nonfatal myocardial infarction, and stroke in diabetic populations in the Prospective Pioglitazone Clinical Trial in Macrovascular Events (PROactive) [13] and in a meta-analysis study [14]. However, a meta-analysis of rosiglitazone trials in diabetic patients showed that rosiglitazone was associated with increased myocardial infarction and cardiovascular death [15]. In an animal study, PIO was found to increase macrophage apoptosis and plaque necrosis in advanced lesions in LDLr-deficient mice [16]. Another animal study found that pioglitazone could induce excessive hepatic triglyceride accumulation and increase the plasma cholesterol [17]. These conflicting and controversial findings highlight the uncertainty regarding the effectiveness of using TZDs to treat atherothrombotic disease. Whether PIO have atheroprotective effects in DES-treated CAD patients remains unknown. Here, we performed a random single-blind placebo controlled clinical study to investigate whether PIO have anti-inflammatory effects in DES-implanted coronary artery disease patients. BODY.2. METHODS.2.1. PATIENTS AND STUDY DESIGN: All subjects were consecutively recruited from the First Affiliated Hospital of Xi'an Jiaotong University. 28 nondiabetic patients with coronary artery disease (CAD) and who underwent DES implantations were randomized into two groups: placebo group (Con, n = 14) and pioglitazone group (PIO, n = 14). The nondiabetic state was determined by a negative history of diabetes mellitus, no treatment with antidiabetic drugs, and assessment of fasting blood glucose and oral glucose tolerance test (OGTT). The diagnosis of CAD was in accorded with the WHO definition. The exclusion criteria included clinical evidence of acute inflammation, tumor and rheumatic condition checked by the elevated CRP and ESR, liver and renal diseases, severe heart failure (NYHA class ≥ II), ejection fraction (EF) < 50%, contraindications to treatment with pioglitazone, and patients who were given immunosuppressants. Table 1 summarized all subjects' demographic data. Study medication (30 mg/d) for 12 weeks was given in addition to optimal standard treatment, including aspirin, clopidogrel, β receptor blockers, angiotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs), and statins. We followed up the patients twice after 4 and 12 weeks. Pioglitazone and placebo were provided by Zhongmei Huadong Pharmaceutical Co., Ltd. (Hangzhou, China). We obtained each patient's medical and family history and general information such as smoking and drinking by medical history interview. This study complies with the Declaration of Helsinki, and the research protocol has been approved by the Ethics Committee of Xi'an Jiaotong University. The informed consents were obtained from the subjects. BODY.2. METHODS.2.2. MONONUCLEAR CELL (MNC) ISOLATION: Peripheral blood MNC samples before medication and at the end of the 12th week after DES stent implantation were collected. Peripheral blood MNC samples were isolated by Ficoll standard density gradient centrifugation. The upper layer containing MNC was harvested and washed with Hanks' balanced salt solution and then with PBS. BODY.2. METHODS.2.3. PLASMA CONCENTRATIONS OF PROINFLAMMATORY CYTOKINES: The concentrations of plasma interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinase-9 (MMP-9) were assayed by ELISA. ELISA was performed by adding 100 μL or 200 μL of each sample to wells in a 96-well plate of a commercially available ELISA Kit (ExCell, Shanghai) according to the manufacturer's instructions. High-sensitivity C-reactive protein (hs-CRP) assays were performed by our hospital's clinical centre laboratory. BODY.2. METHODS.2.4. TOTAL RNA ISOLATION AND MRNA DETECTION: Total RNA was extracted from MNC with the RNAfast kit (Fastgen) according to manufacturer's protocol, and real-time reverse transcription-polymerase chain reaction was performed as previous report [4]. BODY.2. METHODS.2.5. NF-: Nuclear proteins were extracted according to the manufacturer's instructions (Pierce). The NF-κB and PPAR-γ DNA binding activity were measured with NF-κB p65/p50 and PPAR-γ transcription factor assay kit (Abcam) according to the manufacturers' instructions. BODY.2. METHODS.2.6. WESTERN BLOTTING: Polyclonal or monoclonal antibodies (Santa Cruz Biotechnology) were used. Densitometry was performed with the Bio-Rad molecular analyst software, and all values were corrected by loading with Gapdh. BODY.2. METHODS.2.7. STATISTICAL ANALYSIS: Discrete variables were expressed as numbers and percentages and compared by the χ 2 test. Summary values are expressed as mean ± SE. Skewed data were reported as median (interquartile range). Analysis of the changes from baseline was performed by paired t-test. Holm-Sidak two-way repeated-measures ANOVA (TWRMANOVA) method was used for all multiple comparisons between the Con and PIO groups as previous reports [4, 5]. Statistical significance was assumed at the 5% α-error level (P < 0.05). BODY.3. RESULTS.3.1. GENERAL CLINICAL DATA: All 28 patients fulfilled the 12 weeks' follow-up without any drug related side effects. The characteristics of patients were shown in Table 1. There were no differences between the PIO and placebo groups with respect to baseline characteristics. After 12 weeks of treatment, there was no significant change in body mass index (BMI), weight, waist to hip ratio, blood pressure (SBP or DBP), fasting glucose and insulin, NT-pro BNP, and EF of the two groups compared to baseline (Table 2). Considering the unchanged BMI, weight, waist to hip ratio, NT-pro BNP, and EF between two groups, there was no evidence of PIO increasing the occurrence of heart failure in the present study. Total cholesterol, triglycerides, and LDL were reduced significantly after medication; however, no disparities of the reduction between two groups were observed. BODY.3. RESULTS.3.2. PIOGLITAZONE REDUCED THE PLASMA CONCENTRATIONS OF PROINFLAMMATORY CYTOKINES: In the control group, plasma concentrations of IL-6, TNF-α, and MMP-9 were increased by 138 ± 16%, 136 ± 15%, and 110 ± 7%, respectively, compared with the baseline level at 12 wk (paired t-test, P = 0.033, 0.041, and 0.142, resp., Figures 1(a)–1(c)), indicating DES-related proinflammatory status. In the PIO treatment group, plasma concentrations of IL-6, TNF-α, and MMP-9 were decreased by 89 ± 8%, 64 ± 16%, and 86 ± 6% compared with the baseline level at 12 wk (paired t-test, P = 0.204, 0.041, and 0.037, resp., Figures 1(a)–1(c)). Compared to the control group, PIO treatment significantly reduced the plasma IL-6, TNF-α, and MMP-9 concentrations (TWRMANOVA, P = 0.011, 0.008, and 0.012, resp.). Moreover, hs-CRP in both Con and PIO treatment groups were decreased significantly and more drastically in PIO treatment group (TWRMANOVA, P = 0.035, Table 2). BODY.3. RESULTS.3.3. PIOGLITAZONE DOWNREGULATED THE EXPRESSIONS OF PROINFLAMMATORY FACTORS IN MNC: To examine whether the reduction of plasma proinflammatory cytokines by PIO treatment might be associated with downregulation of the mRNA expressions of proinflammatory cytokines in MNC, quantitative real-time PCR was used to calculate the mRNA expression. In the Con group, the mRNA expressions of IL-6, TNF-α, and MMP-9 at 12 weeks' follow-up were increased compared with the baseline (paired t-test, P = 0.009, 0.025, and 0.105, resp., Figures 2(a)–2(c)). Compared with the Con group, PIO treatment significantly reduced the mRNA expressions of IL-6, TNF-α, and MMP-9 (TWRMANOVA, P = 0.031, 0.012, and 0.007 resp., Figures 2(a)–2(c)), indicating that PIO attenuate DES-induced proinflammatory factors expression in MNC. BODY.3. RESULTS.3.4. PIOGLITAZONE REGULATED THE DES-INDUCED NF-: To evaluate whether pioglitazone interacts with the proinflammatory transcription factor NF-κB in MNC, we measured the nuclear NF-κB DNA binding activity in MNCs (Figure 2(a)). As depicted in Figure 2(d), DES enhanced the DNA binding activity of NF-κB (P = 0.023), which were attenuated by PIO (TWRMANOVA, P = 0.017). Next, we detected the expressions of the p50 subunit and p65 subunit of NF-κB in MNCs. As shown in Figure 2(e), PIO treatment significantly reduced DES-induced p50 expression (TWRMANOVA, P = 0.017) but did not alter p65 expression. Moreover, we found that PPAR-γ were dramatically decreased in the DES group (P = 0.038, Figure 3(a)), which were reversed by PIO treatment (TWRMANOVA, P = 0.027, Figure 3(a)), suggesting that pioglitazone regulated the DES-induced NF-κB/PPAR-γ imbalance in vivo. BODY.3. RESULTS.3.5. PIOGLITAZONE REGULATED THE DES-INDUCED PPAR-: To refine the changes of protein and mRNA expressions of PPAR-γ, Western blot and quantitative real-time PCR were performed. As shown in Figure 3(b), DES markedly decreased PPAR-γ protein level, which were blocked by PIO treatment (TWRMANOVA, P = 0.031, Figure 3(b)). We also found that PIO did not alter PPAR-γ mRNA expression, suggesting that PIO may regulate the protein stability in vivo. Furthermore, we detected the ubiquitination of PPAR-γ by Western blot. As depicted in Figure 3(d), DES significantly induced PPAR-γ ubiquitination (P = 0.043), which was blocked by PIO, implying that pioglitazone regulated the DES-induced PPAR-γ ubiquitination and degradation in vivo. BODY.4. DISCUSSION: The present study clearly showed that the circulating inflammatory responses were increased after the implantation of DES in the CAD patients treated with optimal drug combinations, which were blocked by addition of oral pioglitazone. PIO sequentially acts through PPAR-γ activation, NF-κB blockade, and inhibition of inflammatory cytokine expressions. These findings suggested that PIO may have a novel direct protective role in modulating the proinflammatory responses after coronary DES implantation in CAD patients, thus providing further optimizations of the drug therapy in those patients. Vascular inflammation is recognized as the foundation mechanism of atherosclerosis, and proinflammatory mediators including IL-6, TNF-α, and MMP-9 play a pivotal role in atherosclerosis [18]. IL-6 and TNF-α are classic proinflammatory cytokines, which play key roles in vascular disease [19]. Excessive degradation and remodeling of the extracellular matrix, a promoter of the instability of plaques, are the major effect of matrix metalloproteinases (such as MMP-9) [20]. DESs left target vascular intima partially unendothelialized for a sustaining period [21], resulting in intensely inflammation-arousing vessel segments, which may facilitate the release of multiple proinflammatory factors into serum and then proinflammation. It has been reported that PIO may exhibit several antiatherosclerotic effects through multiple mechanisms, including modulation of blood pressure, lipid concentrations, matrix remodeling activation of matrix proteases, and finally induction of inflammation [22]. In the present study, we failed to observe a reduction of blood pressure, blood glucose, insulin, or HbA1c levels in both groups. Firstly, the unchangeable blood pressure and other metabolic parameters may be explained as follows: first, the blood pressure of all subjects was already under control by optimal treatment and the patients included in this study were all nondiabetics. Moreover, it has been reported that PIO treatment had no significant effect on the level of total cholesterol, LDL, and HDL. The improved lipid profile and hs-CRP are mainly attributed to the standard drug therapy. Herein, we found a novel mechanism that PIO enhance PPAR-γ binding activity though inhibiting its ubiquitination and degradation, which may play a key role in PPAR function in vivo [23]. Taken together, our study demonstrated that PIO treatment attenuated the proinflammatory state in circulating MNCs; and our results suggest that PIO treatment may sequentially act through PPAR-γ activation, blocking of NF-κB activation, and inhibition of inflammatory cytokine expressions. These findings suggest that PIO may have a novel direct atheroprotective role by modulating the local and circulating proinflammatory responses in patients with coronary polymer-based drug-eluting stent implantation.

TITLE: A multicentre, randomised, controlled trial to assess the safety, ease of use, and reliability of hyaluronic acid/carboxymethylcellulose powder adhesion barrier versus no barrier in colorectal laparoscopic surgery ABSTRACT.BACKGROUND: Intra-peritoneal adhesions are frequent following abdominal surgery and are the most common cause of small bowel obstructions. A hyaluronic acid/carboxymethylcellulose (HA/CMC) film adhesion barrier has been shown to reduce adhesion formation in abdominal surgery. An HA/CMC powder formulation was developed for application during laparoscopic procedures. ABSTRACT.METHODS: This was an exploratory, prospective, randomised, single-blind, parallel-group, Phase IIIb, multicentre study conducted at 15 hospitals in France to assess the safety of HA/CMC powder versus no adhesion barrier following laparoscopic colorectal surgery. Subjects ≥18 years of age who were scheduled for colorectal laparoscopy (Mangram contamination class I‒III) within 8 weeks of selection were eligible, regardless of aetiology. Participants were randomised 1:1 to the HA/CMC powder or no adhesion barrier group using a centralised randomisation list. Patients assigned to HA/CMC powder received a single application of 1 to 10 g on adhesion-prone areas. In the no adhesion barrier group, no adhesion barrier or placebo was applied. The primary safety assessments were the incidence of adverse events, serious adverse events, and surgical site infections (SSIs) for 30 days following surgery. Between-group comparisons were made using Fisher's exact test. ABSTRACT.RESULTS: Of those randomised to the HA/CMC powder (n = 105) or no adhesion barrier (n = 104) groups, one patient in each group discontinued prior to the study end (one death in each group). Adverse events were more frequent in the HA/CMC powder group versus the no adhesion barrier group (63% vs. 39%; P <0.001), as were serious adverse events (28% vs. 11%; P <0.001). There were no statistically significant differences between the HA/CMC powder group and the no adhesion barrier group in SSIs (21% vs. 14%; P = 0.216) and serious SSIs (12% vs. 9%; P = 0.38), or in the most frequent serious SSIs of pelvic abscess (5% and 2%; significance not tested), anastomotic fistula (3% and 4%), and peritonitis (2% and 3%). ABSTRACT.CONCLUSIONS: This exploratory study found significantly higher rates of adverse events and serious adverse events in the HA/CMC powder group compared with the no adhesion barrier group in laparoscopic colorectal resection. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT00813397. Registered 19 December 2008. BODY.BACKGROUND: Intra-peritoneal adhesions are estimated to occur after 93% to 100% of upper abdominal laparotomies and after 67% to 93% of lower abdominal laparotomies [1]. Adhesions form as a result of surgical trauma or infection/inflammation, and comprise fibrous scar tissue that abnormally connects tissues and organs [2]. They are the most common cause of small bowel obstructions [3–6] and are associated with infertility and possibly chronic pain. Adhesions may also prolong operating time in subsequent surgery, and cause complications such as unintentional enterotomy [2, 6–8]. There is no effective treatment for adhesions, and surgery to deal with the consequences of adhesions, such as small bowel obstruction, often results in further adhesion formation [1, 2]. Adhesion prevention should therefore be considered the best management strategy [2, 9], although this is not widely demonstrated in the literature. A good surgical technique (e.g., minimal tissue trauma, avoiding introduction of foreign materials) can reduce adhesion formation, but is not sufficient for prevention [2, 9]. Adhesions may be reduced by using laparoscopy versus open surgery [10–12], which results in a reduction in adhesion-related complications, such as small bowel obstruction, but does not totally prevent adhesion formation [6]. A number of anti-adhesion products are available for adjuvant use during surgery, with various formulations including films, fabrics, gels, and fluids. These products act as a temporary mechanical barrier to separate organs and tissues for a short time while healing takes place. Despite the number of agents available worldwide, few have demonstrated efficacy in reducing post-surgical adhesions, and limited conclusions can be drawn on the effect of reducing adhesions, as no agent has been shown to improve the myriad negative outcomes commonly associated with adhesions [13]. In abdominal surgery, a hyaluronic acid/carboxymethylcellulose (HA/CMC) film adhesion barrier has been shown to reduce adhesion formation with a favourable safety profile [13–17]. A powder formulation of HA/CMC (HA/CMC powder; SeprasprayTM Adhesion Barrier, Genzyme Corp., Cambridge, MA, USA) was developed for application during laparoscopic procedures. Preclinical animal models indicated that this formulation was effective in reducing adhesion formation and did not disrupt normal wound healing [18, 19]. In a randomised pilot study in women undergoing laparoscopic myomectomy, there was a trend towards a reduction in adhesions with HA/CMC powder versus no adhesion barrier, with a favourable safety profile [20]. The primary objective of this study was to assess the safety of HA/CMC powder versus no adhesion barrier following laparoscopic colorectal and/or small bowel surgery (high risk for morbidity [21]), as determined by the incidence of adverse events, serious adverse events, superficial surgical site infections (SSIs) and deep SSIs, such as fistula, sepsis, abscess, and peritonitis. BODY.METHODS: This was an exploratory, prospective, randomised, single-blind, parallel-group, Phase IIIb, multicentre study conducted at 15 hospitals in France (ClinicalTrials.gov Identifier: NCT00813397). The study comprised a selection visit 1 to 56 days prior to the planned surgery (Day 0) and two postoperative follow-up assessments (day of discharge or 7 ± 3 days post-surgery, and end-of-study assessment 28 to 35 days post-surgery). This follow-up duration was based on postoperative guidelines on infections [22] and the knowledge that HA/CMC is resorbed from the peritoneal cavity within 7 days and fully eliminated from the body in <28 days [23]. The study was carried out in compliance with the Declaration of Helsinki and the principles of the French Good Clinical Practice regulations/clinical research guidelines. The protocol and patient consent forms were approved by an independent ethics committee (reference number 208 R09, Sud-Méditerranée II, Marseille, France). All patients signed an informed consent form. An independent review committee of four independent experts was established to provide real-time expert review of safety reports and assess all safety data at study end. BODY.METHODS.PARTICIPANTS: Men and women ≥18 years of age were eligible if they were scheduled to undergo a laparoscopic colorectal and/or small intestine surgical resection of Mangram contamination class I, II, or III [22] within 8 weeks of selection, whatever the aetiology (including cancer). Participants were also required to have an American Society of Anesthesiologists Physical Status Classification of P1, P2, or P3 and women of childbearing potential were required to use an effective contraceptive method for 1 month after randomisation. The principal exclusion criteria were cancer requiring chemotherapy and/or radiotherapy within 30 days prior to or after surgery; current abdominal abscess and/or peritonitis; pregnancy; clinically significant cardiovascular, hepatic, neurologic, psychiatric, endocrine, or other major systemic disease that would interfere with the study or jeopardize patient outcomes within 30 days; and treatment with heparinic anticoagulants (except prophylaxis for deep vein thrombosis). Additional exclusion criteria applied at the time of surgery were use of another medical device that may interfere with the study (e.g., prosthetic stitch, biological adhesive, haemostatic compress, surgical membrane, or physical barrier to prevent adherence); infection discovered in the abdominal cavity; change to Mangram contamination class IV; and conversion to laparotomy (although mini laparotomy was permitted, based on standard laparoscopic approaches in colorectal surgery). BODY.METHODS.TREATMENT: Participants were randomised to the HA/CMC powder or no adhesion barrier group during the planned surgical procedure (Day 0) in a 1:1 ratio using a centralised randomisation list and an automated Interactive Voice Response System, following re-evaluation of their eligibility. Patients were blinded to their randomisation group throughout the study. Patients assigned to HA/CMC powder received a single application to adhesion-prone areas but not to anastomoses or sutures. HA/CMC powder was applied with a single-use applicator attached to a sprayer to allow precise application to the required sites while minimizing potential dispersion to other sites. The amount applied was at the surgeon's judgement, and ranged from 1 to 10 g, with the maximum determined based on a previous study of HA/CMC film [24]. In patients assigned to the no adhesion barrier group, no adhesion barrier or placebo was applied. In both groups, closure of trocar sites was performed according to the normal routine of the surgeon; peritoneal closure was not performed. BODY.METHODS.SAFETY ASSESSMENTS: The primary objective of this study was to compare the incidence of adverse events, serious adverse events, superficial (incisional) SSIs, and deep SSIs in the HA/CMC powder and no adhesion barrier groups for 30 days following surgery. Any suspected intra-abdominal abscess was investigated by CT-scan/MRI. Any suspicion of sepsis was confirmed by at least one positive blood culture and specific symptoms. Any suspicion of peritonitis was confirmed by positive bacterial cultures from peritoneal swabs of drainage during interventional radiology or surgery. All such SSIs were followed up until resolution. In the HA/CMC powder group, the relationship of adverse events to treatment was judged by investigators as not related, unlikely, possibly, probably, or definitely related. Events that were routinely observed during the postoperative period (e.g., pain, nausea, vomiting) were not reported as an adverse event unless they occurred with unusual severity according to the investigator's judgement, or required unusual or specific management. The duration of hospitalisation after surgery was noted along with intraoperative parameters and perioperative parameters. Exposure to HA/CMC powder was determined from the amount of powder applied (grams) and duration of application (minutes). BODY.METHODS.EASE OF USE, MANAGEABILITY, AND RELIABILITY ASSESSMENTS: Ease of use was evaluated by the surgeon using a 4-point scale (1 = very difficult, 2 = difficult, 3 = easy, 4 = very easy). HA/CMC powder was considered manageable if this item was scored 3 or 4. The ease of attaching the applicator to the sprayer and introducing the HA/CMC powder into the sprayer was assessed by the nurse in charge of surgical instruments in the operating room using the same 4-point scale. HA/CMC powder was considered manageable if both items were scored 3 or 4. Reliability was assessed by the surgeon based on the ability to cover all target areas, apply a homogeneous layer, and deliver the suitable amount of HA/CMC powder using a 4-point scale (1 = very bad, 2 = bad, 3 = good, 4 = very good), with HA/CMC powder being considered reliable if each item was scored 3 or 4. BODY.METHODS.STATISTICAL ANALYSIS: As the study was exploratory, no sample size calculation was performed. Safety analyses were performed on the safety population, comprising all randomised patients who underwent surgery. Surgical and ease of use analyses were performed on the intent-to-treat population, described as all randomised patients who received treatment during surgery as determined by randomisation. Safety data are reported in summary tables and between-group comparisons made using Fisher's exact test. Descriptive statistics are provided for ease of use, manageability, and reliability. A post-hoc analysis of risk factors for deep SSIs and serious adverse events was carried out. Logistic regression was performed for each covariate. The univariate model included treatment effect, covariate effect, and the effect for the interaction between treatment and the covariate. Covariates included gender, age, body mass index, smoking status, smoking frequency, use of drains, adhesiolysis, and type of anastomosis, among others. A significance level of 0.10 (deep SSIs) or 0.20 (serious adverse events) was used to select the individual covariates to be included in the final multivariate model for the backwards stepwise logistic regression analysis. The odds ratio (OR) and 95% confidence intervals (CI) were calculated. BODY.METHODS.ROLE OF THE SPONSOR: The study sponsor was involved in the design of the study. Data were collected by investigators at each site. Patient randomisation and data management were carried out by a research organisation contracted by the study sponsor. Together with the study investigators, the sponsor participated in the analysis and interpretation of data, the writing of the manuscript, and the decision to submit the manuscript for publication. BODY.RESULTS: The study was carried out between September 2008 and July 2009. Patient disposition is summarized in Figure 1. Of 209 patients randomised to the HA/CMC powder (n = 105) or the no adhesion barrier (n = 104) groups, only one patient in each group discontinued prior to study end (one death in each group). Patient characteristics and medical history were similar (Table 1), except for a lower frequency of patients with concomitant allergic diseases (P = 0.031) and a higher frequency of patients taking concomitant penicillin combinations (significance not tested) in the HA/CMC powder group versus no adhesion barrier. Previous or concomitant immunosuppressive drugs were taken by 7/105 (7%) patients in the HA/CMC powder group and 12/104 (12%) patients in the no adhesion barrier group (P = 0.221), and corticosteroids by 15/105 (14%) and 22/104 (21%) patients, respectively (P = 0.193). Within the year prior to study randomisation (but not within 30 days before surgery), 10/105 (19%) of patients in the HA/CMC powder group and 7/104 (15%) in the no adhesion barrier group had received abdominal or pelvic radiotherapy (P = 0.666), and 9/105 (17%) and 8/104 (17%), respectively, had received chemotherapy (P = 0.958). Intraoperative parameters were comparable between groups (Table 2), although adhesiolysis (P = 0.073) and manual anastomosis (P = 0.034) were performed more frequently in the HA/CMC powder group.Figure 1 Patient disposition. ITT, intent-to-treat. Table 1 Patient characteristics and medical history at baseline (intent-to-treat population) HA/CMC powder (n = 105) No adhesion barrier (n = 104) P value Age in years, mean ± SD 57.6 ± 16.3 56.1 ± 16.5 0.531 Men, n (%) 53 (50.5) 51 (49.0) 0.835 BMI in kg/m 2 , mean ± SD 24.7 ± 4.0 24.3 ± 4.2 0.451 Smoking history, n (%) 0.676  Current smoker 16 (15.8) 18 (17.8)  Ex-smoker 19 (18.8) 23 (22.8)  Non-smoker 66 (65.3) 60 (59.4) Previous abdominal/pelvic surgery, n (%) 58 (55.2) 61 (58.7) 0.618 Diabetes, n (%) 10 (9.6) 5 (4.8) 0.180 Pre-operative diagnosis, n (%) 0.566  Cancer 48 (45.7) 44 (42.3)  Diverticulosis 27 (25.7) 36 (34.6)  Crohn’s disease 11 (10.5) 7 (6.7)  Ulcerative colitis 5 (4.8) 7 (6.7)  Polyp (no cancer) 5 (4.8) 5 (4.8)  Other 5 (4.8) 1 (1.0)  Endometriosis 2 (1.9) 3 (2.9)  Polyposis 2 (1.9) 1 (1.0) ASA Physical Status classification, n (%) 0.537  P1 37 (35.2) 41 (39.4)  P2 60 (57.1) 52 (50.0)  P3 8 (7.6) 11 (10.6) Concomitant diseases (≥20% patients in either group), n (%)  Gastrointestinal, hepatic 46 (43.8) 39 (37.5) 0.353  Cardiovascular 31 (29.5) 35 (33.7) 0.521  Metabolic, endocrine, nutritional 38 (36.2) 26 (25.0) 0.079  Allergic 18 (17.1) 31 (29.8) 0.031 Concomitant medications (≥10% patients in either group), n (%)  Heparin 51 (48.6) 47 (45.2) ND  Combination of penicillins* 17 (16.2) 9 (8.7) ND *Including β-lactamase inhibitors. ASA, American Society of Anesthesiologists; BMI, Body mass index; ND, Not determined. Table 2 Intra - operative parameters (intent-to-treat population) HA/CMC powder (n = 105) No adhesion barrier (n = 104) Adhesiolysis, n (%) 36 (34.3) 24 (23.1) Type of resection, n (%)   Sigmoidectomy and/or left colectomy 44 (41.9) 50 (48.1)   Proctectomy 21 (20.0) 25 (24.0)   Right ileocolectomy 31 (29.5) 20 (19.2)   Total proctocolectomy 5 (4.8) 3 (2.9)   Abdominoperineal amputation 2 (1.9) 2 (1.9)   Total colectomy 1 (1.0) 3 (2.9)   Sigmoidectomy and right ileocolectomy 0 1 (1.0)   Left colectomy and small intestine resection (ileum) 1 (1.0) 0 Anastomosis, n (%) 102 (97.1) 99 (95.2)   Manual 28 (27.5)* 15 (15.2)   Mechanical 74 (72.5) 84 (84.8) Stomy, n (%) 27 (25.7) 23 (22.1) Partial omentectomy, n (%) 12 (11.4) 12 (11.5) Classification of surgical area during surgery, n (%)   Clean – contaminated 104 (99.0) 104 (100)   Contaminated 1 (1.0) 0 (0) Exeresis of other organs, n (%) 8 (7.6) 8 (7.7) * P = 0.034. There were no differences between the HA/CMC powder and no adhesion barrier groups in perioperative findings (Table 3) except for number of drains used (P = 0.021) and mean duration of surgery (P = 0.419).Table 3 Summary of perioperative parameters HA/CMC powder (n = 105)No adhesion barrier (n = 104)Estimated blood loss in mL, mean ± SD116.5 ± 197.781.6 ± 95.8Administration of blood products, n (%)7 (6.7)3 (2.9)Blood sediments in units, mean ± SD2.0 ± 1.01.7 ± 0.6Perioperative lavage, n (%)73 (69.5)73 (70.2) Intraperitoneal58 (79.5)54 (74.0) Intraluminal4 (5.5)2 (2.7) Intraperitoneal and intraluminal11 (15.1)17 (23.3)Type of intraperitoneal lavage, n (%) Localized to intervention site52 (76.5)54 (77.1) Whole cavity16 (23.5)16 (22.9)Use of povidone-iodine antiseptic irrigation, n (%)19 (18.6)23 (22.8) Intraperitoneal4 (21.1)5 (21.7) Intraluminal12 (63.2)14 (60.9) Intraperitoneal and intraluminal3 (15.8)4 (17.4)Drain, n (%)50 (47.6)49 (47.1) Vacuum, n (%)37 (74.0)37 (75.5) Number of drains, mean ± SD1.1 ± 0.3*1.6 ± 1.5Surgery duration in minutes, mean ± SD216.2 ± 87.1203.2 ± 81.6Postoperative oxygen therapy, n (%)69 (65.7)56 (53.8)*P = 0.021. BODY.RESULTS.SAFETY OUTCOMES: Adverse events, serious adverse events, and deaths are summarized in Table 4. One patient in each group had an adverse event leading to death: septic shock stemming from aspiration pneumonia in the HA/CMC powder group, and septic shock and multiorgan failure in the no adhesion barrier group; for the patient in the HA/CMC powder group, the investigator assessed the adverse event as not related to treatment.Table 4 Summary of deaths, adverse events, and serious adverse events (events occurring on or after the day of surgery are shown by preferred term) HA/CMC powder (n = 105)No adhesion barrier (n = 104)Deaths1 (1.0)1 (1.0)Any adverse event*, n (%)66 (62.9)† 41 (39.4) Any adverse event considered severe, n (%)§ 14 (13.3)5 (4.8) Most frequently reported adverse events, n (%)§ Hyperthermia6 (5.7)3 (2.9) Incision site abscess5 (4.8)3 (2.9) Pelvic abscess5 (4.8)2 (1.9) Urinary tract infection5 (4.8)1 (1.0) Anastomotic fistula4 (3.8)4 (3.8) Abdominal wall abscess4 (3.8)2 (1.9) Ileus3 (2.9)2 (1.9) Urinary retention3 (2.9)1 (1.0)At least 1 serious adverse event, n (%)29 (27.6)† 11 (10.6) Any serious adverse event considered severe, n (%)§ 9 (8.6)3 (2.9) Serious adverse events occurring in ≥2 patients in either group, n (%)§ Pelvic abscess5 (4.8)2 (1.9) Abdominal abscess4 (3.8)0 Septic shock1 (1.0)2 (1.9) Peritonitis2 (1.9)3 (2.9) Ileus3 (2.9)0 Anastomotic fistula3 (2.9)4 (3.8) Gastrointestinal stoma complication2 (1.9)0*All adverse events coded according to the Medical Dictionary for Regulatory Activities version 11.0. † P <0.001 vs. no adhesion barrier group. §Statistical significance not tested. The overall frequency of adverse events was significantly higher in the HA/CMC powder versus the no adhesion barrier group (66/105 [63%] vs. 41/104 [39%]; P <0.001). The overall frequency of serious adverse events was significantly higher in the HA/CMC powder group compared with the no adhesion barrier (P <0.001). Overall, 10/105 (10%) patients in the HA/CMC powder group experienced at least one adverse event that was considered by the investigator as possibly, probably, or definitely related to the investigational product: abdominal pain (n = 2), flatulence (n =2), ileus (n = 1), impaired gastric emptying (n = 1), intestinal obstruction (n = 1), abdominal abscess (n = 2), intestinal abscess (n = 1), incision site abscess (n = 1), anastomotic fistula (n = 1), incision site haemorrhage (n = 1), and postoperative ileus (n = 1). The frequency of treatment-related serious adverse events in the HA/CMC powder group was 4% (4/105 patients), and these were ileus (n = 1) and the SSIs of abdominal abscess (n = 2), intestinal abscess (n = 1), incision site abscess (n = 1), and anastomotic fistula (n = 1). As this was a single-blind study, the relationship to treatment could not be reported in the no adhesion barrier group. There was no relationship between the amount of HA/CMC powder used and incidence of adverse events or serious adverse events (data not shown). At least one SSI was experienced by 22/105 (21%) of patients in the HA/CMC powder group versus 15/104 (14%) in the no adhesion barrier group (P = 0.216), and at least one serious SSI by 13/105 (12%) versus 9/104 (9%), respectively (P = 0.38; Table 5). There were no numeric differences between the HA/CMC powder and the no adhesion barrier groups in the most frequently reported serious SSIs of pelvic abscess (4.8% and 1.9%, respectively), anastomotic fistula (2.9% and 3.8%), and peritonitis (1.9% and 2.9%; statistical significance not tested).Table 5 Overall frequency of SSIs and serious SSIs, and listing of all SSIs HA/CMC powder (n = 105)No adhesion barrier (n = 104) PvalueAt least 1 SSI, n (%)22 (21.0)15 (14.4)0.216 Deep13 (12.4)8 (7.7)0.260 Incisional13 (12.4)7 (6.7)0.165At least 1 serious SSI, n (%)13 (12.4)9 (8.7)0.380 Deep12 (11.4)8 (7.7)0.359 Incisional2 (1.9)1 (1.0)1.000All SSIs, n (%) Infections and infestations20 (19.0)11 (10.6)– Incision site abscess5 (4.8)3 (2.9)– Pelvic abscess5 (4.8)2 (1.9)– Abdominal wall abscess4 (3.8)2 (1.9)– Abdominal abscess4 (3.8)0– Incision site infection1 (1.0)2 (1.9)– Abscess intestinal1 (1.0)1 (1.0)– Abdominal infection1 (1.0)0– Bacteraemia1 (1.0)0– Postoperative abscess1 (1.0)0– Septic shock01 (1.0)– Subcutaneous abscess1 (1.0)0– Injury, poisoning, and procedural complications6 (5.7)4 (3.8)– Anastomotic fistula4 (3.8)4 (3.8)– Gastrointestinal anastomotic leak1 (1.0)0– Incision site complication1 (1.0)0– Gastrointestinal disorders2 (1.9)5 (4.8)– Peritonitis2 (1.9)3 (2.9)– Colonic fistula01 (1.0)– Gastrointestinal inflammation01 (1.0)–Shown by system organ class and preferred term.–, Statistical significance not tested.SSI, Surgical site infection. In the HA/CMC powder group, the mean ± SD amount of powder applied was 2.7 ± 1.4 g, with 40% of patients receiving 4 to 6 g, 37% receiving 2 to 3 g, and 23% receiving only 1 g. The mean ± SD duration of application was 5.6 ± 3.4 min. The mean ± SD duration of hospitalisation after surgery was 9.7 ± 6.3 days in the HA/CMC powder group compared with 7.5 ± 3.4 days in the no adhesion barrier group (P = 0.009). Adverse events by preoperative diagnosis are summarized in Table 6. A greater frequency of adverse events among patients with cancer was observed than in patients with other diagnoses in both groups; however, the rate of overall adverse events and serious adverse events was higher in the HA/CMC powder group than in the no adhesion barrier group in patients with cancer (33% vs. 17% and 16% vs. 6%, respectively; significance not tested). Similarly, adverse events were more frequent in the HA/CMC powder group versus the no adhesion barrier group in patients with Crohn's disease and ulcerative colitis (Table 6; significance not tested).Table 6 Frequency of adverse events, serious adverse events, and serious SSIs by preoperative diagnosis Preoperative diagnosisHA/CMC powder (n = 105)No adhesion barrier (n = 104)Cancer, n (%) Adverse event35 (33.3)18 (17.3) Serious adverse event17 (16.2)6 (5.8) Serious SSI4 (3.8)5 (4.8)Diverticulosis, n (%) Adverse event11 (10.5)12 (11.5) Serious adverse event2 (1.9)2 (1.9) Serious SSI2 (1.9)2 (1.9)Crohn's disease, n (%) Adverse event7 (6.7)2 (1.9) Serious adverse event4 (3.8)1 (1.0) Serious SSI4 (3.8)1 (1.0)Ulcerative colitis, n (%) Adverse event5 (4.8)4 (3.8) Serious adverse event3 (2.9)1 (1.0) Serious SSI2 (1.9)0Polyp (no cancer), n (%) Adverse event4 (3.8)4 (3.8) Serious adverse event1 (1.0)1 (1.0) Serious SSI00Endometriosis Adverse event1 (1.0)1 (1.0) Serious adverse event1 (1.0)0 Serious SSI1 (1.0)0Polyposis Adverse event1 (1.0)0 Serious adverse event00 Serious SSI01 (1.0)Other Adverse event2 (1.9)0 Serious adverse event1 (1.0)0 Serious SSI00SSI, Surgical site infection. Subgroup analysis also indicated a greater frequency of adverse events among patients who had undergone previous abdominal/pelvic surgery than in those who had not in both groups; however, the rate of adverse events and serious adverse events was higher in the HA/CMC powder group than in the no adhesion barrier group, regardless of previous abdominal/pelvic surgery status (data not shown). BODY.RESULTS.RISK FACTOR ANALYSIS: For the serious adverse events risk factor analysis, the covariates included were age, smoking status, smoking frequency, use of corticoids, surgical risk (National Nosocomial Infections Surveillance Index; NNIS), previous cancer, fluorouracil used during the most recent chemotherapy and abdominal or pelvic radiation therapy administration. The probability of a serious adverse event was greater in the HA/CMC powder versus the no adhesion barrier group (OR = 4.08; 95% CI, 1.67–9.95; P = 0.002), in younger patients (for age in years, OR = 0.94; 95% CI, 0.91–0.98; P = 0.002), and in patients who smoked frequently (OR = 1.06; 95% CI, 1.02–1.10; P = 0.006). The probability of a serious adverse event was also greater in patients with a higher level of surgical risk: NNIS index 0 vs. ‒1 (OR = 1.33; 95% CI, 0.53–3.36; P = 0.027) and NNIS index 1 vs. ‒1 (OR = 9.87; 95% CI, 1.82–53.53; P = 0.027), in those with previous cancer (OR = 3.46; 95% CI, 1.02–11.70; P = 0.046) and in those having used fluorouracil during their last chemotherapy (OR = 7.12; 95% CI, 1.52–33.42; P = 0.013). For the deep SSIs risk factor analysis, age and smoking frequency were included as covariates. The probability for a deep SSI was greater in younger patients (for age in years OR = 0.97; 95% CI, 0.94–1.00; P = 0.0362) and in patients who frequently smoked (OR = 1.05; 95% CI, 1.01–1.09; P = 0.010). No significant effect of treatment was observed (OR = 0.58; 95% CI, 0.22–1.53; P = 0.269). BODY.RESULTS.EASE OF USE AND RELIABILITY: HA/CMC powder was considered to be manageable by nurses in 98% (103/105) of procedures, based on ease of use assessment (Figure 2A). Surgeons considered HA/CMC powder manageable and reliable in 94% (99/105) and 79% (83/105) of procedures, respectively (Figure 2A,B).Figure 2 Ease of use and reliability. (A) Ease of use of HA/CMC powder, as assessed by surgeons and nurses; (B) Reliability of HA/CMC powder, as assessed by surgeons. Overall manageability/reliability rates determined by overall number of cases scoring 3 or 4 on a 4-point scale (3 = easy or good, 4 = very easy or very good). For multi-component items, all were required to achieve a score of 3 or 4. BODY.DISCUSSION: In this exploratory study evaluating safety outcomes, there were statistically significant differences between the HA/CMC powder and the no adhesion barrier groups in the frequency of adverse events and serious adverse events (P <0.001). The occurrence of abdomino-pelvic abscess was more frequent in the HA/CMC powder group than in the no adhesion barrier group, although there were no statistically significant differences between groups in the frequency of SSIs or serious SSIs. Surgeons considered HA/CMC powder easy to use and reliable, and nurses also considered HA/CMC powder to be manageable in the majority of procedures. Based on the observed safety findings, the use of HA/CMC powder is no longer being pursued in colorectal laparoscopic surgery. The most frequent types of adverse events were those often encountered in patients undergoing colorectal or intestinal resection ('infections and infestations' and 'gastro-intestinal disorders'). Among the adverse events and serious adverse events that were considered by investigators to be related to treatment, there was not one particular type of event or safety issue that was more frequently reported than others. Frequency appeared to be independent of the quantity of HA/CMC powder applied. There was a trend towards a higher frequency of adverse events in patients with cancer versus other aetiologies, and a higher frequency of adverse events in the HA/CMC powder group versus the no adhesion barrier group in patients with cancer and those with inflammatory pathologies, although the study was not powered to detect a significant difference for such a subgroup analysis. As this was an exploratory study, no sample size calculation was made, but the enrolment of approximately 100 patients per group would have provided a 95% CI of 25.6 to 44.4 for the detected difference, based on an expected overall rate of serious adverse events of 35% [21]. Other studies report that SSIs occur following colorectal surgery with a frequency ranging from 5% to 45% [25–33]; the frequency of SSIs observed in this study falls within this range. The risk factor analysis in our study found no significant effect of treatment on risk for deep SSIs. Factors that appeared to be associated with a greater risk were lower age and frequent smoking. There was a significant treatment effect on risk of serious adverse events; other factors that appeared to be associated with a greater risk were lower age, frequent smoking, high level of surgical risk, previous cancer, and having used fluorouracil during the last chemotherapy. However, given the exploratory design of this study, these results must be considered with caution. The findings of our study were unexpected given that no safety issues were identified in a pilot study by Fossum et al. assessing the use of HA/CMC powder in laparoscopic myomectomy [20]. In their study, Fossum et al. observed no overall difference in adverse event frequency between the HA/CMC powder and the no adhesion barrier groups (67% vs. 60%, respectively), and only one patient experienced a serious adverse event (leukaemia, in the no adhesion barrier group). No adverse events directly related to HA/CMC powder were identified, as determined by the surgeon, and there were no reports of SSIs or intra-abdominal abscess. Furthermore, in animal models, HA/CMC powder has been shown to be effective in preventing adhesions without affecting wound healing [18, 19]. In a rat model of anastomotic healing, there were no statistically significant differences between the HA/CMC powder and the no adhesion barrier groups in number of deaths or short-term complications of abscess formation, bowel obstruction, proximal colonic dilatation, and wound dehiscence [19]. In the present study, an important consideration was that the patient population enrolled had a high level of comorbidity and a high probability of postoperative morbidity. This population was intended to be heterogeneous, comprising a broad range of patients undergoing various abdominal surgeries for a variety of diagnoses, including cancer. The risk of postoperative infection is high in patients undergoing gastrointestinal surgery as a result of opening the bowel, whereas gynaecologic surgery is usually a 'clean' (Class I) wound in comparison [34]. Although direct comparison with other studies is not possible, the overall rate of adverse events in the no adhesion barrier group in this study (39%) was similar to overall postoperative comorbidity rates in prospective studies of open or laparoscopic colorectal surgery (~25% to 35%) [21, 35]. However, this does not explain the significantly higher frequency of adverse events and serious adverse events in the HA/CMC powder group versus the no adhesion barrier group. Intraoperative and perioperative parameters that differed between groups were adhesiolysis and manual anastomosis (both performed more frequently in the HA/CMC powder group), number of drains (lower in the HA/CMC powder group), and mean duration of surgery (longer in the HA/CMC powder group). As the safety of the film formulation has been confirmed in clinical studies of gynaecological and abdominal surgery [13, 14, 36, 37], the powder formulation of HA/CMC used in this study is likely to be an important contributing factor for the increased frequency of adverse events and serious adverse events. One study noted that wrapping a new bowel anastomosis with HA/CMC film adhesion barrier increased the risk of anastomotic leak and related events such as fistula, peritonitis, abscess, and sepsis [24], possibly by interfering with anastomosis healing; this practice is therefore contraindicated. For adhesion barriers with a liquid or gel formulation, greater diffusion of product across the peritoneal surface may play a role in increasing adverse event risk as a result of application occurring away from the wound site. Gels may also have a propensity to pool away from the wound site. A study of 0.5% ferric hyaluronate gel adhesion barrier in open colorectal surgery was suspended owing to significantly greater morbidity versus the control group (distilled water, 65% vs. 27%; P = 0.031). There was a higher rate of anastomotic dehiscence (5/17 vs. 1/15, respectively; not significant) [34] but investigators were unable to determine whether anastomotic healing was disrupted directly by the adhesion barrier or by infection associated with gel use. Furthermore, icodextrin 4% fluid is indicated for use only in patients undergoing gynaecological laparoscopic adhesiolysis in the USA owing to occurrence of serious complications following laparotomy and bowel resection/repair [38]. Pre-clinical data suggested that HA/CMC powder demonstrated more rapid dissolution and wider diffusion than HA/CMC film [19], with a greater capacity for fluid absorption and release of a higher concentration of polymer into solution within the first few hours of hydration than the equivalent amount of HA/CMC film [18]. As application of HA/CMC film to anastomoses is contraindicated owing to the potential for an increase in anastomotic leak-related events [24], great care was taken in our study to practise avoidance of anastomoses with HA/CMC powder. Nevertheless, due to the potential for greater diffusion of the powder formulation, the authors speculate that migration away from the application site to anastomoses could have occurred in some cases. Furthermore, over-hydration of the HA/CMC powder might have resulted in pooling of the resulting gel away from the application site, raising the possibility of migration onto an anastomosis or provision of a nidus for abscess; such migration to anastomoses might increase the rate of SSIs. There is no current evidence of a biological effect of HA/CMC [39–41], although hyaluronan is known to promote cell proliferation and migration [42]. Histological analysis of tissues from pre-clinical studies indicated that macrophage response and rate of remesothelialisation appeared to be similar for both film and powder formulations [18]. There may be potential for powder and gel formulations to act as sites of origin for abscess formation, and/or to be associated with septic complications [19]. An animal study assessing the effect of adhesion barriers on the progression of bacterial infection in the peritoneum found that HA/CMC films had no effect when compared with control groups (saline), whereas some modified gel formulations appeared to increase mortality [43]. Furthermore, testing of HA/CMC powder in a rat model of sepsis involving simultaneous exposure to variable doses of Escherichia coli and sterile caecal contents identified a safety signal on repeat testing (unpublished data, Genzyme Biosurgery), suggesting adverse effects in the presence of active infection or gross caecal contamination. Pre-clinical studies observed an infection potentiation resulting from a physical interaction of HA/CMC powder with caecal material and bacteria during the initial stages of hydration within the first 4 h of implantation (unpublished data, Genzyme Biosurgery). Although our study excluded patients with pathology requiring class IV contamination surgery and those with abdominal abscesses and/or peritonitis or abdominal cavity infection, it is possible that in some patients the presence of HA/CMC powder in combination with luminal contents resulted in adverse events owing to peritoneal bacterial contamination. The main limitation of this trial is its exploratory nature and lack of efficacy assessment. The study population enrolled was a heterogeneous and difficult-to-treat population with a high risk of morbidity and a broad range of aetiologies; therefore, these results cannot be extrapolated to other patient populations. The key strength of the study was the care taken to standardize the application technique according to the instructions provided. The findings suggest that the observed safety signals are most likely related to the specific formulation (powder); therefore, these results should not be extrapolated to other formulations of adhesion barriers. BODY.CONCLUSIONS: This exploratory study found significantly higher rates of adverse events and serious adverse events in the HA/CMC powder group compared with the no adhesion barrier group, indicating a global safety signal in the laparoscopic application of HA/CMC powder in colorectal and small bowel resection. Thus, further development of HA/CMC powder is no longer being pursued for use in patients undergoing colorectal and small bowel laparoscopic surgery, given that this population has an elevated risk for postoperative comorbidity.

TITLE: Comparison of Complications and Short Term Results of Conventional Technique Versus New Technique During Graft Ureteral Stent Insertion in Bari Technique at Emam Khomeini Hospital, Urmia ABSTRACT.INTRODUCTION:: There is debate on renal graft stenting during ureteroneocystostomy, patients with ureteral stents may encounter several complications such as encrustation, stent crustation which can lead to loss of kidney, and complications related to stent extraction: pain and UTI increasing related to cystoscopy for stent extraction accompanying excess expenses. This study designed to reduce complications related to stent extraction. ABSTRACT.MATERIAL AND METHODS:: 90 patients prepared for renal transplantation during 1 year randomly classified to groups, study group: patients with attached stent to Foley catheter, control group: patients with conventional technique (stent separated from Foley) then in their follow up; UTI, stent crustation, luts severity compared to each other. ABSTRACT.RESULTS:: Second week and fourth week UTI reported 25.6%, 2.3% in study group versus 34.9%, 4.7% in control group (P value 0.48 and 0.5). Urinary leakage was 3.3% overall, that all of them occurred in separate stent group, 37.5% vs. 0% in the linked stent group. Stent crustation in separate stent was 25% compared with 0% in the linked stent. ABSTRACT.CONCLUSION:: Low complications rate in linked stent group, despite the lack of significant statistical differences, but indicate the effectiveness and success of the new technique. BODY.1. INTRODUCTION: Double J. stents have different uses in urology in renal implantation is used for reducing ureteral leakage in preventing ureteral stenosis to bladder (Rajaian & Kumar, 2010; Mangus & Haag, 2004; Fayek et al., 2012a, 2012b) Many complications following a kidney transplant are due to poor technique of anastomosis or ureteral ischemia, although the routine use of stent in ureteral anastomosis to bladder in the extracorporeal urinary is routinely discussed (Rajaian & Kumar, 2010) but it has been observed that the use of stent is associated with reduced complications compared with those without a stent (Mangus & Haag, 2004; Fayek et al., 2012a). But using stent causes complications such as increasing infections and stoning stents (Rajaian & Kumar, 2010) and the difficulties imposed on the patient during removing the stent such as the use of anesthesia or general anesthesia to remove the stent that these problems causes tend to use the stent is reduced. There are different techniques to remove stent that include: with the help of fluoroscopy (Robertson, Edwards, & Harden, 1993) associated with radiation exposure to the patient's body and cannot be used in some patients, the next with the help of the preceding flexible or rigid cystoscopy that due to low tolerance for pain, some patients require regional or general anesthesia, which is common in most centers to remove the stent (Evans & Ralph, 1991; Haluk Söylemez et al., 2011). Finally, non-aggressive technique in which with the help of Foley ureteral stent linked with Foley is removed. In this non-aggressive method ureteral stent is removed usually earlier and without aggressive methods and anesthesia or general anesthesia. In this method due to short time survival, stent is decreased in the probable location of complications and due to less impose of complications to the patient is expected to be an acceptable method (Morris Stiff, 1998; Jonathon Olsburgh, 2004; Christian, 2012; Haluk Söylemez et al., 2011). Given that kidney transplant center of Urmia is one of the active centers all over the country and in this study unlike other studies complications are compared separately it is expected in the project that is performed for the first time in the area effects and benefits of this approach are reviewed as short-term. BODY.2. METHODS: 90 patients that are supposed to undergo renal transplantation were randomly divided into two groups. Techniques and processes similar in both groups after vascular anastomosis is usually renal vein to external iliac vein and renal artery to internal iliac artery. Ureteral anastomosis to bladder technique is Bari in both groups except that the first group during anastomosis of ureter to the bladder Double J stent was inserted that in the common technique one end of stent in the ureteral and the other end is inserted into the bladder, but in the second group new technique intra-end bladder of stent to the Foley catheter that is inserted into the bladder initially fixed with 0-4 vicryl suture then anastomosis is continued in both groups and the steps are the same. Patients in both groups are hospitalized one month in the section and in the first group stent are removed in the fourth week with cystoscopy but in the second group after a week stent are removed with the help of removing Foley. Patients are follow-up after discharge 1 to 2 times per week for 3 months, then every 1 to 2 weeks to 6 months, then every 4 to 6 weeks to 12 months. In this case, inpatient and outpatient patients with complications such as urinary leakage and urinary infection and stoning stent and difficult removing of stent and severity of urinary symptoms, patients are examined based on IPSS questionnaire. According to the similar studies, in this study that is conducted with RCT method, 90 patients with inclusion criteria were randomly assigned to two groups of control and study using a random numbers table. For this study, SPSS 16 software is used for data analysis. (Noruzy et al., 2013). BODY.3. RESULTS: A total of 90 patients were transplanted in this study, 4 patients were excluded due to the rejection of transplantation, 2 patients were in attached stent group, and 2 patients were in the separated stenting procedure. Finally, the study was performed with 86 transplanted patients in two control group (43 patients with separated stent and 43 patients with attached stent connected to catheter duct) and study group were enrolled. In the control group (separated stent procedure), 27 patients (62.8%) were male and 16 patients (37.2%) were female. In the study group (attached stent procedure) 26 patients (60.5%) were male and 17 patients (39.5%) were female. The mean age of the study group was 15.63 ± 44 and the control group was 15.20 ± 40.60 years. BODY.4. CONCLUSIONS: Cause of transplant in the control group, 6 patients (14%) PCKD, 1 patient (2.3%) SLE, 15 patients (34.9%) HTN and 9 patients (20.9%), diabetes, 2 patients (4.7%), reflux, 5 patients (11.6%), kidney stones, 3 patients (7%), nephrotic syndrome and in 2 patients (4.7%) were associated with idiopathic etiology. Patients in the study group, 6 patients (14%) PCKD, 1 patient (2.3%) SLE, 26 patients (60.5%) HTN, 4 patients (9.3%), 2 patients (4.7%) reflux, 2 patients (4.7%) kidney stones, and in 2 patients (4.7%) were associated with idiopathic etiology. Number of transplants: In the control group, 39 patients (90.7%) were transplanted for the first time and in 4 patients (9.3%) were transplanted for the second time. In the study group, 38 patients (88.4%) were transplanted for the first time and in 5 patients (11.6%) were transplanted for the second time. Frequency of second week urinary infection: Prevalence of urinary tract infection in the control group during the second week, 15 patients (34.9%) were positive and 28 patients (65.1%) were negative and in the second week of the study group, the prevalence of urinary tract infection in 11 patients (25.6%) were positive and in 32 patients (74.4%) were negative. According to Chi-square test, there was no significant differences between the two groups in terms of infection in admission (P=0.48) (Table 1). Table 1 Distribution of absolute and relative frequency of infection in the two groups Stenting procedure Infection Positive Negative Separate 15(34.9%) 28(65.1%) Attached 11(25.6%) 32(74.4%) Total 26(30.2%) 60(69.8%) Surveying infection during discharge time (one month after transplant) patients, results showed that of 43 patients in the control group, 2 patients (4.7%), urinary tract infection was positive and in 41 patients (95.3%) was negative and of 43 patients in the study group 1 patient (2.3%), urinary tract infection was positive and in 42 patients (97.7%) were negative. According to the Fisher Exact test there was no statistically significant differences between the two groups regarding urinary tract infection during discharge (P=0.5) (Table 2). Table 2 Distribution of absolute and relative frequency of urinary tract infection one month after transplantation in both groups Stenting procedure Urinary tract infection one month after transplantation Positive Negative Separate 2(4.7%) 41(95.3%) Attached 1(2.3%) 42(97.7%) Total 3(3.5%) 83(96.5%) Number of brain death frequency: Of the 43 patients in the control group, transplantation in 2 patients (4.7%) were of brain death and in 41 patients (95.3%) were from living donors. Of the 43 patients in the study group, only 1 patient (2.3%) was transplanted from brain death and in 42 patients (97.7%) had received transplants from living donors. Urinary symptoms of patients during the first month was analyzed according to Luts results showed that: of 43 patients in the control group 27 patients (62.8%) were mild, in 12 patients (27.9%) were mod and in 4 patients (9.3%) were severe. Of the 43 patients in the study group, 30 patients (69.8%) AuA Mild and in 12 patients (27.9%) mod and in 1 patient (2.3%) were severe. According to Chi-square test there was no significant difference between the two groups of patients between Luts severity of patients. (P=0.37) (Table 3). Table 3 Distribution of absolute and relative frequency of urinary symptoms in the first month according to Luts in the two groups Stent Urinary symptoms in the first month according to Luts Mild Mod Severe Separate 27(62.8%) 12(27.9%) 4(9.3%) Attached 30(69.8%) 12(27.9%) 1(2.3%) Total 57(66.3%) 24(27.9%) 5(5.8%) The complications between the two groups, of 43 patients in the control group, 8 patients (19%) had complications after stenting and 37 patients (81%) had no complications after stenting. Of the 43 patients in the study group, 4 patients (9.3%) had complications after stenting and 39 patients (90.7%) had not stent complications. Overall, 12 patients had complications and side effects and according to our findings is as follows. In the control group: 3 cases urinary leakage (37.5%), 2 cases (25%) difficult removing of stent due to unusual positioning of the stent, ureteral obstruction in 2 cases (25%) due to forgetting to close the stent and stoning requiring reoperation and removing stent, 1 patient (12.5%) collection that was lymphoceles nature. (In 3 cases urinary leakage problem was solved with ureteroneocystostomy). 4 cases of complications in the attached group include lack of functioning of Foley and removing Foley earlier than a week was reported in 2 cases (25%), one case was due to the lack of functioning (25%) and one cases was due to the depletion of cuff liquid, 1 (25%) cases was due to distal ureteral stenosis caused by a blood clot and treated with expectant treatment, in 1 patient (25%) collection (peritoneum leakage) was reported. According to statistical analysis, Fisher Exact Test, there was no significant difference between the complications, which led to the departure of stent, and the two groups. (P=0.17) (Table 4). In the study group, 1 patient had ureteral obstruction problem that was fixed with nephrostomy. Table 4 Absolute and relative frequency of complications (lack of Foley function) in the two groups Complications (lack of Foley functioning Stenting procedure Collection Distal stenosis Cuff liquid depletion Lack of Foley functioning Ureteral obstruction Difficult removing Ureteral leakage 1(12.5%) 0(0%) 0(0%) 0(0%) 2(25%) 2(25%) 3(37.5%) Separate 0(0%) 1(25%) 1(25%) 1(25%) 1(25%) 0(0%) 0(0%) Attached 1(8.3%) 1(8.3%) 1(8.3%) 1(8.3%) 3(25%) 2(16.7%) 3(25%) Total According to the test results obtained by Kaplan-Meier, P. value showed that the follow-up period had no effect on removing stent. (P=0.64) BODY.5. DISCUSSION: Ureteral stent in urologic surgeries to reduce complications has long been considered. In recent years, despite differences in terms of using stent in transplant surgeries, the ureteral stent is mounted. Ureteral stents in renal transplantation center, Urmia routinely used in transplantation surgeries (Mohamadi et al., 2011). Given underlying conditions of transplant patients such as immunodeficiency or other underlying diseases long-term survival of stent in transplant patients can be associated with complications which can lead to transplanted kidney failure. However, removing the stent in patients with conventional methods (using cystoscopy) increased infection rates and costs and pain while removing stent that for reducing these complications in this study, we used G-Morris et al. (Evans & Ralph, 1991) that was conducted as Pilot in 1998. G-Morris study was aimed at reducing urinary sepsis. Urinary leakage is one of the most important complications in transplant patients, which can cause kidney failure. In a study of Hafez et al. (2011) a population of 101 people, the technique Lich-gregoir anastomotic was used and in all patients stent was used. Urinary leakage rate was 2%. In another study by M. EL-MEKRESH et al. (2001) conducted on 1,200 patients that various techniques of urethral anastomosis to bladder are used that the preferred technique was Lich-gregoir and without stenting leakage rate have been reported in 35 cases. Also in the same study (Sameh et al., 2012) Sameh A. urinary leakage rate in without stent group was 2.8% and in the stent group was 1.8% that in our study that this rate was reported in 3 cases (3.3%) that all 3 cases were in separated stent group, with the exception of ureteral anastomosis technique used in our study was Bari. And all cases of leakage, observed in the separated stent that two cases in the second week, and 1 case after the first month, referred with hydronephrosis. In the group that stenting was separated from ureter that these findings were in contrast with the results expected because despite the longer stent, this complication was due to attached stent group that due to independent factors such as ischemia of distal ureteral and technical errors. All 3 cases were improved by re-treatment, whereas we expected based on studies that did not use stents higher leakage was created that the difference could be related to our anastomosis technique. Forgotten stent is also one of the important complications that in our study 2 cases were in separated group that one case after 3 months and one case after 1.5 months was referred with stone stent that underwent TUL and stent was removed. The other 2 patients were because of the difficulty in removing the stent due to stent migration upward with local procedure underwent general anesthesia and by ureteroscopy stent was removed. But in the attached stent group, there was no problem in removing stent during removing Foley. In attached stent group there was one distal ureteral stricture case that improved after embedding nephrostomy and expect treatment. But a similar complication was not seen in the separated stent group, while in Tavakoli et al. (2007) study in non-stent group stenosis was 7.7% compared with 0% in the stent group. In this study time period of stent was 2 weeks that was lower than our study (4 weeks), which could be indicative of the difference. As the transplant patients infected with immunodeficiency are exposed to more urinary infection. In Shrestha BM (Shrestha, 2006) study total rate of urinary infection in transplant patients was 43%, but statistically there was no significant difference between stent and non-stent groups. In Chordia et al. study, 212 patients that ureteral stent was used during anastomosis with 183 non-stent patients in terms of amount of bacteriuria at the first 6 weeks (stent was in ureter) and then 6-12 weeks that stent was removed and after 1 year was compared that the difference was not significant. In this study the long-term viability of stent was not a risk factor for UTI instead long-term urethral catheter, causing bacteriuria. In the study of W. Parapiboon (W. Parapiboon, 2012) the rate of bacteriuria in the first week was the highest amount which is more associated with longer survival (7 days) urethral catheter, but at the end of the first month infection rate have been reported 23%. In this study we compared ureteral infection in 2 times in second week (after removal of the catheter lumen) and the end of the first month in 2 group that 26 cases (33.2%) in the second week, and 3 cases (3.5%) one month after transplant urinary tract infection were positive. And separately in 5 cases (34.9%) out of 43 patients with urinary tract infection in separated stenting procedure and 11 cases (25.6%) out of 43 patients in the attaches stenting procedure, urinary tract infection were positive, one month after transplantation, 2 cases (4.7%) in the separated stenting procedure and 1 case (2.3%) in the attached stenting procedure were positive for UTI. In this study, for the removal of the effects of urethral catheter in bacteriuria, we compared urine culture in the second and fourth weeks (after catheter removal). Symptoms of lower urinary tract in patients with urologic associated with the presence of the ureteral stent, perhaps due to defying the trigone removing stent with activity and stimulate the lining of the bladder (Rane et al., 2001; Sur et al., 2008). In the study by Ricardo et al. (2009) severity of luts stimulation in patients, who underwent stent for various reasons, had been investigated. Luts was in 80% of patients with a preferred frequency and urgency in 60-70% of cases. In this study severity of Luts depending on, mild, mod and severe compared between the two groups that in our study, there was no significant difference between the severity of luts in the group that stent was removed earlier with the group that stent was removed later (P = 0.37) (urinary symptoms in separate stenting procedure, 27 cases (62.8%) had mild, 12 cases (27.9%) mod and 4 cases (9.3%) severe and in the attached stenting procedure, 30 (69.8%) had mild, 12 cases (27.9%) mod and 1 cases (2.3%) severe. Since in the patients transplanted the new ureteral orifice is usually away from trigone thus the probability of stent and trigone contact is less also disability of patients' associates can cause retentive symptoms of Luts. Cystoscopy for removing stent in transplanted patients compared with cystoscopy for removing stent in patients with ureteral orifice is more difficult in the natural place because in in patients with ureteral orifice in the ceiling and sometimes the tip of the stent is placed in the bladder neck usually causes difficulty in the procedure. Due to imposed pain on the patient, removing the stent is performed in some centers in the operating room under local anesthesia or general anesthesia. The average time required to remove stent in patients with orifice ureter at the normal place from preparing patients and cystoscopy equipment in women less than 1 min and in men 1.5 minutes and in patients transplanted in women 1.5 minutes and in men 1.5 to 2 minutes, which can indicate the difficulty of procedure in these patients. (This time was obtained by making a senior associate in 10 patients who were treated with endoscopic stone and stent transplant compared with patients who had been admitted to remove the stent), while the stents in patients who were linked to the Foley removing stent is possible with deflation Foley cuff that it also can be done by nurses. However, the cost of patients referred to an outpatient center to remove stent with insurance was averaged 8 dollars and uninsured 43 dollars. While the patients to remove the stent under anesthesia with insurance was 22 dollars and uninsured 172 dollars, while in patients with attached stent does not impose any additional costs to patients. BODY.6. DESIGN LIMITATIONS: Small sample size to investigate the complicationsLack of awareness of the severity of LUTS patients before stentingFailure to differentiate between men and women with severe LUTSFailure to analyze and compare patients in terms of satisfaction from cystoscopy under local anesthesia or general anesthesia compared with local procedure BODY.7. CONCLUSION: Results of this study showed a low rate of complications such as infection and urinary leakage in patients with stent attached to the Foley compared with the separated stent is (although was not statistically significant) and the other costs imposed on patients and pain in patients during removing the stent is less that attached stent. These results indicate the usefulness and alternativeness of the conventional technique with this method, which required similar studies with larger sample sizes.

TITLE: Feasibility of TEE-guided stroke risk assessment in atrial fibrillation—background, aims, design and baseline data of the TIARA pilot study ABSTRACT.BACKGROUND: Antithrombotic management in atrial fibrillation (AF) is currently based on clinical characteristics, despite evidence of potential fine-tuning with transoesophageal echocardiography (TEE). This open, randomised, multicentre study addresses the hypothesis that a comprehensive strategy of TEE-based aspirin treatment in AF patients is feasible and safe. ABSTRACT.METHODS: Between 2005 and 2009, ten large hospitals in the Netherlands enrolled AF patients with a moderate risk of stroke. Patients without thrombogenic TEE characteristics were randomised to aspirin or vitamin K antagonists (VKA). The primary objective is to show that TEE-based aspirin treatment is safe compared with VKA therapy. The secondary objective tests feasibility of TEE as a tool to detect echocardiographic features of high stroke risk. This report compares randomised to non-randomised patients and describes the feasibility of a TEE-based approach. ABSTRACT.RESULTS: In total, 310 patients were included. Sixty-nine patients were not randomised because of non-visualisation (n = 6) or TEE risk factors (n = 63). Compared with non-randomised patients, randomised patients (n = 241) were younger (65 ± 11 vs. 69 ± 9 years, p = 0.004), had less coronary artery disease (9 vs. 20%, p = 0.018), previous TIA (1.7 vs. 7.2%, p = 0.029), AF during TEE (25 vs. 54%, p < 0.001), mitral incompetence (55 vs. 70%, p = 0.038), VKA use (69 vs. 82%, p = 0.032), had a lower mean CHADS2 score (1.2 ± 0.6 vs. 1.6 ± 1.0, p = 0.004), and left ventricular ejection fraction (59 ± 8 vs. 56 ± 8%, p = 0.016). ABSTRACT.CONCLUSIONS: This study shows that a TEE-based approach for fine-tuning stroke risk in AF patients with a moderate risk for stroke is feasible. Follow-up data will address the safety of this TEE-based approach. BODY.INTRODUCTION: Atrial fibrillation (AF) is an independent risk factor for stroke, and patients with AF have a 5% annual risk for thromboembolic stroke, i.e. five times more than in patients without AF [1, 2]. Therapy with vitamin K antagonists (VKA) reduces the risk of thromboembolism dramatically: ischaemic stroke by 67% and peripheral thromboembolism by 42% [3]. Alternatively, aspirin reduces the risk of ischaemic stroke by 22%, but has a significantly lower risk of bleeding [4]. It is recommended to stratify the risk of ischaemic stroke in patients with AF using straightforward clinical characteristics [5]. However, many AF patients classified as having a high or moderate risk for thromboembolism may actually be at low risk, as was shown in the Stroke Prevention in Atrial Fibrillation (SPAF) III study. In SPAF III, a subset of patients with a very high risk of stroke underwent transoesophageal echocardiography (TEE) [6]. Complex plaques in the aorta and signs of atrial stasis (spontaneous echo contrast, low peak emptying velocities and presence of thrombus in the left atrium or left atrial appendage) appeared strongly associated to a high risk of ischaemic stroke and peripheral thromboeobolism [6–8]. Conversely, patients with a normal TEE had an extremely low rate of thromboembolism despite the fact that they had just used aspirin. Up till now no prospective study on the clinical safety and feasibility of a TEE-based risk assessment has been performed. In the TEE-guIded randomized comparison of AspiRin and VKA in patients with AF and an increased risk of stroke (TIARA) pilot study we hypothesise that a comprehensive strategy of TEE-based aspirin treatment is safe and feasible in AF patients who are eligible for vitamin K antagonist therapy on the basis of conventional risk assessment. Our primary objective is to show that—compared with VKA therapy—TEE-based aspirin treatment is safe with respect to a composite endpoint of major cardiovascular and cerebrovascular events, in patients who do not harbour predefined high-risk features on TEE. Our secondary objective is to test the feasibility of TEE as a tool to assess all four echocardiographic features of high stroke risk, i.e. complex atheromatous plaques in the thoracic aorta and signs of atrial stasis including dense spontaneous echo contrast, thrombus or low blood flow velocity. Here we report the study design and feasibility of TEE-guided stroke risk assessment. In addition we compare the baseline characteristics of randomised patients with those of patients who were not randomised because of thrombogenic TEE abnormalities. BODY.METHODS.PATIENTS: Subjects were eligible for participation if they had documented paroxysmal or permanent AF, and a conventional indication for VKA treatment (history of hypertension, age >60 years with diabetes mellitus or coronary artery disease, age >75 years). Subjects were excluded if they had an indication for VKA treatment other than AF, secondary AF, a contraindication for treatment with VKA or aspirin or a contraindication for TEE [9]. Also patients with a presumed very high risk of stroke and thromboembolism (previous ischaemic stroke, transient ischaemic attack (TIA) or systemic thromboembolism, heart failure or significant left ventricular systolic dysfunction, mitral valve stenosis, hypertrophic cardiomyopathy) were not eligible. The Institutional Review Boards of all participating hospitals approved the study and written informed consent was obtained from all patients. Since the risk of stroke in patients who suffered from TIA but not from stroke is uncertain and its mechanisms vary, the steering committee changed the exclusion criteria of the study accepting also patients with a history of TIA for screening. This protocol amendment was implemented in March 2006. BODY.METHODS.DESIGN: The TIARA pilot study was an open randomised, multicentre trial in which a risk stratification based on TEE was examined in AF patients with a moderate risk of stroke. The study was performed in ten large hospitals in the Netherlands between 2005 and 2009. All centers are highly experienced in echocardiography. Most patients were enrolled in the outpatient clinic, but also hospitalised patients were included. The trial has been registered under ClinicalTrials.gov # NTC 00224757. Patients underwent a TEE and were randomly assigned to aspirin or VKA treatment if TEE did not show high-risk abnormalities. Patients assigned to aspirin received a once-daily dose of 100 mg and patients assigned to VKA received adjusted-dose acenocoumarol or phenprocoumon (target INR 2.5 to 3.5). Obviously, patients with one or more abnormalities on TEE were excluded from randomisation and received adjusted-dose VKA and were followed in the high-risk observational group. The Netherlands' regional Thrombosis Service routinely checked the INR. All patients were followed up for at least 1 year. Figure 1 depicts the flow chart of screening, randomisation and follow-up of included patients in the TIARA pilot study. Fig. 1Overview of the screening, randomisation and follow-up of the TIARA pilot study. Eligible patients were planned for TEE to assess presence or absence or echocardiographic features of high risk of stroke i.e. signs of atrial stasis and complex aortic plaques. VKA: vitamin K antagonists. TEE: transoesophageal echocardiography. Positive TEE: presence of at least one of the echocardiographic features of high risk of stroke. Negative TEE: absence of echocardiographic features of high risk of stroke. QoL: Quality of life scores using SF-36, EuroQol 5-D, Minnesota and AFSS questionnaires BODY.METHODS.ECHOCARDIOGRAPHIC PROCEDURES: All TEEs were performed and interpreted by certified cardiologists in a standard fashion, as recommended by the American Society of Echocardiography [9]. All echocardiograms were acquired by commercially available equipment: HP Sonos 5500 imaging system (Hewlett-Packard Co., Andover, MA) or Vingmed System V (General Electric Company, Fairfield, Connecticut) equipped with a 5-MHz omniplane probe for TEE. When in doubt, the echocardiograms could be sent to the echo core laboratory (Maastricht University Medical Center) for review. TEE was performed with a 5-MHz omniplane transducer. The oropharynx was anaesthetised by use of topical lidocaine spray and viscous lidocaine solution prior to insertion of the probe. The left atrial appendage (LAA) was imaged by placing the probe at the level of the mid-oesophagus. The plane of the multiplane probe was adjusted to achieve a short-axis view of the aortic valve (45°); to obtain an optimal view of the left atrial appendage, the shaft of the scope was rotated anticlockwise in 5 to 10° increments from 0 to 180°. Multiple views of the LAA were then obtained; cine loops were acquired [10]. The imaging plane and gain settings were adjusted for optimal visualisation of thrombi and spontaneous echo contrast (SEC) in the left atrium (LA), LAA, right atrium (RA) and right atrial appendage (RAA). For measurement of the blood flow velocities in the LAA, the sample of the pulsed wave Doppler was placed at the orifice of the LAA and the profile of the velocities was recorded over at least five cardiac cycles. A maximum velocity was then calculated by averaging these five cardiac cycles [11]. In order to image complex plaques, the thoracic aorta was carefully visualised by retracting and rotating the probe. BODY.METHODS.DEFINITION OF HIGH-RISK ECHOCARDIOGRAPHIC FEATURES: The following definitions were used for echocardiographic abnormalities. Spontaneous echo contrast was defined as a persistent pattern of a slow swirling motion of intracavitary echo density throughout the LA and LAA at normal gain [6]. A thrombus was defined as an intracavitary echodense mass with sharp edges, echogenicity different from adjacent structures, some degree of mobility, and present in more than one plane [12]. Low blood flow velocity was defined as peak emptying velocity of less than 25 cm/s on TEE, measured at the orifice of the LAA. Complex atheromatous plaque was defined as a mobile, pedunculated or ulcerated plaque, or plaque thickness ≥4 mm in the thoracic aorta [7]. BODY.METHODS.RANDOMISATION: Randomisation was performed by permuted block design with stratification for each participating hospital. Eligible patients were randomised to either treatment with aspirin 100 mg once daily or treatment with adjusted-dose VKA. In patients randomised to VKA, the INR was checked by the regional Thrombosis Service on a routine basis. BODY.METHODS.FOLLOW-UP: Patients visited the outpatient clinic at 3, 6 and 12 months after randomisation and at the end of the study. All patients were followed up for at least 1 year. If needed, patients were seen between regular visits. At every visit, symptoms and signs of the composites of the primary endpoint were checked. Also INR values were collected. TEE was not repeated on a regular basis in the included patients. Within the study period it was not expected that aortic plaque or left atrial low flow states would develop if not present on the first examination. A TEE was, however, repeated when clinically indicated. If electrical cardioversion was indicated, aspirin patients were temporarily switched to VKA. One month after the shock, irrespective of the rhythm, aspirin was reinstituted. BODY.METHODS.QUALITY OF LIFE QUESTIONNAIRES: The quality of life was studied using the EuroQol 6-D, the Short Form (SF)-36 Health Survey questionnaire and the Toronto Atrial Fibrillation Severity Scale (AFSS) at inclusion and after 12 months of follow-up. SF-36 contained items to assess physical health and mental health, AFSS secured AF-specific complaints. The quality of life assessment was completed with a multidimensional index that contains five subscales: general, physical, activity, motivation and cognition. BODY.METHODS.DATA COLLECTION: Data were collected using an electronic case report form accessed at a secure Internet site. Data were transferred through the Internet to the central database in Maastricht. Data could be either entered online, or offline with frequent data transfer. By using a validation plan, integrated in the data entry software, data were continuously checked for missing or contradictory entries and values out of the normal range. The study-monitoring nurse performed additional edit checks. The data analysis was done at the Maastricht University Medical Center. Patient identification was registered in the participating centres, and was not transferred to the central database. Questionnaires for quality of life were processed by the Center for Data and Information Management (MEMIC), a subdivision of the department of the Faculty of Health, Medicine and Life Sciences (Maastricht University). BODY.METHODS.ENDPOINTS: The primary endpoint was a composite of (1) stroke including haemorrhagic strokes, (2) systemic embolism, (3) major bleeding, (4) acute coronary syndrome (unstable angina and myocardial infarction) and (5) all-cause mortality. The Central Independent Adjudication Committee (CIAC) independently adjudicated all events using definitions as mentioned below. In case of doubt the members held a conference for an unanimous adjudication. Definitions of primary endpoints are described in the Appendix. BODY.METHODS.STATISTICAL ANALYSIS: Sample size calculation was driven by the primary endpoint on safety, i.e. the composite endpoint. The incidence of the composite safety endpoint with aspirin treatment could be estimated from the available literature. In patients with a normal TEE, the estimated yearly incidences of ischaemic stroke and systemic thromboembolism, major bleeding, acute coronary syndrome and death with aspirin treatment were 1.1%, 0.7%, 0.8% and 1.8%, respectively. Therefore, the estimated yearly incidence of the composite primary endpoint was 4.4% with aspirin. With VKA treatment (INR 2.0–3.0) the yearly incidences of ischaemic stroke and systemic thromboembolism, major bleeding, acute coronary syndrome and death were 1.0%, 1.7%, 1.0% and 1.8%, respectively. As a result, the estimated yearly incidence of the composite endpoint with VKA treatment was 5.5%. With an estimated average follow-up duration of 1.5 years, the estimated incidences of the composite safety endpoint were 6.6% for the aspirin group and 8.3% for the VKA group, respectively. The primary safety objective of this pilot study is to show that applying TEE-based aspirin prophylaxis does not lead to unacceptable high frequency of the safety endpoint. Inclusion of 300 patients (150 per randomisation arm) would rule out an excess of 7% with a power of 80% and a type I error of 5%. The difference in incidence of the safety endpoint in the randomised groups was calculated and described with an exact one-sided 95% confidence interval. A Kaplan-Meier curve will be used to visualise the occurrence of events over time. Hazard ratios will be calculated using Cox regression. Similar analyses will be done for the separate components of the safety endpoint. The secondary—feasibility—objective will be evaluated by calculating the number of patients eligible for the TEE approach in whom the TEE cannot be performed or does not yield unequivocal information on echocardiographic thromboembolism risk markers. Data that are not normally distributed will be log-transformed and presented as geometric means. Analyses were done using the t-test for independent groups for continuous variables and Fisher's exact test for categorical variables. SPSS was used for all statistical calculations. Analyses will be done using the intention-to-treat principle, that is, patients will be analysed in the group to which they were randomly assigned, regardless of the treatment they actually received. A P value less than 0.05 is considered statistically significant. All tests are two-tailed. BODY.RESULTS.BASELINE CHARACTERISTICS: The inclusion in the TIARA pilot trial was slower than expected and the follow-up period longer than foreseen. As a consequence inclusion was stopped after 310 patients and a projected mean follow-up duration of 1.6 years. Recruitment was diminished predominantly due to refusal of eligible patients to undergo a TEE examination because of fear for the TEE. Analysis of the patient screening log showed that 35% of patients eligible for participation in the TIARA pilot study refused TEE for this reason. In the group randomised to aspirin, three patients withdrew their informed consent directly after randomisation. Therefore they will be discarded from further analysis in respect to follow-up. For the secondary (per protocol) analysis, patients not meeting the inclusion (n = 1 for VKA group, n = 2 for aspirin group), or exclusion criteria (n = 3 for VKA group), and patients randomised despite TEE abnormalities (n = 1 for VKA group), will be discarded from analysis. We had included 310 patients by July 2008, of which 241 (78%) patients were randomised. In total, 69 patients were not randomised on the basis of an abnormal TEE showing either predefined echocardiographic markers of a high stroke risk or another abnormality considered highly thrombogenic. Summaries of clinical and echocardiographic data are expressed as means or frequencies with 95% confidence intervals (CIs) (Table 1). Non-randomised observational patients had a slightly higher CHADS2 score due to on average higher age, higher prevalence of diabetes mellitus and previous TIA. The prevalence of hypertension was very high and similar in both groups. On the other hand, the prevalence of TIA was low, while patients with stroke and heart failure were excluded. Non-randomised observational patients were more often in AF at the time of TEE and had mitral incompetence of any grade more frequently. They also more often used a vitamin K antagonist at entry. Distribution of type of AF (paroxysmal or permanent) did not differ among groups. Table 1Characteristics of randomised and non-randomised patientsRandomised patientsNon-randomised patients (observational group)p-valuen = 241n = 69Clinical characteristicsAge (years)65 ± 1169 ± 90.004Male,%66610.479Hypertension,%90860.388Coronary artery disease,%9200.018Diabetes,%10190.057Transient ischaemic attack,%1.77.20.029CHADS2 score1.2 ± 0.61.6 ± 1.0<0.001- Score 0,%2.14.3- Score 1,%76.353.6- Score ≥2,%21.642.1Time since 1st diagnosis AF (years)4.9 ± 9.04.6 ± 6.10.754Type of AF0.320- Paroxysmal,%8578- Permanent,%1522Body mass index (g/m2)28 ± 528 ± 50.458Systolic blood pressure (mmHg)136 ± 18137 ± 180.615Diastolic blood pressure (mmHg)80 ± 982 ± 100.288Antithrombotic drug at baseline,%99990.637- VKA at baseline,%69820.032- Aspirin at baseline,%32180.023EchocardiographyAF during TEE,%2554<0.001Left atrial diameter (mm)41 ± 1142 ± 120.389Left ventricular ejection fraction (%)59 ± 856 ± 80.016Left ventricular mass (g/m2)124 ± 83130 ± 1020.571End-diastolic septum width (mm)9.6 ± 2.610.1 ± 3.20.204End-diastolic posterior wall width (mm)9.1 ± 2.49.5 ± 2.60.211Mitral incompetence,%55700.038Data are expressed as mean ± SD unless otherwise specifiedAF atrial fibrillation, VKA vitamin K antagonists, TEE transoesophageal echocardiography BODY.RESULTS.FEASIBILITY OF TEE-GUIDED STROKE RISK ASSESSMENT: Of all 310 patients planned for TEE, 69 were not eligible for randomisation because of non-visualisation (n = 5), technical failure (n = 1) or abnormal TEE (n = 63) (Table 2). At least one echocardiographic marker for thromboembolism could not be visualised with certainty in five patients; mostly due to incomplete imaging of the LAA and therefore LAA flow velocities could not be measured (n = 4). In the remaining patient, the aorta could not be visualised. In one patient, TEE could not be performed because of technical reasons; the patient was not capable of swallowing the probe and therefore none of the markers for thromboembolism were visualised. Furthermore, 63 patients had abnormalities on TEE, of which 60 patients (19.7%) had at least one predefined risk marker on TEE. Thrombi were detected in ten patients (3.3%), low LAA flow velocities were seen in 33 patients (10.9%), spontaneous echo contrast in 22 patients (7.2%), and complex plaques in 13 patients (4.3%). The majority had only one risk marker (n = 49, Table 2); LAA flow velocities, SEC, complex plaques and thrombi were seen in 20, 11, 9 and 6 patients, respectively. In 13 patients, two abnormalities were detected with TEE; seven patients had low LAA flow velocities with SEC, three patients had a thrombus with low LAA flow velocities, and three patients had SEC with complex plaques. Only one patient had all four markers of thromboembolism risk on TEE. Table 2Reasons for non-randomisationn = 69Technical reasons/patient refusal1At least one TEE risk factor not clearly visible5At least one TEE risk factor present63- 1 TEE abnormality49- 2 TEE abnormalities13- 3 TEE abnormalities0- 4 TEE abnormalities1TEE transoesophageal echocardiography Surprisingly, other cardiac abnormalities were observed in three patients who had no other risk markers for stroke; two patients had Lambl's excrescences on the aortic valve and one patient had a myxoma in the left atrium. Lambl's excrescences are not considered to be a primary source of cardioembolism in patients with sinus rhythm, but the importance in atrial fibrillation remains uncertain [13]. These patients were therefore not randomised. The patient with a myxoma refused surgery, and therefore was not randomised because of the need for permanent anticoagulation. Thus, TEE could provide accurate information on thromboembolic risk in 304 out of 310 patients (98.1%). BODY.DISCUSSION: The TIARA pilot study is a randomised comparison of aspirin and vitamin K antagonists for stroke prevention in patients with a moderate stroke risk who do not harbour predefined high-risk features on TEE. It was constructed as a pilot study to show feasibility and to prepare a larger study later on. Considering the high number of equivocal TEE imaging results (98.1% of patients) and the low number of technical failures of TEE, we feel that a TEE-guided approach is feasible in clinical practice. If our study shows that TEE-based aspirin treatment is also safe, this may form the basis for a large TEE-guided stroke risk assessment trial. We found that in only six out of the 310 included patients (1.9%) irrefutable information about the thrombogenicity could not be obtained by TEE. In one patient a TEE could not be performed due to technical reasons, and in the remaining five patients the left atrial appendage and aorta could not be visualised accurately. This high technical success rate may relate to the fact that all participating centers in the TIARA pilot study were highly trained in performing TEE. However, TEE is now well incorporated in the daily practice of any cardiology clinic. We hence believe that a TEE-based approach for fine-tuning stroke risk in AF patients with a moderate risk for stroke is feasible, not only in this trial but also in real-life. In patients using VKA before TEE, VKA was not stopped prior to performing TEE. Of note, VKA does not influence the presence of SEC and LAA flow velocities, or complex plaques since it does not alter the underlying haemodynamic mechanism [14–16], nor aortic plaque anatomy and complexity [17], respectively. On the other hand, continued VKA therapy may have precluded finding thrombi on TEE. However, we reasoned that the incidence of thrombi after stopping VKA in patients randomised to aspirin would be low, especially since high LAA flow velocities and absence of SEC virtually exclude thrombus [15, 18]. In this respect, of all three echocardiographic atrial thrombosis markers, LAA flow velocity measurement is the most robust concerning sensitivity and specificity for thrombosis risk [8, 19]. Therefore, to prevent unjust randomisation as much as possible we used a higher than usual cut off of LAA flow velocity of 25 cm/s. Notwithstanding the above, temporary discontinuation of stable VKA treatment for e.g. 4 weeks to assess stroke risk markers with TEE off VKA may seem feasible since most patients eligible for the TIARA approach have a relatively low stroke risk. Although the stroke risk during 4 weeks of interrupted VKA is low, we did not feel justified to do so, especially since the guidelines advise keeping discontinuation of VKA (e.g. to perform elective surgery) as short as possible. The overall mean CHADS2 score was fairly low and median CHADS2 score was only 1, mainly because we did not consider patients with a previous stroke or heart failure. In contrast, patients included in RACE [20] and AFFIRM [21] were older and were presumably in a higher CHADS2 risk category mostly due to previous stroke and heart failure, although hypertension was less frequent. Obviously, the patients enrolled in the ACTIVE trial [22] and RELY study [23] had higher CHADS2 scores because patients at higher risk for thromboembolism were included, whereas the TIARA pilot study intended to focus on patients with an intermediate risk, i.e. a lower CHADS2 score. Patients who were excluded from randomisation (non-randomised observational group) because of TEE abnormalities had, expectedly, a higher mean CHADS2 score than randomised patients, mainly due to higher average age and higher prevalence of diabetes. Nevertheless, the median CHADS2 score is 1 in both randomised and non-randomised groups. We can conclude that the average patient randomised in the TIARA pilot study was the younger patient with well-controlled hypertension and paroxysmal AF without other risk factors of the CHADS2 risk score (i.e. heart failure, previous stroke or TIA, diabetes, and older age). The relatively high number of women included in the TIARA study is remarkable, considering that women are generally underrepresented in clinical trials. In the non-randomised observational group, ten thrombi were found (3.3% of patients); eight of these patients were on VKA and two on aspirin. The number of thrombi as well as stroke risk category in our study is comparable with several recent studies showing prevalences of thrombi between 0.6% and 3.6% [24–26]. Remarkably, also three patients (0.97%) with a CHADS2 score of 0 had at least one TEE stroke risk factor. Scherr et al. also report a prevalence of 0.3% thrombi in patients with a CHADS2 score of 0 [25]. This underscores our hypothesis that current risk stratification schemes—which are based merely on clinical characteristics (such as the CHADS2 score)—are unable to detect all patients with a high risk of stroke with certainty. Surprisingly, three patients had no thromboembolic markers but other abnormalities with an indication for treatment with VKA. These patients were asymptomatic beforehand, and they were subsequently treated with VKA and followed in de observational group. To our knowledge, this is the first study to show that routine TEE yields unexpected findings in AF patients, leading to a change of antithrombotic treatment. Performing TEE in AF patients with a low to average thromboembolism risk (CHADS2 score of 0 and 1) is useful since it may enhance aspirin or no antithrombotic treatment in some of these patients which may reduce avoidable bleeding. In addition, in patients considered at low risk, TEE may detect high-risk features warranting VKA therapy, protecting these patients from unexpected strokes. In this respect it is noteworthy that out of 310 patients with an average CHADS2 risk score of 1, 63 patients had an abnormal TEE (20.3%) necessitating continuation of or switch to VKA therapy. Most noteworthy in this respect were the three patients with a CHADS2 risk score of 0 who appeared to have thrombogenic features on TEE. BODY.CONCLUSIONS: In summary, the TIARA pilot study showed that a TEE-based risk stratification in patients with atrial fibrillation and a moderate risk for stroke is feasible. Decreasing the threats and inconvenience of VKA is a desirable goal. Identifying patients in whom VKA (or other antithrombotic drugs) can be avoided may reduce bleeding and improve quality of life without an increase in stroke complications or costs of care. Application of a new echo-guided antithrombotic strategy may help to reduce bleeding whilst stroke prevention is maintained. If so, this study may significantly change daily clinical practice of antithrombotic management of atrial fibrillation. These results may form the basis for a larger multi-centre study.

TITLE: Effect of pelvic floor muscle exercises on pulmonary function ABSTRACT: [Purpose] This study aimed to determine the correlation between pelvic floor muscle strength and pulmonary function. In particular, we examined whether pelvic floor muscle exercises can improve pulmonary function. [Subjects] Thirty female college students aged 19–21 with no history of nervous or musculoskeletal system injury were randomly divided into experimental and control groups. [Methods] For the pulmonary function test, spirometry items included forced vital capacity and maximal voluntary ventilation. Pelvic floor muscle exercises consisted of Kegel exercises performed three times daily for 4 weeks. [Results] Kegel exercises performed in the experimental group significantly improved forced vital capacity, forced expiratory volume in 1 second, PER, FEF 25–75%, IC, and maximum voluntary ventilation compared to no improvement in the control group. [Conclusion] Kegel exercises significantly improved pulmonary function. When abdominal pressure increased, pelvic floor muscles performed contraction at the same time. Therefore, we recommend that the use of pelvic floor muscle exercises be considered for improving pulmonary function. BODY.INTRODUCTION: The pelvic floor muscles (PFM) primarily protect the endopelvic organs but are also involved in breathing during speaking, deep breathing, and coughing1). Speaking, deep breathing, and coughing occur as the diaphragm rises due to increased abdominal pressure. Here the abdominal and PFM generate pressure through their co-contraction2). PFM contraction protects the endopelvic organs against increased abdominal pressure under the body's certain reactions such as deep breathing and coughing and helps breathing by relieving anal and urethral obstructions3). Therefore, PFM exercises should be included in efforts to improve pulmonary function. Although the PFM have breathing-related functions, most related studies have investigated urological diseases4, 5). However, a few studies have examined the effects of PFM reinforcement on pulmonary function. Most studies have reported that Kegel exercises reinforce the PFM6). Meanwhile, a recent study by Park7) attempted to identify the effects of one-time Kegel exercises on pulmonary function and reported that the vital capacity was improved in certain categories. However, one-time Kegel exercises may not be sufficient for reinforcing the PFM. Therefore, the purpose of this study was to examine the effects of self-disciplined Kegel exercises performed for 4 weeks on PFM strength and determine whether PFM reinforcement could improve pulmonary function. BODY.SUBJECTS AND METHODS: The subjects of this study were 30 female students at S University in Busan City who fully understood the study's purpose and methods and agreed to participate in it. This study complied with the ethical standards of the Declaration of Helsinki, and written informed consent was obtained from each participant. Selection criteria were: no past history of nervous, musculoskeletal, or heart-lung system disease (any of which could affect our experiment); no history of participating in pulmonary function promotion exercise programs; and agreeing not to perform any other exercises during the experiment. The subjects were randomly divided into a Kegel exercise group and a control group that received no treatment (n = 15 each). Two individuals in both the test and control groups did not participate in the second round of measurements; therefore, their measured values were excluded from the collected data. The control group had an age of 20.23 ± 0.78 years, height of 159.13 ± 3.62 cm, and weight of 55.20 ± 6.67 kg, while the experimental group had an age of 20.41 ± 0.84 years, height of 158.30 ± 4.70 cm, and weight of 54.14 ± 5.88 kg. A digital Pony FX microspirometer (Cosmed Inc, Italy) was used to measure pulmonary function by determining the amount and velocity of air that enters and exits the lung. This study measured forced vital capacity (FVC) and maximal voluntary ventilation (MVV). The FVC measurement was made while the subjects were sitting on a chair with their limbs and shoulders straight and legs spread shoulder-width apart and perpendicular to the ground. They were then instructed to close their nose using a nose clamp and hold the measuring instrument with one hand and let it nip the mouth. Measurements were taken while the subjects breathed normally three or four times and then took a deep and fast inspiration followed by a fast expiration. The expiratory breath was maintained for 6 seconds. The MVV was then measured with the subjects in the same position. The subjects were led to perform inspiration and expiration for 12 seconds in the deepest and fastest manner. Each measurement was taken three times, and the average value of each was used in the analysis. The first round of measurements was taken before the start of the Kegel exercises, while the second round of measurements was taken after the end of the 4-week Kegel exercises course. Kegel exercises have been shown to help reinforce the PFM8). The ready position of Kegel exercises was lying on the floor with the knees bent and the soles touching (frog position). Next, without contracting the abdominal, lumbar, gluteal, and lower-limb muscles, the subjects slowly contracted only the PFM for 5 seconds; at the fifth second, they performed a maximum contraction of the muscle. The subjects relaxed at the sixth second and performed a fast contraction and relaxation between the seventh and ninth seconds, then rested at the tenth second. One set of exercises included performing ten sets of the above exercise twice. A 40-second break was provided between sets. The statistical program SSPWIN (ver 21.0) was used for the analysis, and the statistical significance level was set at α = 0.05. This study used paired t-tests to compare the test and control groups before and after the exercises, and an analysis of covariance test was conducted to identify differences in variations between the two groups. BODY.RESULTS: The changes in pulmonary functions of the test group before and after Kegel exercises were as follows. Among the lower-level categories of FVC, the FVC increased from 2.75 L to 3.13 L (p < 0.05), FEV1 increased from 2.39 L to 2.78 L (p < 0.05), PEF increased from 4.48 L to 5.97 L (p < 0.05), FEF 25–75% increased from 2.83 L to 3.48 L (p < 0.05), and FVC increased from 1.53 L to 1.81 L (p < 0.05). These increases were all statistically significant. On the other hand, while the FEV1/FVC increased from 87.15% to 89.19%, this result was not statistically significant. The MVV increased from 84.88 L to 112.62 L, a difference that was statistically significant (p < 0.05) (Table 1Table 1.Pulmonary function changes by groupGroupPre-exercisePost-exerciseFVCFVC(L)*Experimental2.75±0.473.13±0.47Control3.06±0.312.98±0.36FEV1 (L)*Experimental2.39±0.512.78±0.38Control2.48±0.252.33±0.49PEF (L/s)*Experimental4.48±1.425.97±1.49Control4.66±0.774.46±1.34FEV1/FVC (%)Experimental87.15±7.9389.19±6.64Control81.32±7.0578.44±14.55FEF 25–75% (L/s)Experimental2.83±0.983.48±0.77Control2.50±0.562.94±0.87IC (L)Experimental1.53±0.371.81±0.30Control1.75±0.211.77±0.28MVVMVV* (L/min)Experimental84.88±13.23112.62±19.92Control92.41±8.8091.48±7.74Mean ± SD, *p < 0.05). Changes in pulmonary functions of the control group were also examined between the first and second measurements rounds. The FVC decreased from 3.06 in the first round of measurements to 2.98 in the second round of measurements, FEV1 declined from 2.48 to 2.33, FEV1/FVC decreased from 82.50 to 75.81, PEF decreased from 4.66 to 4.46, FEF decreased from 2.57 to 2.50, and FIC decreased from 1.65 to 1.64. However, these changes were not statistically significant. While the MVV increased from 85.56 to 90.80, this result was also not statistically significant (p > 0.05) (Table 1). Comparison of changes in pulmonary functions of the test and control groups showed the following. The test group showed increases in FVC (p < 0.05), FEV1 (p < 0.05), and PEF (p < 0.05), whereas the control group exhibited decreases in the same variables. These results were statistically significant. For FEV1/FVC, the test group showed an increase and the control group exhibited a decrease, but these changes were not statistically significant. In addition, both the test and control groups revealed increased FEF 25–75% and FIC without statistical significance. For MVV, the test group showed an increase and the control group exhibited a decrease. These differences were statistically significant (p < 0.05) (Table 1). BODY.DISCUSSION: In normal breathing, inspiration refers to the entry of external air into the body as the diaphragm contracts and moves downwards, while expiration refers to the relaxation and upward movement of the contracted diaphragm. However, when forced expiration or coughing occurs, the anterior and lateral abdominal muscles contract, thereby generating pressure that strongly moves the diaphragm upward. Here PFM contraction helps maintain the abdominal pressure. From this viewpoint, the PFM and deep abdominal muscles are involved in breathing through their concerted contractions9). This study used Kegel exercises to reinforce the PFM8). The reinforcement of the PFM through Kegel exercises improved the lower-level FVC categories. This result verifies that PFM reinforcement through Kegel exercises has positive effects on improving the FVC, which requires increases in intra-abdominal pressure. This finding coincides with those of a study by Sapsford10), which showed that PFM contraction generated forced expiration when conditions requiring increases in abdominal internal pressure occurred, such as nose blowing, coughing, and sneezing. In addition, the FVC measured in the present study included the process of forced expiration after maximal inspiration. Eventually, the PFM that was reinforced when intra-abdominal pressure increased during the process of forced expiration may have also performed co-contraction, thereby reinforcing forced expiration, which subsequently increased the FVC. The MVV, another variable tested in this study, also increased. The MVV is measured after breathing for 12 seconds in the deepest and fastest manner; thus, it increases intra-abdominal pressure. Since increased intra-abdominal pressure activates the PFM, the MVV may have increased after Kegel exercises. This study has a certain limitation. It was conducted based on the assumption that a 4-week Kegel exercise program reinforced the PFM, but it did not directly confirm this hypothesis. In other words, this study measured the vital capacity based on the result of preceding research that Kegel exercises performed for 4 weeks reinforced the PFM. Therefore, additional studies are required to verify that Kegel exercises directly reinforce the PFM and then identify the correlation between PFM reinforcement and pulmonary function.

TITLE: Long-Term Outcomes of Internet-Based Self-Management Support in Adults With Asthma: Randomized Controlled Trial ABSTRACT.BACKGROUND: Long-term asthma management falls short of the goals set by international guidelines. The Internet is proposed as an attractive medium to support guided self-management in asthma. Recently, in a multicenter, pragmatic randomized controlled parallel trial with a follow-up period of 1 year, patients were allocated Internet-based self-management (IBSM) support (Internet group [IG]) or usual care (UC) alone. IBSM support was automatically terminated after 12 months of follow-up. In this study, IBSM support has been demonstrated to improve asthma-related quality of life, asthma control, lung function, and the number of symptom-free days as compared to UC. IBSM support was based on known key components for effective self-management and included weekly asthma control monitoring and treatment advice, online and group education, and communication (both online and offline) with a respiratory nurse. ABSTRACT.OBJECTIVE: The objective of the study was to assess the long-term effects of providing patients 1 year of IBSM support as compared to UC alone. ABSTRACT.METHODS: Two hundred adults with physician-diagnosed asthma (3 or more months of inhaled corticosteroids prescribed in the past year) from 37 general practices and 1 academic outpatient department who previously participated were invited by letter for additional follow-up at 1.5 years after finishing the study. The Asthma Control Questionnaire (ACQ) and the Asthma Quality of Life Questionnaire (AQLQ) were completed by 107 participants (60 UC participants and 47 IG participants). A minimal clinical important difference in both questionnaires is 0.5 on a 7-point scale. ABSTRACT.RESULTS: At 30 months after baseline, a sustained and significant difference in terms of asthma-related quality of life of 0.29 (95% CI 0.01-0.57) and asthma control of -0.33 (95% CI -0.61 to -0.05) was found in favor of the IBSM group. No such differences were found for inhaled corticosteroid dosage or for lung function, measured as forced expiratory volume in 1 second. ABSTRACT.CONCLUSIONS: Improvements in asthma-related quality of life and asthma control were sustained in patients who received IBSM support for 1 year, even up to 1.5 years after terminating support. Future research should be focused on implementation of IBSM on a wider scale within routine asthma care. ABSTRACT.TRIAL REGISTRATION: International Standard Randomized Controlled Trial Number (ISRCTN): 79864465; http://www.controlled-trials.com/ISRCTN79864465 (Archived by WebCite at http://www.webcitation.org/6J4VHhPk4). BODY.INTRODUCTION: Asthma is a common chronic disease with a prevalence of approximately 6% among adults [1]. It is characterized by chronic inflammation and/or structural changes of the airways, which leads to recurrent episodes of wheezing, coughing, difficulty breathing, and/or chest tightness [2,3]. According to clinical guidelines [2,3], treatment strategies for asthma should be aimed at minimization of symptoms, optimization of lung function, and prevention of symptom aggravation with few medication side effects. Even though effective therapies are widely available, many patients do not achieve these treatment goals [1,4]. As a consequence, asthma still imposes a significant burden of disease on the individual patient. A proactive patient-centered approach consisting of education, treatment goals, self-monitoring, and an action plan, accompanied by guidance and regular review by a health care provider, has the potential to improve outcomes in asthma [5], including improved quality of life and a reduced number of hospitalizations and unscheduled doctor visits. In spite of the prominent role within guidelines, adoption of this "guided self-management" is lacking [6]. While many practices do offer patients a routine medical review, only a minority of patients are provided with an action plan by their health care provider [7,8]. Usage of action plans by patients could be enhanced if action plans are part of a patient-professional partnership and when they are tailored to the needs of the individual patient [9]. Provision of Internet technology has been proposed as an appealing medium for asthma management [10-14]. Indeed, in the study by Van der Meer et al [15] in patients with mild to moderate persistent asthma, it was demonstrated that provision of an Internet-based self-management (IBSM) support program for 1 year leads to improved asthma-related quality of life, asthma control, lung function, and the number of symptom-free days as compared to usual care (UC) alone. A post hoc analysis of this study [16] demonstrated that patients with asthma that was not well controlled benefited the most from IBSM support. In addition, this study showed that at 12 months of follow-up, about 60% of the patients were still using the program of their own initiative. However, it is unknown whether the benefits are sustained over a long-term period. We hypothesized that the benefits of providing 1 year of IBSM support are sustained over a long-term period. In this paper, we aim to assess the long-term effects of providing patients 1 year of IBSM support as compared to UC alone. BODY.METHODS.PARTICIPANTS: Two hundred patients who previously participated in a 12-month multicenter, nonblinded, pragmatic randomized controlled parallel trial were invited for additional follow-up 1.5 years after finishing the study. Full details of the study methodology and subjects for the Self-Management of Asthma Supported by Hospitals, ICT, Nurses and General practitioners (SMASHING) study have been published elsewhere [15]. Briefly, patients were recruited from 37 general practices in the Leiden and The Hague area and from the outpatient clinic of the department of Respiratory Medicine of the Leiden University Medical Center (LUMC), the Netherlands. Eligibility criteria were adult age (18-50 years), physician-diagnosed asthma, prescription of inhaled corticosteroids ≥3 months in the previous year, access to Internet at home, and the ability to understand written and oral Dutch instructions. Patients who received a maintenance dose of oral corticosteroids were excluded. All participants were trained in a group educational session to measure lung function as forced expiratory volume in 1 second (FEV1) by using a handheld electronic spirometer (PiKo-1, Ferraris Respiratory). After this session, patients were asked to report during a 2-week period FEV1 (daily), day and night symptom score (daily), and to fill in at least once weekly an Asthma Control Questionnaire (ACQ) [17] on a specifically designed website or by mobile phone text messaging (SMS). The ACQ is a validated 7-item questionnaire for assessment of actual level of asthma control, consisting of 6 questions on asthma symptoms in the previous 7 days and an FEV1 measurement. Optimal cut-point for "well-controlled" is ≤0.75 and a value of ≥1.50 confirms "uncontrolled" asthma [18]. During these 2 weeks, patients did not receive feedback on their actual level of asthma control. After the 2-week period, all patients were randomized to either IBSM support adjacent to UC, that is, Internet group (IG), or to UC alone. Strategy allocation of patients on a 1:1 ratio was conducted by JKS using a computer-generated permuted block scheme. Patients were stratified on care provider (general practice vs outpatient clinic) and asthma control at baseline. The SMASHING study was powered to detect a difference in the primary outcome asthma-related quality of life, as measured by the Asthma Quality of Life Questionnaire (AQLQ) score [19] between the two groups. Due to the nature of the intervention and its pragmatic character, researchers were not blinded for group allocation. This study was approved by the ethical committee of the LUMC, the Netherlands, and was conducted in concordance with the principles of the Declaration of Helsinki [20], as amended in Seoul 2008. The trial conformed to the Consolidated Standards of Reporting Trials (CONSORT)-eHealth Checklist (Multimedia Appendix 1) [21]. BODY.METHODS.INTERNET-BASED SELF-MANAGEMENT SUPPORT PROGRAM: The IBSM support program is based on focus groups [22], the Chronic Care model [23], and known key components for effective self-management [5]. The program was aimed at supporting patients in conducting self-management activities and developing a patient-provider partnership in asthma care [3]. Focus groups were conducted to explore barriers for conducting self-management skills and to identify the potential role of an IBSM support tool. In particular, patients with asthma that was not well controlled (ACQ>0.75) were motivated to use novel information and communication technologies for management of their disease. The Chronic Care model is aimed at improving health care outcomes for patients with a chronic disease by means of a proactive patient-professional partnership that addresses both organizational factors (eg, decision support systems) and resources (eg, self-management support). We incorporated modules for electronic monitoring of asthma control and lung function (weekly ACQ and FEV1), a personal action plan, communication with a respiratory nurse (RN), and education. During 12 months of follow-up, IG patients had access to IBSM support (Multimedia Appendix 2); after this period, IBSM support (including access to the website) was automatically terminated. Patients were instructed on how they could log in by using a personal username and password and how to use their personal action plan. The program included reminder options for monitoring activities (ie, ACQ, day and night symptom score, lung function), which were initially sent once weekly by either email or mobile phone SMS text messaging, but during follow-up frequency could be adjusted according to the preferences of the individual patient. Patients received immediate feedback (to maintain, step up or step down in medication, and/or to contact a health care professional) on self-monitoring outcomes according to a treatment algorithm (Figure 1) and a predefined action plan based on 6 medication steps (Table 1). Five respiratory physicians, two general practitioners with a particular focus on respiratory diseases, and two respiratory epidemiologists participated in the development of this algorithm. Action plans of patients were based on their actual medication at the time of study enrollment. Treatment steps corresponded with (inter) national guidelines [3,24] on asthma management. Briefly, asthma medication can be aimed at (1) decreasing of airway narrowing (ie, beta2-agonists), and/or (2) decreasing airway inflammation (ie, glucocorticosteroids, leukotriene modifiers). A traffic light display (Figure 2) was used to indicate the level of asthma control: green (well-controlled, ACQ≤0.5), yellow (0.5<ACQ<1.0), orange (1.0<ACQ<1.5), and red (uncontrolled, ACQ score≥1.50). After each medication change, a 4-week evaluation period commenced during which no advice to change treatment was given, except in the case of symptom deterioration. E-messaging, telephone, or Web-based communication allowed patients to interact with the RN. Additionally, the RN supported patients by nurse-initiated communication characterized by a supportive style to give positive feedback on achieved successes (eg, step-down in medication) or to inquire for reasons on not following treatment advice (eg, side effects). The RN reminded patients to fill in research questionnaires at 12 months of follow-up. On average, the RN spent 1-2 hours per week on patient- and nurse-initiated communication for all IG patients. Education components of IBSM support were provided both online (eg, educational pages, newsfeed) and offline. Offline education consisted of a group education session that dealt with topics related to asthma self-management, usage of the IBSM tool, and designing an action plan based on current medication. Online information was based on information provided by the Lung Foundation Netherlands. Newsfeed content was kept up to date and contained items related to asthma and management of chronic diseases (eg, healthy lifestyle). During the study, neither major content/functionality changes nor bug fixes were required. Program content was developed by JKS in close collaboration with the departments of Public Health and Primary Care (LUMC), Respiratory Medicine (LUMC, Amsterdam Medical Center), and Haga Teaching Hospital, The Hague. Software was developed by Furore BV, Amsterdam. Table 1 Medication treatment steps. Step a Medication 1 As needed rapid-acting beta2-agonist b 2 Low-dose inhaled glucocorticosteroids 3a Low-dose inhaled glucocorticosteroids + long-acting beta2-agonist 3b Medium-dose inhaled glucocorticosteroids 3c High-dose inhaled glucocorticosteroids 4a Medium-dose inhaled glucocorticosteroids + long-acting beta2-agonist 4b High-dose inhaled glucocorticosteroids + long-acting beta2-agonist 4c Contact RN or other health care provider: consider addition of leukotriene modifier 5 Contact RN or other health care provider: consider addition of oral glucocorticosteroids a Step numbers correspond with GINA guideline treatment steps [ 3 ]. b Applies to all treatment steps as this is reliever medication. Figure 1Treatment algorithm. Figure 2Traffic light display. BODY.METHODS.USUAL CARE: Patients allocated to UC received care as usual by their health care provider. According to the Dutch College of General Practitioners [24], each patient should be provided with a (paper-based) action plan and be invited for a medical review at least once a year. BODY.METHODS.ADDITIONAL FOLLOW-UP 30 MONTHS AFTER BASELINE: Patients who previously participated were invited, by a letter containing information on the follow-up measurements, to attend the LUMC for follow-up measurements at 30 months after baseline (Table 2). Nonresponding patients received a reminder letter within 2-4 weeks and an additional telephone call. All participants gave written informed consent during this visit, prior to obtaining measurements. Patients were asked to report on their daily dose of inhaled corticosteroids (ICS) and to complete 2 paper-based questionnaires, namely an ACQ (including FEV1) and an AQLQ [19], a validated 21-item questionnaire for assessment of asthma-related quality of life. The minimal clinical important difference for the ACQ is -0.5 and for the AQLQ is 0.5 [25,26]. Both questionnaires have a 7-point scale. Patients were asked to withhold short-acting beta2-agonists for 6-8 hours prior to FEV1 measurement. Questionnaires were sent in the mail to patients who were unable or unwilling to attend the LUMC, and an additional home visit was scheduled in case of unavailability of a Piko-1 meter. Inhaled corticosteroid doses were reported as fluticasone equivalents. BODY.METHODS.STATISTICAL ANALYSIS: Differences in characteristics at baseline (null months) were analyzed between participants and nonparticipants of both groups (UC and IG) with unpaired t tests. ACQ and AQLQ scores, FEV1, and daily ICS dose were compared between participants from both groups by applying linear mixed-effect models. ICS doses were reported as fluticasone equivalents. Within- and between-group differences were analyzed with paired and unpaired t tests, respectively. Statistical analyses were carried out by intention to treat. For analysis, Stata 9.2 was used. Subgroup analyses were conducted for patients with well-controlled (ACQ≤0.75) and uncontrolled asthma (ACQ>0.75) [17] at baseline. Table 2 Outcome measures. Characteristics IBSM support (IG only) Baseline 3 months 12 months 30 months/additional follow-up Clinical Asthma control (ACQ) X X X X Lung function (FEV 1 ) X X X X Daily inhaled corticosteroid X X X X Quality of life Asthma-related quality of life (AQLQ) X X X X BODY.RESULTS.CHARACTERISTICS: In total, 107 out of 200 (54%) invited patients consented to participate for additional follow-up at 1.5 years after finishing the SMASHING study (Figure 3), of whom 60 patients were previously allocated to UC and 47 patients to the IG. Participants in the IG differed in baseline ACQ scores from nonparticipants (0.93 vs 1.29; P=.009). Apart from these differences, no other differences in clinical characteristics at baseline between participants and nonparticipants in each group were identified. Table 2 gives an overview of baseline characteristics of study participants. Baseline characteristics of both groups were similar (Table 3). BODY.RESULTS.CLINICAL OUTCOMES: Twelve months after baseline, significant improved outcomes in favor of the IG were demonstrated for asthma-related quality of life (AQLQ) at 0.37 (95% CI 0.14-0.61) and asthma control of -0.57 (95% CI -0.88 to -0.26) as compared with UC. For those who participated at 30 months, a difference in ACQ score between baseline and 12 months was 0.43 (95% CI 0.21-0.66) in favor of the IG. For those who did not participate at 30 months, a difference between baseline and 12 months of 0.34 (95% CI 0.06-0.61) in favor of the IG was detected. However, there was no significant difference in effect (in ACQ score between 0 and 12 months) between participants (0.43) and nonparticipants (0.33) at 30 months. At 30 months after baseline, a significant and slightly attenuated improvement was shown for both AQLQ (adjusted between-group difference 0.29 [95% CI 0.01-0.57]) and ACQ (adjusted difference of -0.33 [95% CI -0.61 to -0.05]) scores in favor of the IG (Figures 4 and 5). No such differences were demonstrated for ICS dosage and lung function measured as FEV1 (Figures 6 and 7). Patients with uncontrolled asthma at baseline (ACQ≤0.75) had significant better outcomes at 30 months for AQLQ (adjusted within-group difference of 0.52 [95% CI 0.10-0.95]) and asthma control (adjusted difference -0.44 [95% CI 0.04-0.85]) in favor of the IG. Table 3 Baseline characteristics. Characteristics Internet group n=47 Usual care group n=60 Age, mean (SD) 36 (8.7) 37 (8.0) Gender, n (%) Male 12 (26) 19 (32) Female 35 (74) 41 (68) Smokers, n (%) 25 (53) 27 (45) Inhaled corticosteroids (µg/day), mean (SD) 455 (279) 476 (338) AQLQ score, mean (SD) a 5.88 (0.74) 5.84 (0.82) ACQ score , mean (SD) b 0.93 (0.60) 0.97 (0.65) Prebronchodilator FEV 1 , mean (SD) c 3.26 (0.80) 3.41 (0.96) Prebronchodilator FEV 1 (% predicted), mean (SD) c 96.8 (20) 95.5 (18) a Score range (worst-best), 1-7; minimal clinical important difference (MCID): 0.5. b Score range (worst-best), 6-0; MCID: 0.5. c Number of available observations in the Internet group is 26 and in the usual care group 37. Figure 3Flowchart of study participants. Figure 4Mean Asthma Quality of Life Questionnaire score for the Internet and Usual care group as measured at 0, 3, 12, and 30 months of follow-up. Figure 5Mean Asthma Control Questionnaire score for the Internet and Usual care group as measured at 0, 3, 12, and 30 months of follow-up. Figure 6Mean ICS dosage for the Internet and Usual care group as measured at 0, 3, 12, and 30 months of follow-up. Figure 7Mean FEV1 for the Internet and Usual care group as measured at 0, 3, 12, and 30 months of follow-up. BODY.DISCUSSION.PRINCIPAL FINDINGS: This study indicates that provision of IBSM support for 1 year leads to sustained benefits in terms of asthma control and asthma-related quality of life as compared with usual care, even up to 1.5 years after terminating support. IBSM support was additional to usual care and consisted of education, an action plan, self-monitoring, and regular medical review. Therefore, this study illustrates that sustained health improvements in health can be achieved by a structured care approach and self-management support as outlined by the Chronic Care model [23]. To our knowledge, this is the first study on long-term outcomes of Internet-based comprehensive self-management delivered to patients with asthma in primary care. In the study by Thoonen et al [27], it was demonstrated that guided self-management to patients with asthma in Dutch general practice for 2 years leads to small but sustained benefits in asthma control (except for lung function) and asthma-related quality of life as compared to usual care. BODY.DISCUSSION.LIMITATIONS: The original SMASHING study had several strengths, which have been discussed at length elsewhere [15]. Briefly, the study had a strong randomized controlled design without major baseline differences between patients in both groups and was characterized by a pragmatic attitude [28]. Nevertheless, results of this follow-up measurement need to be interpreted with some caution. First, our response rate was relatively low compared to other long-term outcome studies [29], which might limit the generalizability of our results. Second, even though a sustained effect for both asthma control and asthma-related quality of life in favor of the IG was demonstrated, these differences did not reach the threshold of a clinical important difference (MCID). However, we included patients with well-controlled asthma at baseline. In contrast, the subgroup of patients with uncontrolled asthma at baseline did show a clinical relevant difference in terms of asthma-related quality of life. Patients with worse asthma control have a larger room for improvement and could therefore be more willing to participate in self-management activities [30]. Even though IG patients had better outcomes in terms of asthma control and asthma-related quality of life as compared with UC patients, this difference was not accompanied with a higher ICS dosage for IG patients. Whether this can be attributed to the ability of the individual IG patient to tailor treatment to his or her need remains unclear. Moreover, the propensity to change in-treatment steps could have been reduced as guidance by IBSM support was terminated. To what extent demonstrated long-term benefits can be attributed to the mode of delivery, beyond incorporating components (education, monitoring, action plan, and regular review) for effective self-management, remains unclear. More specifically, whether the IBSM approach has led to improved self-management skills can only be postulated as we did not collect data on self-management outcomes, such as self-efficacy or compliance. If self-management skills have been improved, this is due to more intensive support in the start-up phase, since at 12 months after baseline only about 60% of the patients were still using the program on their own initiative [16]. This illustrates that patients do not seem to develop dependence on modern technology, a known barrier for both patients and professionals for using modern technology for asthma self-management support [31]. Moreover, it also suggests that intensity of a self-management support program should be tailored to the individual patient; once a patient has met his or her personal objective, the intensity should be adjusted. Recently, Ryan et al [32] compared a comprehensive self-management approach for adults with poorly controlled asthma (ACQ≥1.5) in general practice in which the monitoring module was either paper-based or mobile phone-based. After 6 months follow-up, both patient groups had improved in terms of asthma control and self-efficacy, but no difference was demonstrated between groups. Clearly, this study does illustrate that there is room for improvement in provision of routine asthma care, since in both groups self-management was delivered in concordance with asthma guidelines (consisting of education, self-monitoring, action plan, and guidance by a professional). Nevertheless, this does not imply that modern technology might not be important since it does offer opportunities to enhance adoption of self-management support within routine care. BODY.DISCUSSION.CONCLUSIONS: From a patient perspective, there is an increasing demand to use modern technology in the management of chronic disease. In asthma, this is illustrated by a growing number of available apps for asthma "self-management" [33]. Unfortunately, these apps are characterized not only by their lack of all components for effective self-management but also by a lack of reliable content. Given the sustained benefits, cost-effectiveness becomes more favorable for IBSM, since major intervention costs, such as equipment and education sessions, apply to the first year, while maintenance costs can be spread over a longer period and could be improved by an increased number of users. For successful adoption of IBSM within routine care and into a patient's daily life, several preconditions need to be identified and addressed among stakeholders [34,35]. For instance, IBSM support should address daily routines of patients, for example, frequency of monitoring should be able to be adjusted to the needs of the individual patient. From an organizational point of view, an adequate infrastructure for asthma care (eg, routine consultations) should be available within practices. Moreover, technology should be integrated within the current available digital infrastructure. At a professional level, (Internet-based) self-management support requires a proactive role from the health care provider, which allows for a patient-professional partnership. Finally, from an economic point of view, financial incentives such as adequate reimbursements of lung function meters and consultations by health care insurance companies could be considered. More research is needed on the question of how to embed self-management support by means of modern technology within routine practice. A comprehensive Internet-based self-management approach leads to sustained benefits in terms of asthma-related quality of life and asthma control, even up to 1.5 years after terminating support, particularly for patients with asthma that was not well controlled at baseline. Future research is needed to gain insight on long-term outcomes, cost-effectiveness, and strategies for integration of self-management support by modern technology in real-life settings.

TITLE: Effects of various food ingredients on gall bladder emptying ABSTRACT.BACKGROUND/OBJECTIVES:: The emptying of the gall bladder in response to feeding is pivotal for the digestion of fat, but the role of various food ingredients in contracting the gall bladder postprandially is not well understood. We hypothesized that different food ingredients, when consumed, will have a different effect on stimulating gall bladder emptying. To investigate this we designed two randomized, investigator-blind, cross-over studies in healthy subjects using magnetic resonance imaging (MRI) to measure gall bladder volumes serially and non-invasively. ABSTRACT.SUBJECTS/METHODS:: Study 1: exploratory study evaluating the effects of 10 different food ingredients on gall bladder emptying in eight healthy subjects. The choice of ingredients varied from common items like coffee, tea and milk to actives like curcumin and potato protease inhibitor. Study 2: mechanistic study investigating the cholecystokinin (CCK) dose response to the best performer ingredient from Study 1 in 21 healthy subjects four ways. ABSTRACT.RESULTS:: The largest gall bladder volume change in Study 1 was observed with fat, which therefore became the dose-response ingredient in Study 2, where the maximum % gall bladder volume change correlated well with CCK. ABSTRACT.CONCLUSIONS:: These serial test-retest studies showed that the fasted gall bladder volume varied remarkably between individuals and that individual day-to-day variability had wide coefficients of variation. Improved knowledge of how to stimulate bile release using food ingredients will be useful to improve in vitro–in vivo correlation of bioavailability testing of hydrophobic drugs. It could improve performance of cholesterol-lowering plant stanol and sterol products and possibly aid understanding of some cholesterol gallstone disease. BODY.INTRODUCTION: The gall bladder stores and concentrates bile salts during fasting and releases them into the duodenum in response to gastric emptying of a meal.1 The flow of bile into the duodenum is controlled by gall bladder muscular contraction, gall bladder tone, hepatic secretory pressure and relaxation of the sphincter of Oddi.2 This process is mostly regulated by the hormone cholecystokinin (CCK) that is released by the proximal intestine in response to products of macronutrients digestion, particularly fat.3, 4, 5, 6, 7, 8 Although there is good knowledge of other secretory responses to dietary composition, such as pancreatic exocrine secretion, the effect of various food ingredients on gall bladder emptying has been explored to a much lesser extent. Specific food ingredients could be effective to trigger gall bladder emptying including dietary protein that stimulates CCK secretion,9, 10 curcumin that increases gall bladder emptying11 and protease inhibitors that potentiate the CCK response to nutrients.12 Widely consumed beverages such as coffee can also be effective,13 but which food ingredients are most effective in contracting the gall bladder is not well understood. Increased knowledge would be desirable for many reasons. Bile secretion in the duodenum is key to lipid digestion.14, 15 The presence of mixed micelles of bile salts16 with lipolysis products may have an impact on the performance of highly hydrophobic, low solubility (classes II and IV biopharmaceutics drug classification system, BCS,17) compounds;18, 19 it also has an impact on the ability of plant stanol and sterol products to block dietary cholesterol and the re-circulation of endogenous cholesterol20, 21 thereby lowering levels of plasma cholesterol. Further, impaired gall bladder emptying has been linked with the pathogenesis of cholesterol gallstones.22 The main hypothesis here was that different food ingredients, when consumed, will have a different effect on stimulating gall bladder emptying. To investigate this we designed two randomized, investigator-blind, cross-over studies in healthy subjects using magnetic resonance imaging (MRI) to measure gall bladder volumes serially and non-invasively. Study 1 was an exploratory study evaluating the effects of 10 different food ingredients on gall bladder emptying in eight healthy subjects. The ingredients selected were all approved for use in the EU, a mix of technology and occasion driven food ingredients, and were chosen based on their potential ability to stimulate gall bladder emptying. Study 2 was a mechanistic study aiming at investigating the CCK dose response to the best performing ingredient from Study 1. These studies would also provide insights on normal fasting gall bladder volume ranges and their coefficients of variation. BODY.SUBJECTS AND METHODS.STUDY DESIGN: Both studies obtained local University Medical School Research Ethics Committee approval, and all subjects gave informed written consent. The study was carried out according to Good Clinical Practice principles. All test products, Case Report Forms and study monitoring were provided by the sponsor. The inclusion criteria were male or female, 18–45 years of age, healthy, with body mass index (BMI) 20–30 kg/m2. Exclusion criteria comprised pregnancy, any history of serious acute or chronic illnesses, alcohol and/or substance abuse, the presence of implant or devices in the body that are contraindicated for MRI (for example, pace makers, metal implants), use of medication that may interfere with gastrointestinal physiology and the use of sterol/stanol margarine and/or fiber supplement 30 days before and/or during the course of the study. The subjects were instructed to fast overnight for at least 8 h before the study period. On each study day, they filled in a questionnaire to confirm they followed study restrictions and eligibility. The operator who processed the data was kept blinded to the randomization code by the investigator who dispensed the test meals and saved the image files from the MRI scanner in a blind manner. The code was broken only after data processing and blind data review were completed. The MRI images were used to calculate the volume of the gall bladder at each specified time interval as described below, and the percentage (%) of gall bladder volume change from baseline and the area under the curve (AUC) between t=−5 and t=65 min were derived. The primary end point was the maximum % gall bladder volume change between t=−5 and t=65 min by test product. This was chosen as primary end point, as it takes into account the maximum gall bladder volume change occurring over the whole postprandial period investigated and also normalizes each subject's gall bladder to the starting fasted time point on each study day, making it easier to carry out comparisons in these multiple ways studies. The secondary end points were the gall bladder volume for each individual time point by test product, the % gall bladder at volume change at t=45 min, the AUC for gall bladder volume and the time to maximum gall bladder volume change. BODY.SUBJECTS AND METHODS.STUDY 1: Eight subjects were recruited and they all completed the 10-way study. They were five men and three women, 26.5±8 years old with BMI 22.5±0.6 kg/m2. The subjects consumed one of 10 test products at each of the 10 test visits (one week apart from each other) based on a computer-generated randomization schedule. The products comprised emulsions, yogurt, coffee, tea and semi-skimmed milk as specified in Supplementary Table 1. At each test visit, an MRI scan of the abdomen encompassing the gall bladder was obtained at fasted baseline before consumption of the food product (t=−5 min) and after that at times t=5, 15, 25, 35, 45, 55 and 65 min post ingestion of the test product, for a total of eight scans at each visit. Additional analysis investigated the correlation of maximum % gall bladder volume change between t=−5 and t=65 min with fat, protein and volume characteristics of the test products. BODY.SUBJECTS AND METHODS.STUDY 2: Twenty-two subjects were recruited. One subject withdrew after two occasions, and hence 21 subjects completed the 4-way study. They were 11 men and 10 women, 27±8 years old with body mass index (BMI) 22.5±0.6 kg/m2. Four of these 21 subjects failed to give at least one blood sample (for example because a blocked cannula) so there were n=17 completed subjects available for the plasma CCK analysis. The subjects consumed one of four test products at each of the four test visits (1 week apart from each other) based on a computer-generated randomization schedule. The products comprised milk-based, ready-to-drink beverages based on the results of Study 1. They were 150 ml milk-based drinks that contained the same amount of protein (9 g) and the same amount of plant stanol ester (2.23 g) but increasing amounts of fat (1.5 g, 3.5 g, 6.5 g, 10 g fat, respectively). At each test visit, an intravenous cannula was inserted in a forearm vein by an experienced research nurse. A MRI scan of the abdomen encompassing the gall bladder was obtained at fasted baseline before consumption of the food product (t=−5 min) and after that at times t=5, 15, 25, 35, 45, 55 and 65 min post-ingestion of the test product, for a total of eight scans at each visit. After each MRI scan, a blood sample was drawn from an intravenous cannula for CCK analysis. Additional secondary end points for this study were maximum plasma CCK between t=5 min and t=65 min, plasma CCK at t=45 min and correlation between maximum CCK levels and maximum % gall bladder volume change between t=5 min and t=65 min. BODY.SUBJECTS AND METHODS.MAGNETIC RESONANCE IMAGING: MRI was carried out on a 1.5T Philips Achieva (Philips Healthcare, Eindhoven, The Netherlands) whole body scanner using the whole body Transmit/Receive coil. Each subject was positioned supine in the scanner. A coarse scout scan was run to locate the position of the gall bladder and plan the position of the image planes for the following data acquisition scan. Coronal, single-shot, turbo spin-echo MRI images were then acquired with spectral presaturation with inversion recovery fat suppression. This imaging sequence was heavily T2-weighted so that liquid appears very bright and the rest of the surrounding organs appear quite dark. They comprised 30 contiguous slices with no gap, echo time TE=4.4 ms with echo spacing 4.4 ms, acquisition matrix 268 × 198 and flip angle 90° and an acquired in-plane resolution of 1.5 × 1.5 mm2 and a slice thickness of 6 mm (reconstructed 0.78 × 0.78 × 6 mm3). They were acquired in a single breath-hold of 26 s. The MRI scanning schedule allowed interleaving two subjects per morning thus increasing throughput and reducing cost. BODY.SUBJECTS AND METHODS.PLASMA COLLECTION AND CCK ASSAY: On arrival an 18-gauge BD Venflon Pro (Becton Dickinson Infusion Therapy, Helsingborg, Sweden) intravenous cannula was placed in a forearm vein of the subjects. 6.5-ml blood samples were collected fasted and at each postprandial scanning time point. The blood was transferred immediately into chilled blood collection tubes containing 0.3 ml ethylenediamine tetra-acetic acid and 5000 KIU aprotinin. The samples were cooled in an ice-bath immediately. The plasma was then readily separated by centrifugation for 10 min at 3000 g at 4 °C. The separated plasma was collected using a plastic pastette and transferred to clean tubes for immediate storage at −80 °C until assayed using radioimmuno assay with commercially available kits according to the manufacturer's instructions (EURIA-CCK, Euro-Diagnostica, obtained from Immunodiagnostic Systems, Ids Ltd., Tyne and Wear, UK) as described previously.8 BODY.SUBJECTS AND METHODS.DATA ANALYSIS, STATISTICAL METHODS AND POWER OF THE STUDIES: The image data set from each subject and each time point was recalled on a workstation and commercial software (Analyze 6, Biomedical Imaging Resources, Mayo Clinic, Rochester, MN, USA) was used to trace around the gall bladder a region of interest on each image slice using the 'Smart Edge' function. This function is commonly used as it searches for areas of high gradient to adjust the path of the region of interest, thus aiding the operator semi-automatedly. The gall bladder volume was then calculated by summing across all region of interests for that given subject and time point. These were tabulated and plotted in the Microsoft Excel to determine time courses and to calculate the AUC using the trapezoidal method. The % gall bladder volume change was calculated as (1−gall bladder volume at time t/gall bladder volume at baseline) × 100 expressed in % and with time t defined as the time of imaging postprandially (that is, between 5 and 65 min). As shown by Shapiro Wilk's test, the vast majority of the data were normally distributed and were expressed as mean±s.e.m. Accordingly, parametric statistic tests were used to assess the significance of the effect of test products and time on the end point measures, followed by post hoc Dunnett's test corrected for multiple comparison comparing all test products versus the 4-ml high-fat emulsion in Study 1 and versus the least fatty sample in Study 2. As this was a cross-over study design, each subject acted as his/her own control. The CCK plasma values were not normally distributed even after log transform, and hence for these a 2-way ANOVA was not performed. Differences were being significant at P<0.05. GraphPad Prism version 4 (GraphPad Software Inc, La Jolla, CA, USA) was used throughout. Study 1 was an exploratory study, and hence a power calculation of sample size was not available. On the basis of the results of Study 1 in which the semi-skimmed milk drink achieved a mean maximum % gall bladder volume change of 41% with an s.d.of 9%, it should be possible to detect a difference in maximum gall bladder volume change of 15% with P<0.05 and a 90% power using n=22 subjects. BODY.RESULTS: The study procedures were well tolerated and all subjects completed the studies, but one subject in Study 2 withdrew after two study days for personal reasons. Imaging subjects with an MRI scanner in-built body coil was very rapid, requiring no positioning of additional abdominal receivers on the subjects or parallel imaging calibration scans, while providing images of good quality for gall bladder volume measurements as shown by an example in Figure 1. BODY.RESULTS.STUDY 1: The primary end point was the maximum % gall bladder volume change between t=−5 and t=65 min. This was significantly different between test products as shown in Figure 2 (P<0.0001). The 20 ml high-fat emulsion yielded the largest gall bladder volume change (42%), and this was significantly larger than for all the other test products (P<0.05) apart from the 250 ml semi-skimmed milk (41% volume change, P>0.05) and the 4 ml high-fat emulsion (27% volume change, P>0.05). Comparing drinks with equal volumes showed that the semi-skimmed milk achieved a significantly higher maximum % gall bladder volume change than coffee (22%) and tea (18%) (P<0.05 for both). All data are summarized in Supplementary Table 1. Looking at the other outcomes, there was a significant effect on gall bladder volumes by meal (P<0.0001). There was a significant effect of meal type on gall bladder emptying as measured by AUC of the volume curves (P<0.0001) and also as % gall bladder volume change at t=45 min (P<0.0006). The maximum % gall bladder volume change correlated significantly with fat content (r=0.80, P<0.0052) but not with protein content (r=0.41, P<0.2) or drink volume (r=0.13, P<0.7). The slope of the line to the maximum gall bladder volume change was the highest for the milk, indicating the fastest emptying response, while the high-fat emulsion meals yielded a lower slope value, indicating a slower (albeit deeper) gall bladder emptying. BODY.RESULTS.STUDY 2: We confirmed previous work showing that, of all the ingredients tested, fat produced the strongest gall bladder response with a maximum % gall bladder volume change dose response to fat (P<0.003). The least amount of fat (1.5 g) achieved already a mean maximum percentage gall bladder volume change of 33%. The drinks containing 6.5 g and 10 g fat achieved significantly higher maximum % gall bladder volume change (P<0.01 for both). The mean maximum % gall bladder volume change data are plotted in Figure 3 and the other data are summarized in Supplementary Table 2. Looking at the other outcomes, there was a significant effect of fat content on gall bladder volumes (P<0.0001). There was a significant effect of meal fat on gall bladder emptying as measured by AUC of the volume curves (P<0.01) and also as % gall bladder volume change at t=45 min (P<0.0008). There was a dose response of the effect of meal fat on maximum plasma CCK levels (P<0.007). There was a significant effect of meal fat on plasma CCK as measured by overall AUC (Figures 4, P<0.004) and on plasma CCK at t=45 min (P<0.0008). The maximum % gall bladder volume change correlated significantly with the maximum plasma CCK (Spearman's r=0.42, P<0.0001). BODY.RESULTS.FASTING GALL BLADDER VOLUMES NORMAL RANGES: The test-retest repeated Studies 1 and 2 yielded also a unique collection of fasting gall bladder volumes that enable assessment of size and inter- and intra-subject variability. The individual data are shown in Figure 5. The mean volume of the fasted gall bladder per subject showed wide variability between 12 ml and 52 ml with test-retest coefficients of variation ranging between 3% and 42%. The overall mean of the individuals' mean fasted gall bladder volumes was 26±2 ml. There was a strong correlation of mean gall bladder size with subjects' weight (r=0.68, P<0.0001), height (r=0.50, P<0.006) and BMI (r=0.48, P<0.008) but not with age (r=0.12, P<0.5). BODY.DISCUSSION: The emptying of the gall bladder has been studied using gamma scintigraphy or ultrasound techniques.3, 4, 23, 24, 25, 26, 27, 28, 29 In recent years, MRI has been shown to measure serially the emptying of the gall bladder accurately, non-invasively and with high spatial resolution.30, 31, 32, 33, 34 In these studies, serial MRI performed well and it was an effective, non invasive and accurate way of monitoring the effects of various food products on gall bladder emptying. The main hypothesis of Study 1 that different food ingredients will have a different effect on stimulating gall bladder emptying was proven to be correct. The choice of ingredients varied from common (occasion driven) items like coffee, tea and milk to actives like curcumin and potato protease inhibitor (food manufacturing driven). Semi-skimmed milk performed well but it is difficult at this stage to discriminate whether its composition of fat and protein would have an additive effect in contracting the gall bladder. The protein contribution would need to be the subject of future studies. The largest gall bladder volume change was observed with the positive fat-containing control (20 ml of high-fat emulsion). However, gall bladder emptying induced by amounts of fat as low as 7 g (perfused in the jejunum)35 had been observed previously. Our data show that a very low amount of fat (2 g fat in 4 ml of high-fat emulsion) nevertheless achieved a marked gall bladder volume change of 27%. The potato protease inhibitor achieved a modest gall bladder volume change. The same potato protease inhibitor did not significantly affect healthy volunteers' plasma CCK at twice the dose we used.36 The 80 mg curcumin stimulus was the fourth best performer achieving a mean 23% maximum gall bladder volume change. This was, however, much lower than the 40% volume change reported at 1 h in a previous ultrasonography study.37 Whether the reason for this difference might rest on the different imaging technologies used or in the delivery vehicle (a yogurt drink in our study versus plain curcumin ingested with a glass of water in that report) is unclear. When looking at the correlation with meal components, the maximum % gall bladder volume change correlated positively and significantly with the fat content of the samples (with or without the stanol included in the calculation, Pearson's P<0.0052 and P<0.0012, respectively) but neither with the protein nor with the volume of the meals. The main conclusion from this initial scouting study was that long-chain fat with a high degree of emulsification was the best performer in stimulating gall bladder emptying, likely to be explained with current knowledge2 via the cholecystokinin (CCK) duodenal response and to be tested in our second study. Semi-skimmed milk performed well and this was one of the reasons driving the choice of a milk-based beverage as vehicle for increasing amounts of emulsified fat, while keeping protein and volume the same, in Study 2. Protein is also a potent stimulant of CCK, whereas carbohydrate is not.10 Gastric emptying of stable fat emulsions is slow8 that may be a factor contributing to the longest time to maximum gall bladder volume change shown by the 20 ml high-fat emulsion. The main hypothesis of Study 2 that the best performer of Study 1 (emulsified fat in this case) will have a dose response effect on maximum % gall bladder volume change was correct. This correlated well with increased plasma CCK levels in keeping with the well-established role of fat in releasing CCK, which depends on fatty acid chain length,38, 39 and the role of CCK in regulating gall bladder emptying.40 Interestingly the milk-based beverage with the lowest amount of fat (1.5 g) achieved already a marked mean maximum percentage gall bladder volume change of 33% over the length of time investigated. Our meals were all low calorie (for example, 90 kcal for the 20 ml high-fat emulsion). Much larger volume changes have been observed with higher calorie meals. A 70% gall bladder volume change was reported 45 min after a 465 kcal mixed solid-liquid meal41 and 70% gall bladder volume change 70 min after a 675 kcal fat emulsion test meal8 in healthy subjects. From the test-retest studies, we have also observed that the fasted gall bladder volume varied remarkably between individuals and that individual day-to-day variability can have wide coefficients of variation of up to 42%. An influence of gender, age and body size on gall bladder volume has been reported from large surveys.42, 43, 44 Our mean fasted value of 26 ml is higher than previously reported values in the 18-ml range,37 and this could be because of imaging technology and/or subjects' cohort differences. One possible explanation for the large intra-individual variation in fasting gall bladder volumes is the cephalic phase response to food. The subjects may have been exposed to food-related sensory stimulation at home or on their way to the study site. Cephalic, pre-ingestive neural signals at the thought, sight or smell of food can trigger a number of physiological and secretory responses,45 while sham feeding clearly induces gall bladder emptying.46 Although in this study it was not possible to control the subjects' environment overnight before the morning measurements, future studies could consider this option in the study design. Gall bladder volume may also fluctuate with the cyclic variations of the migrating motor complex and motilin levels.47 We observed a correlation between gall bladder fasting volume size and individuals' body frame but this was not surprising.43 Because of the different volume, taste/aroma, color and texture of the samples, we could not exclude the presence of a possible ingestive cephalic component of gall bladder emptying in these data. One thought for future studies would be to manufacture the new test samples as equal in volume/texture/flavor/color as possible. Cephalic effects could also help to explain the wide variation in time to maximum gall bladder volume change, which varied not just inter-subjects but also between study days. It is worth noting that in some of the experiments carried out the time to maximum gall bladder volume change was 65 min, indicating that the volume change could possibly have been greater at later times than the period investigated. The time period investigated is a limitation of this study, and it would be desirable in future studies to extend the monitoring time window to at least 120 min to fully capture the time course of the emptying of the gall bladder. Intravenous infusion of CCK48 could be used as a gall bladder emptying standard for comparison. In conclusion, various food ingredients are capable of inducing varying degrees of gall bladder emptying. Among the various food ingredients and actives tested here, fat and semi-skimmed milk induced the most profound volume changes. Fat showed the strongest effect, which correlated with plasma CCK levels. Improved knowledge of how to stimulate bile release using food ingredients will be useful to improve in vitro/in vivo correlation of bioavailability testing of hydrophobic drugs. It could improve performance of cholesterol-lowering plant stanol and sterol products and possibly aid understanding of some cholesterol gallstone disease.

TITLE: Betahistine as an add-on: The magic bullet for postoperative nausea, vomiting and dizziness after middle ear surgery? ABSTRACT.PURPOSE:: Patients undergoing middle ear surgery experience variable degrees of postoperative nausea and vomiting (PONV) despite prophylaxis and treatment with ondansetron or other 5HT3 receptor antagonists. Furthermore vertigo or dizziness are not well controlled perioperatively. Role of betahistine was tested as an add-on to ondansetron in control of PONV and vertigo in middle ear surgery cases. ABSTRACT.MATERIALS AND METHODS:: We conducted a prospective, randomized, double-blind, placebo controlled study, enrolling one hundred patients undergoing middle ear surgery under local anesthesia into two groups consisting of fifty (n = 50) patients each. Group A patients were given betahistine 16 mg plus ondansetron 8 mg and placebo plus ondansetron 8 mg were given to group B or placebo group, orally 3 hours before starting operation. The incidence of nausea, vomiting, and dizziness was noted during the intraoperative and postoperative 24 hours period. Chi-square test, unpaired 't' test, and Fisher's exact tests were performed for statistical analysis using SPSS version 16 and Open Epi version 2.3.1 softwares. ABSTRACT.RESULTS:: Complete response was obtained in 90% patients in the betahistine group as compared to 66% in the placebo group. Vomiting in the intraoperative and postoperative period was noted in 4% and 8% cases, respectively, in the betahistine group as compared to 18% and 26%, respectively, in the placebo group. Overall, vertigo was 10% versus 32% in betahistine group and placebo group, respectively. ABSTRACT.CONCLUSION:: Betahistine as an add-on to ondansetron can significantly attenuate PONV and perioperative vertigo, following middle ear surgeries. BODY.INTRODUCTION: Postoperative nausea and vomiting (PONV) are among the most common adverse events following surgery, anesthesia, and opioid analgesia, with an estimated incidence of 25-30%.[1] The aetiology is multifactorial that includes patient factors, type of surgery, and anesthetic technique.[2] Middle ear surgeries disturb the vestibular system and are associated with high incidence of PONV and vertigo, which are further aggravated by use of opioids. A 62-80% incidence of PONV following middle year surgery has been reported.[34] Reduction in PONV and opioid-induced emesis is more with use of ondansetron and other 5HT3 receptor antagonists (5HT3 RA) than other groups of antiemetics, but many patients still experience PONV and vertigo. No reduction of PONV occurs in patients with a history of motion sickness.[4] Involvement of multiple types of receptors and factors like disturbances in the inner ear from surgical stimulation may be a reason for inadequate control of PONV with a single agent. Betahistine, a histamine agonist at H1 receptor and antagonist at H3 receptor, is widely used in Meniere's disease and other types of vertigo where disturbance or imbalance in vestibular system is considered as an etiological factor.[5] We noticed that some of our patients, on betahistine pre-operatively for some indication, suffered from less incidence of PONV. Hyoscine (scopolamine), used for suppression of motion sickness due to the activity on the vestibular system, has been reported to successfully control PONV in middle ear surgeries.[3] In this pilot study, we evaluated the effectiveness of betahistine as a routine add-on pre-medication in control of PONV and vertigo in middle ear surgeries. BODY.MATERIALS AND METHODS: After approval from the Hospital Research and Ethics committee, a prospective, randomized, double-blind, placebo controlled study was conducted on adult patients undergoing tympanoplasty or mastoidectomy under local anesthesia. Written informed consent was taken from all the patients and their legal guardian for participation in the study. One hundred patients scheduled for either tympanoplasty or mastoidectomy (from July 2010 to November 2011) was divided in two groups of fifty each using web-based randomization. All patients consented to undergo surgery under local anesthesia (LA). The baseline data of hospital anxiety and depression was taken for all patients with Hospital Anxiety Depression Scale (HAD), a validated scale for this purpose. Only the patients with a baseline HAD data <7 were included in the study.[6] Group A patients were given betahistine 16 mg and ondansetron 8 mg (orally) 180 min before the start of surgery. Group B patients were given placebo and ondansetron 8 mg (orally) 180 min before starting surgery. Riboflavin tablet 20 mg was used as placebo. Inclusion criteria included American Society of Anesthesiologists (ASA) grade I and II patients, both males and females, aged 20-60 years, scheduled for tympanoplasty and/or mastoidectomy and with a HAD score <7. Patients not consenting for surgery under local anesthesia, lack of valid consent, patients with gastrointestinal disease, Meniere's disease, vertigo, dizziness, pregnant, menstruating, patients on regular anti-emetics, anti-vertigo, anti-psychotic drugs, having severe hypertension, coronary insufficiency, psychiatric disorders, and communication problems were excluded from the study. After admission, detailed history was taken from the patients to exclude any other disease and they were subjected to routine investigations. History was taken regarding the regular use of antiemetics, motion sickness, or vomiting within 24 hours before operation. All patients were advised to be nil per oral for solid food for 6 hours and for water for 2 hours before operation. Ranitidine 300 mg and lorazepam 1 mg were administered orally 4-5 hours before surgery. About 180 min before starting the surgery, group A patients were given betahistine 16 mg and ondansetron 8 mg orally, while group B patients were given placebo and ondansetron 8 mg orally. About 15 min prior to the surgery, all patients were given local block with bupivacaine 0.25%. Skin infiltration with lignocaine 0.1% with adrenaline (1 in 1,00,000) was done in all patients to reduce bleeding from the wound. The total volume of local anesthetic used was 10-12 ml. All patients were given midazolam 2 mg intravenous (IV) for sedation and anxiolysis. Monitoring of non-invasive blood pressure, heart rate, and pulse oximetry was done. Postoperative pain was treated with diclofenac 1 mg/kg intramuscular (IM) in all patients (n = 100). Nausea in the operative period was recorded whenever complained voluntarily. Patients were asked to report any nausea and vertigo during the operative procedure. Patients were assessed intraoperatively and up to 24 hours postoperatively for the subjective complaints like nausea, vomiting (including retching), and vertigo by a trained nurse unaware of the study. Nausea in the postoperative period was recorded in a 100 mm VAS scale at 3, 6, 12, and 24 hours postoperatively. Vertigo was evaluated by categorical response of the patients to the question, "Are you feeling vertigo, yes or no?" or if the patient complained voluntarily. Incidences of postoperative severe nausea, retching, and vomiting occurring after 6 hours were treated with ondansetron 4 mg IV. Prochlorperazine 10 mg was given IM in case of vomiting during surgery in the first 6 hours after surgery and in case patient complained of dizziness and vertigo postoperatively. Adverse reaction to any drug was noted during the study period. The primary efficacy parameters were complete response (CR), which included patients free from PONV and no rescue antiemetic in a defined period. Incidence of vomiting in the operative, postoperative 24 hours, and overall period (operation and postoperative 24 hours) were measured. The secondary efficacy parameter was incidence of nausea and/or vertigo during operation, at post operative 24 hours, and after overall period. Any association between nausea, vomiting, and vertigo was also to be noted. Use of rescue antiemetics and anti-vertigo medication was also noted. The demographic data like age, sex, body weight, history of motion sickness, and duration of surgery in two groups were compared using unpaired 't' test and Chi square tests using statistical software SPSS, version 16. Data on complete response, nausea, vomiting, and vertigo were compared and analyzed using uncorrected and Yates corrected Chi-square tests and Fisher's exact tests with statistical software (SPSS version 16 and Open Epi version 2.3.1). A P < 0.05 was considered statistically significant. BODY.RESULTS: There was no significant difference in the age, sex distribution of the patients of both the groups, and duration of anesthesia or operation between two groups. ± (Group data were presented as mean ± SD) [Table 1]. Table 1 Demographic figures of the patients Number of patients with CR, incidences of nausea, vomiting, and vertigo are shown in [Table 2]. The detail of the patients who had clinically significant nausea (VAS >50 mm), vomiting, and vertigo during surgery and first 24 hours of postoperative period in two groups and (shown and compared) in [Figures 1–3]. Any patient with a VAS score for nausea >50 were considered as clinically significant and score >75 was taken as severe nausea.[7] In the betahistine group, CR was observed in 90% cases as compared a CR 66% in the other group (P <0.5) [Figure 4]. Rescue antiemetics were administered to all such patients (n = 24) [Table 2; Figure 5]. Table 2 The incidences of nausea, vomiting, and vertigo in two groups of patients during surgery and then up to 24 hours of postoperative period Figure 1Comparative incidences of vomiting. Higher incidences were noted in the placebo group Figure 2Comparative nausea in both groups of patients. Higher number of patients experienced nausea in the placebo group at all three time frames Figure 3Comparative vertigo in both groups of patients. More number of patients experienced vertigo in the placebo group Figure 4Complete response in two groups during surgery, postoperative 24 hours, and overall period. Numbers of patients are plotted along the "Y" axis (vertical axis) and time frame along the "X" axis (horizontal axis). The number of patients who had complete response was higher in the betahistine group in all the three time frames as compared to placebo group Figure 5Rescue antiemetic use in two groups. (Vertical axis depicting number of patients and horizontal axis depicting different time frame) In the placebo group, 9 patients complained of nausea, retching, and vomiting during operation and 13 patients during 12 hours of postoperative period, however, emesis was present in 15 patients overall in the placebo group, indicating repeated occurrence of vomiting and nausea in same patients. A strong association was noted between vertigo and emesis in these middle ear surgery cases. Most of the patients who had experienced vertigo or dizziness during surgery or within 24 hours after surgery also experienced clinically significant nausea and vomiting. No adverse effects to any of the study drugs were noted in the study period. BODY.DISCUSSION: Addition of betahistine in the PONV prophylaxis regimen for middle ear surgery was superior to the placebo group in the control of nausea, vomiting and vertigo. Most patients who experienced vertigo in the operative or post-operative period eventually developed PONV. Use of rescue antiemetic was less when betahistine was used in combination with ondansetron for prophylaxis of nausea and vomiting. Tympanoplasty and mastoidectomy are two of the most common procedures performed on the middle ear. Both these surgeries are associated with a high incidence of PONV. Patients experiencing PONV consume more resources and require additional healthcare professional time.[8] A wide range of drugs are used to treat/prevent PONV that includes phenothiazines, anticholinergics, anti-histamines, butyrophenones, substituted benzamides, corticosteroids, 5-HT3 receptor antagonists (5HT3RA), and NK1 receptor antagonists.[9] Drug like hyoscine, primarily used in motion sickness and have been found to be effective in control of PONV and dizziness following middle ear surgery.[310] Ondansetron, a 5HT3RA, is one of the most commonly used drugs for PONV prophylaxis and control. Combination of drugs is recommended for PONV, as it is superior to monotherapy.[11] Although Ondansetron controls PONV effectively in diverse situations, it is not effective in motion sickness and vertigo.[12] Betahistine, a structural analog of histamine with H1 agonist and H3 antagonist activity, is effective in vertigo of both central and peripheral origin.[13] H1-mediated vasodilatation improves microcirculation in the inner ear and vestibular nuclei, a conventionally postulated theory for the mechanism of anti-vertigo action of betahistine. H3 antagonism increases histamine synthesis and release from the vestibular nucleus, further augmenting H1 action. Spike generation from the neurons of lateral and medial vestibular nucleus is also reduced. These actions along with a less specific action on alertness are supposed to improve vestibular compensation.[1415] Uncontrolled change in resting discharge of vestibular nuclei, which is responsible for vertigo, is suppressed both by betahistine and its active metabolite.[16] Betahistine has an established role as an anti-vertigo agent in Meniere's disease.[17–20] It's use has also been found to benefit in motion sickness.[21] However, there are no studies supporting the role of betahistine in PONV. The exact mechanism of nausea, vomiting, and vertigo in middle ear surgery is not known, but physical stimulation in the form of drilling and irrigation in the bones adjacent to the inner ear at the time of surgery is a postulated mechanism. Physical stimulation leads to the generation of noises of different frequencies, producing low frequency sound (LFS) stimulation. LFS is a major contributing factor for instability in Meniere's disease, chronic suppurative otitis media (CSOM) with vertigo, and vertigo of peripheral origin. This sound induced disequilibrium or "Tullio phenomenon" is due to activation of vestibulospinal response. Similarly, sound waves generated during middle ear surgery may lead to expression of "Tullio phenomenon."[22] There is some similarity in the underlying mechanism of vertigo-dizziness in middle ear surgery and that due to Meniere's disease or vertigo of peripheral origin. Drugs that suppress vestibular stimulation or improve vestibular compensation might have some role in the control of dizziness and vomiting associated with middle ear surgery. Hyoscine, a drug that controls motion sickness, was found to be effective in control of PONV in middle ear surgeries.[39] Many patients of middle ear surgery are noticed to complain of dizziness and vertigo in the intra and postoperative period and this could possibly be attributed to LFS and physical stimulation. There is considerable association between occurrences of vertigo-dizziness with nausea-vomiting in these cases. Complaints of vertigo and dizziness by a group of patients who eventually ended up with vomiting probably indicate some cross-link between vertigo-dizziness and nausea-vomiting in middle ear surgery cases. Betahistine probably acts by intersecting at this point. Choosing local anesthesia provided some additional benefit in planning the study. Local anesthesia for middle ear surgery is feasible, cheap, safe and is one of the popular methods employed in such surgeries in some parts of the world.[23] Free verbal communication with the patients is possible in the intra-operative period, which helped in assessment of symptoms of nausea, vomiting, and vertigo during this period and early postoperative period, which would have been impossible if patients are administered a general anesthetic. The weakness of the study is a small sample size. Non-inclusion of cases under general anesthesia can be considered another limitation. Further the role of betahistine as an individual agent was also not assessed in this study. The use of prophylactic betahistine preoperatively in conjunction with ondansetron for middle ear surgery was found to significantly reduce PONV and dizziness in patients after middle ear surgery, and it was also associated with superior control of perioperative vertigo and nausea and vomiting. Further study in this field is suggested with a larger sample size including both local and general anesthetics. Any possible direct antiemetic action of betahistine also needs further research. Larger sized randomized controlled trials may be conducted to justify the role of betahistine.

TITLE: Targeted therapy for high-grade glioma with the TGF-β2 inhibitor trabedersen: results of a randomized and controlled phase IIb study ABSTRACT: This randomized, open-label, active-controlled, dose-finding phase IIb study evaluated the efficacy and safety of trabedersen (AP 12009) administered intratumorally by convection-enhanced delivery compared with standard chemotherapy in patients with recurrent/refractory high-grade glioma. One hundred and forty-five patients with central reference histopathology of recurrent/refractory glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) were randomly assigned to receive trabedersen at doses of 10 or 80 μM or standard chemotherapy (temozolomide or procarbazine/lomustine/vincristine). Primary endpoint was 6-month tumor control rate, and secondary endpoints included response at further timepoints, survival, and safety. Six-month tumor control rates were not significantly different in the entire study population (AA and GBM). Prespecified AA subgroup analysis showed a significant benefit regarding the 14-month tumor control rate for 10 μM trabedersen vs chemotherapy (p= .0032). The 2-year survival rate had a trend for superiority for 10 μM trabedersen vs chemotherapy (p = .10). Median survival for 10 μM trabedersen was 39.1 months compared with 35.2 months for 80 μM trabedersen and 21.7 months for chemotherapy (not significant). In GBM patients, response and survival results were comparable among the 3 arms. Exploratory analysis on GBM patients aged ≤55 years with Karnofsky performance status >80% at baseline indicated a 3-fold survival at 2 and 3 years for 10 μM trabedersen vs chemotherapy. The frequency of patients with related or possibly drug-related adverse events was higher with standard chemotherapy (64%) than with 80 μM trabedersen (43%) and 10 μM trabedersen (27%). Superior efficacy and safety for 10 μM trabedersen over 80 μM trabedersen and chemotherapy and positive risk–benefit assessment suggest it as the optimal dose for further clinical development in high-grade glioma. BODY: The high-grade gliomas, mainly anaplastic astrocytoma (AA; WHO grade III) and glioblastoma multiforme (GBM; WHO grade IV), represent about 60% of all primary malignant brain tumors. Prognosis in these cases has only marginally improved despite technical advances in neurosurgery and radiotherapy and the approval of novel anticancer agents. Chemotherapy with the alkylating agent temozolomide (TMZ) has shown a clinically meaningful and statistically significant survival benefit in patients with newly diagnosed GBM with a median survival of 14.6 months with a combination of radiotherapy and TMZ vs 12.1 months with radiotherapy alone.1 The respective 5-year survival rates were 9.8% vs 1.9%.2 However, overall survival (OS) is still poor, especially in patients with recurrent or refractory tumors with a median survival of 11.3 months for patients with AA and 7.4 months for patients with GBM.3 The transforming growth factor 2 (TGF-β2) is overexpressed in more than 90% of high-grade gliomas, and its levels are closely related to tumor progression.4,5,6 Inhibition of TGF-β2 in tumor tissue leads to reversal of tumor-induced immune suppression as well as inhibition of tumor growth, invasion, and metastasis.7,8,9 Trabedersen (AP 12009) is a synthetic antisense phosphorothioate oligodeoxynucleotide complementary to the mRNA of the human TGF-β2 gene, developed as a novel, targeted treatment for patients with high-grade glioma.10 The safety and efficacy of trabedersen has been established through various pharmacokinetic and toxicology studies, both in vitro and in vivo.11 In 3 phase I/II dose escalation studies, adult patients with recurrent or refractory AA or GBM and evidence of tumor progression were treated with trabedersen.12 The maximum tolerated dose was not reached despite up to a 64-fold increase in the administered concentration. Long-lasting complete remissions were observed at doses of 10 and 80 μM trabedersen. The current randomized and controlled phase IIb study evaluated the efficacy and safety of 2 doses (10 and 80 μM) of trabedersen in comparison with standard chemotherapy in patients with recurrent high-grade glioma of either AA or GBM. BODY.METHODS: This study was conducted in accordance with the International Conference on Harmonisation (ICH) Consolidated Good Clinical Practice (GCP) Guideline E6 (Note for Guidance CPMP/135/95), the ethical principles of the Declaration of Helsinki, and the rules of the EU Directive 2001/20/EC. The study protocol was fully approved by regulatory authorities and Independent Ethics Committees/Institutional Review Boards. Written informed consent was obtained from all patients. An independent Data and Safety Monitoring Board periodically reviewed safety and efficacy data. BODY.METHODS.STUDY DESIGN: The study was multinational, open-label, randomized, and active controlled. Patients were randomized in a 1:1:1 ratio using a centralized randomization procedure to treatment with either 10 μM trabedersen, 80 μM trabedersen, or standard chemotherapy. Main inclusion criteria were: male or female patients between 18 and 75 years of age with a reference neuropathology-confirmed diagnosis of recurrent/refractory AA or GBM, Karnofsky performance status (KPS) ≥70% at baseline, with tumor lesions ≤50 cm3, and a measurable enhancing tumor lesion in MRI with a diameter ≤4.5 cm. Main exclusion criteria were: patients with tumor surgery within the last 2 weeks, radiotherapy within 8 weeks, chemotherapy within 4 weeks, or a baseline MRI showing a significant mass effect. The diagnosis of AA or GBM was confirmed by central neuropathology assessment of tissue specimens (Institute for Neuropathology, German Brain Tumor Reference Center, University Hospital Bonn, Germany). BODY.METHODS.TREATMENT: The planned duration of treatment in the study was approximately 6 months in all 3 treatment arms. The survival status of all patients was followed for at least 48 months. The standard chemotherapy control was either TMZ (150–200 mg/m2 on days 1–5 in 28-day cycles) or procarbazine/CCNU (N-(2-chloroethyl)-N′-cyclohexyl-N-nitrosourea; lomustine)/vincristine (PCV; lomustine 110 mg/m2 on day 1, procarbazine 60 mg/m2 on days 8–21, and vincristine 1.4 mg/m2 on days 8 and 29 in 56-day cycles) depending on patient's previous therapy: TMZ if the patient had not been treated with TMZ before (35 patients) and PCV if prior TMZ treatment had failed (10 patients). Study treatment for patients of the standard chemotherapy group included up to 6 treatment cycles with TMZ or 3 treatment cycles with PCV. Patients showing a response to therapy might have continued treatment at the discretion of the investigator. Patients in the trabedersen groups received up to 11 treatment cycles (14 days per cycle). One cycle consisted of trabedersen for 7 days with a flow rate of 4 μL/min, followed by isotonic saline for 7 days with a flow rate of 1 μL/min. The trabedersen dose per cycle was 2.48 mg (10 μM group) or 19.81 mg (80 μM group). Trabedersen was infused intratumorally using convection-enhanced delivery (CED) via a subcutaneous port access system connected to an external pump allowing outpatient treatment. The CED technique allows a highly efficient, homogeneous, and targeted administration of the drug, bypassing the blood–brain barrier.13,14 In contrast to other CED studies,15,16 a single catheter was placed into the solid and contrast-enhancing area of the largest tumor lesion. The drug delivery system was implanted 2 days before the start of the infusion. BODY.METHODS.STATISTICAL ENDPOINTS AND ANALYSIS: This randomized and controlled phase II study was designed to be hypothesis generating. The primary endpoint was the comparison of the 2 trabedersen dose groups for the tumor control rate after 6 months using Fisher's exact test at a 2-sided significance level of 5%. In addition, as the internal control, the trabedersen groups were compared descriptively with the standard chemotherapy group. Tumor assessments were done via central, blinded MRI readings of 2 independent radiologists and 1 adjudicator using the classical Macdonald criteria.17 The tumor control rate was defined as the percentage of patients with complete response (CR) + partial response (PR) + stable disease (SD). Assuming a tumor control rate of 50% in the 80 μM trabedersen group and of 20% in the 10 μM trabedersen group, 32 evaluable patients per arm were needed to give 80% power to reject the null hypothesis of no difference between the 2 trabedersen groups at an α-level of 10%. Assuming a dropout rate of 18%, 39 GBM patients per arm were planned to be randomized. About 8 AA patients were expected to be included in each treatment arm, resulting in a total of 141 patients to be randomized. Secondary endpoints included response at time points other than 6 months, with 14 months as the last time point with sufficient MRI data available for interpretable analysis. Further secondary endpoints included survival, progression-free survival, and safety. The OS was calculated from the date of randomization to date of death and analyzed using the Kaplan–Meier methodology. Patients who had not died at the time of analysis or had been lost to follow-up were censored at the time of their last survival assessment. Exploratory analyses were planned to be done separately for the prespecified subpopulations of AA and GBM patients. An additional exploratory analysis was done on GBM patients with the prognostic factors age ≤55 years and KPS >80%. Adverse events (AEs) were graded according to the National Cancer Institute–Common Toxicity Criteria (NCI-CTC), version 2.0. BODY.RESULTS: Altogether 145 patients were randomized, 103 with recurrent GBM and 42 with recurrent AA. Eight trabedersen patients discontinued before catheter-port implantation and 2 standard chemotherapy patients discontinued before start of treatment, thus the safety population included 135 patients. One trabedersen patient discontinued after catheter-port surgery but before start of treatment. Therefore, a total of 134 patients were treated with study medication (40 with 10 μM trabedersen, 49 with 80 μM trabedersen, and 45 with standard chemotherapy: 35 with TMZ and 10 with PCV) and represented the primary efficacy population. During the treatment period, 83 patients discontinued the study due to disease progression (57), AEs (8), patient request (6), death (5), study drug toxicity (2), loss to follow-up (1), and other reasons (4). All patients had previous first-line tumor surgery, and 91% had previous first-line radiotherapy (Table 1). The 10 μM trabedersen group included more patients >55 years (43%) than the 80 μM trabedersen (18%) or the standard chemotherapy (29%) group. Patients of the 10 μM trabedersen group also had the largest total tumor volume at baseline (Table 1). Almost all patients had brain edema at baseline. Table 1.Patient baseline characteristics (primary efficacy population, n= 134): all patients, GBM patients, and AA patients (primary efficacy population)Patient characteristics10 μM trabedersen80 μM trabedersenStandard chemotherapy (TMZ/PCV)All patientsn= 40n= 49n= 45 Median age (years)46.544.045.0 Patients >55 y (%)17 (43%)9 (18%)13 (29%) Female (%)13 (33%)12 (25%)22 (49%) Median duration of disease (months)7.06.07.0 Previous radiation therapy (%)35 (88%)46 (94%)41 (91%) Previous surgery (%)40 (100%)49 (100%)45 (100%) Previous chemotherapy (%)22 (55%)26 (53%)28 (62%) Karnofsky performance status 90–100 (%)30 (75%)34 (69%)30 (67%) 70–80 (%)10 (25%)15 (31%)14 (31%) Median tumor volume (mm3)24,15921,51015,834GBM patientsn= 28n= 34n= 33 Median age (years)56.545.552.0 Patients >55 y (%)15 (54%)7 (21%)12 (36%) Female (%)7 (25%)10 (29%)16 (49%) Median duration of disease (months)7.06.07.0 Previous radiation therapy (%)23 (82%)32 (94%)29 (88%) Previous surgery (%)28 (100%)34 (100%)33 (100%) Previous chemotherapy (%)14 (50%)21 (62%)21 (64%) Karnofsky performance status 90 or 100 (%)22 (79%)23 (68%)20 (61%) 70 or 80 (%)6 (21%)11 (32%)12 (36%) Median tumor volume (mm3)27,07226,88517,664AA patientsn= 12n= 15n= 12 Median age (years)39.040.036.5 Patients >55 y (%)2 (17%)2 (13%)1 (8%) Female (%)6 (50%)2 (13%)6 (50%) Median duration of disease (months)5.57.08.0 Previous radiation therapy (%)12 (100%)14 (93%)12 (100%) Previous surgery (%)12 (100%)15 (100%)12 (100%) Previous chemotherapy (%)8 (67%)5 (33%)7 (58%) Karnofsky performance status 90–100 (%)8 (67%)11 (73%)10 (83%) 70–80 (%)4 (33%)4 (27%)2 (17%) Median tumor volume (mm3)19,97614,34615,701AA, anaplastic astrocytoma; GBM, glioblastoma multiforme; PCV, procarbazine/CCNU (lomustine)/vincristine; TMZ, temozolomide. Patients received a median of 7 and 6 treatment cycles in the 10 and 80 μM trabedersen groups, respectively. TMZ-treated patients (n= 35) received a median of 6 treatment cycles and PCV-treated patients (n= 10) a median of 1 treatment cycle. The median overall treatment duration was 91 days in the 10 μM and 78 days in the 80 μM trabedersen groups, and 145 days for TMZ vs 29 days for PCV. The short treatment duration in PCV-treated patients could be explained by their higher median age at baseline (54 years) compared with TMZ-treated patients (43 years) and the associated inferior bone marrow capacity and prognosis. BODY.RESULTS.EFFICACY IN THE ENTIRE STUDY POPULATION: Tumor control rate at 6 months (Table 2) in the entire study population was nonsignificantly higher in the 10 μM trabedersen group (33%) compared with the 80 μM trabedersen group (20%, p= .1298) and the standard chemotherapy group (27%, p= .6219). After 14 months, the 10 μM trabedersen group had the highest tumor control rate (23%) compared with the 80 μM trabedersen group (8%, p= .1197) and the standard chemotherapy group (7%, p= .0529) (Table 2). Median survival was 12.0 months (95% CI: 7.0–26.6) with the 10 μM trabedersen group compared with 13.1 months with the 80 μM trabedersen group (95% CI: 9.3–14.8, p= .5153), and 11.0 months (95% CI: 8.7–13.7, p= .5119) with the standard chemotherapy group. The OS rate was similar in all 3 groups at 12 months (Supplementary Material, Table S1), but starting from 24 months, survival rates were nonsignificantly higher in the 10 μM trabedersen group (39%, 95% CI: 24–54) compared with the 80 μM trabedersen group (29%, 95% CI: 17–42, p= .2186) and the standard chemotherapy group (22%, 95% CI: 12–35, p= .0592). Table 2.Tumor response: all patients, GBM patients, and AA patients (primary efficacy population)Tumor responseNumber of patients (%)10 μM trabedersen80 μM trabedersenStandard chemotherapy (TMZ/PCV)All patientsn = 40n = 49n = 45 6 months Tumor control rate (CR + PR + SD)13 (33%)10 (20%)12 (27%) Overall response rate (CR + PR)2 (5%)3 (6%)3 (7%) Progressive disease19 (48%)34 (69%)23 (51%) Missing MRI data8 (20%)5 (10%)9 (20%) 14 months Tumor control rate (CR + PR + SD)9 (23%)4 (8%)3 (7%) Overall response rate (CR + PR)6 (15%)3 (6%)2 (4%) Progressive disease22 (55%)31 (63%)32 (71%) Missing MRI data9 (23%)14 (29%)10 (22%)GBM patientsn = 28n = 34n = 33 6 months Tumor control rate (CR + PR + SD)4 (14%)4 (12%)5 (15%) Overall response rate (CR + PR)01 (3%)0 Progressive disease16 (57%)26 (77%)19 (58%) Missing MRI data8 (29%)4 (12%)8 (24%) 14 months Tumor control rate (CR + PR + SD)2 (7%)1 (3%)3 (9%) Overall response rate (CR + PR)1 (4%)02 (6%) Progressive disease20 (71%)25 (74%)25 (76%) Missing MRI data6 (21%)8 (24%)5 (15%)AA patientsn = 12n = 15n = 12 6 months Tumor control rate (CR + PR + SD)9 (75%)6 (40%)7 (58%) Overall response rate (CR + PR)2 (17%)2 (13%)3 (25%) Progressive disease3 (25%)8 (53%)4 (33%) Missing MRI data01 (7%)1 (8%) 14 months Tumor control rate (CR + PR + SD)7 (58%)3 (20%)0 Overall response rate (CR + PR)5 (42%)3 (20%)0 Progressive disease2 (17%)6 (40%) 7 (58%) Missing MRI data3 (25%)6 (40%) 5 (42%)AA, anaplastic astrocytoma; GBM, glioblastoma; PCV, procarbazine/CCNU (lomustine)/vincristine; TMZ, temozolomide; CR, complete response; PR, partial response; SD, stable disease. Case reports of 1 GBM patient and 1 AA patient treated with 10 μM trabedersen are presented in Supplementary Material, Figures S1 and S2. BODY.RESULTS.EFFICACY IN GBM AND AA SUBGROUPS: As AA and GBM patients differ in their prognoses,18,19 efficacy analyses were repeated on an exploratory basis separately for the 2 subpopulations of AA and GBM patients. BODY.RESULTS.EFFICACY IN GBM AND AA SUBGROUPS.GBM PATIENTS: The primary efficacy population included 95 patients with recurrent/refractory GBM: 28 were treated with 10 μM trabedersen, 34 with 80 μM trabedersen, and 33 with standard chemotherapy. Baseline characteristics are shown in Table 1, and safety results are given in Supplementary Material, Table S2. In GBM patients, the tumor control rates at 6 months were comparable in all 3 groups (Table 2): 14% (10 μM trabedersen), 12% (80 μM trabedersen), and 15% (standard chemotherapy). Tumor control rates subsequently decreased and were 7% (10 μM trabedersen), 3% (80 μM trabedersen), and 9% (standard chemotherapy) after 14 months. Median survival was 7.3 months (95% CI: 5.0–12.0) with 10 μM trabedersen compared with 10.9 months with 80 μM trabedersen (95% CI: 5.6–13.9, p= .9370), and 10.0 months (95% CI: 7.0–13.0, p= .7310) with standard chemotherapy. At 24 months, OS was nonsignificantly higher with 10 μM trabedersen (20%, 95% CI: 7–36) compared with 80 μM trabedersen (18%, 95% CI: 7–32, p= .6783) and standard chemotherapy (15%, 95% CI: 6–29, p= .4861) (Supplementary Material, Table S1). The median of the prognostic factor age for GBM patients of the 10 μM trabedersen group was higher than in the other 2 treatment groups (56.5 vs 45.5 and 52.0 years) (Table 1) as well as the number of patients of age >55 years (54% vs 21% and 36%), which may have biased the survival results for this group. Furthermore, as it is well known that high-grade glioma patients with lower age and higher KPS have a better prognosis,18,20 an additional exploratory analysis was performed for GBM patients with the prespecified prognostic factors age ≤55 years and KPS >80%. This subgroup included 45 patients (primary efficacy population): 9 were treated with 10 μM trabedersen, 21 with 80 μM trabedersen, and 15 with standard chemotherapy. Both trabedersen groups showed favorable survival results, especially the 10 μM trabedersen group. Median survival for this group was 12.0 months (95% CI: 5.9 to not defined [ND]) compared with 10.1 months (95% CI: 7.8–13.7, p= .2972) for standard chemotherapy. At 12 months, OS was 40% (95% CI: 6–74) with 10 μM trabedersen and 40% (95% CI: 15–65) with standard chemotherapy. Of note, at 2 and 3 years, the OS rate was 3-fold higher for 10 μM trabedersen (40%, 95% CI: 6–74) compared with standard chemotherapy (13%, 95% CI: 0–31). BODY.RESULTS.EFFICACY IN GBM AND AA SUBGROUPS.AA PATIENTS: The primary efficacy population included 39 patients with recurrent AA: 12 were treated with 10 μM trabedersen, 15 with 80 μM trabedersen, and 12 with standard chemotherapy. Baseline characteristics are shown in Table 1, and safety results are given in Supplementary Material, Table S2. In AA patients, the tumor control rate was nonsignificantly higher with 10 μM trabedersen at 6 months (75%) compared with 80 μM trabedersen (40%, p= .1302) and standard chemotherapy (58%, p= .6668) and significantly higher vs standard chemotherapy at 14 months (58% vs 20% with 80 μM trabedersen, p= .1534, and 0% with standard chemotherapy, p= .0032) (Fig. 1A, Table 2). The median duration of response was 29.1 months (95% CI: 4.1–49.8) with 10 μM trabedersen, 24.0 months (95% CI: 2.3–44.9) with 80 μM trabedersen, and 8.0 months (95% CI: ND) with standard chemotherapy. The median time to progression was 22.4 months (95% CI: 1.2–42.0) with 10 μM trabedersen, 3.4 months (95% CI: 1.2–7.8) with 80 μM trabedersen, and 13.0 months (95% CI: 3.7–14.3) with standard chemotherapy. Fig. 1.Efficacy results in patients with AA (primary efficacy population, n= 39). (A) Tumor control rates (complete response + partial response + stable disease) at months 6 and 14. *Comparison between 10 μM trabedersen and standard chemotherapy: p= .0032. (B) Kaplan–Meier plot for patients treated with 10 μM trabedersen (n= 12) vs 80 μM trabedersen (n= 15). (C) Kaplan–Meier plot for patients treated with 10 μM trabedersen (n= 12) vs standard chemotherapy (n= 12). (D) Overall survival rates. Median survival in AA patients was considerably longer in the trabedersen groups: 39.1 months (95% CI 31.6 to ND) for 10 μM trabedersen and 35.2 months (95% CI: 13.9–43.1) for 80 μM trabedersen compared with 21.7 months (95% CI: 10.1–37.4) for standard chemotherapy (Fig. 1B and C), resulting in a median survival benefit of 17.4 months for 10 μM trabedersen compared with standard chemotherapy (not significant). After 12 months, the 10 μM trabedersen group had the highest survival rate (92% vs 73% and 67%) (Fig. 1D, Supplementary Material, Table S1). After 2 years, the 10 μM trabedersen group had a higher survival rate (83%, 95% CI: 48–96) compared with the 80 μM trabedersen group (53%, 95% CI: 26–74, p= .2071) and the standard chemotherapy group (42%, 95% CI: 15–67, p= .1038). At 3 and 4 years, the survival rate remained highest for 10 μM trabedersen (Fig. 1D, Supplementary Material, Table S1). BODY.RESULTS.SAFETY: A total of 98% of patients had at least one AE (Table 3). AEs of NCI-CTC grade 3 or 4 were reported for 76% of patients with 10 and 80 μM trabedersen, and 60% with standard chemotherapy (Table 4). Most of these events were nervous system disorders in all 3 groups (66% for 10 μM trabedersen, 59% for 80 μM trabedersen, and 31% for standard chemotherapy), with convulsion, brain compression, hemiparesis, and brain edema as the most common. Most AEs were related to the underlying disease. Table 3.AE overview (safety population, n= 135)AENumber of patients (%)10 μM trabedersen (n = 41)80 μM trabedersen (n = 49)Standard chemotherapy (TMZ/PCV) (n = 45)At least 1 AE40 (98%)48 (98%)44 (98%)AEs leading to discontinuation of study drug19 (46%)24 (49%)10 (22%)AEs drug related or possibly drug related11 (27%)21 (43%)29 (64%)SAEs32 (78%)37 (76%)18 (40%)SAEs drug related or possibly drug related03 (6%)1 (2%)SAEs procedure related14 (34%)14 (29%)n.a.*SAEs with outcome death11 (27%)17 (35%)10 (22%)AE, adverse event; PCV, procarbazine/CCNU (lomustine)/vincristine; SAE, serious adverse event; TMZ, temozolomide.*n.a., not applicable, no medical device was used. Table 4.Patients with NCI-CTC toxicity grade 3/4 AE in ≥10% of patients in any group (safety population, n= 135)AEsNumber of patients (%)10 μM trabedersen (n = 41)80 μM trabedersen (n = 49)Standard chemotherapy (TMZ/PCV) (n = 45)Patients with NCI-CTC toxicity grade 3 or 4 AEs31 (76%)37 (76%)27 (60%) Blood and lymphatic system disorders2 (5%)4 (8%)5 (11%) General disorders and administration site conditions10 (24%)3 (6%)7 (16%) Infections and infestations brain abscess3 (7%)6 (12%)0 Injury, poisoning, and procedural complications7 (17%)8 (16%)0 Investigations6 (15%)4 (8%)5 (11%) Karnofsky scale worsened4 (10%)4 (8%)4 (9%) Nervous system disorders27 (66%)29 (59%)14 (31%) Aphasia6 (15%)5 (10%)2 (4%) Brain compression3 (7%)5 (10%)2 (4%) Brain edema11 (27%)10 (20%)2 (4%) Convulsion5 (12%)4 (8%)3 (7%) Depressed level of consciousness5 (12%)2 (4%)0 Headache4 (10%)5 (10%)3 (7%) Hemiparesis11 (27%)11 (22%)1 (2%) Intracranial pressure increased8 (20%)7 (14%)2 (4%) Neurological symptom7 (17%)4 (8%)2 (4%) Psychiatric disorders5 (12%)3 (6%)1 (2%)AE, adverse event; NCI-CTC, National Cancer Institute–Common Toxicity Criteria; PCV, procarbazine/CCNU (lomustine)/vincristine; TMZ, temozolomide. AEs leading to permanent discontinuation of treatment were more common in the trabedersen groups: 46% (10 μM) and 49% (80 μM) compared with 22% for standard chemotherapy (Table 3), but AEs considered possibly drug related were more common with standard chemotherapy: 64% compared with 27% (10 μM trabedersen) and 43% (80 μM trabedersen). These events were most commonly blood and lymphatic disorders in the standard chemotherapy group and nervous system disorders as defined above in the trabedersen groups. Only in 4 patients were drug-related or possibly drug-related serious adverse events (SAEs) reported: meningitis, hyponatremia and brain edema, and thrombocytopenia for 3 patients in the 80 μM trabedersen group and cerebral disorder for 1 standard chemotherapy patient. Procedure-related SAEs were reported in 34% of patients receiving 10 μM trabedersen and 29% of patients receiving 80 μM trabedersen; for those respective groups, application site infection occurred in 12% and 10% of patients and complications associated with catheter placement occurred in 7% and 2%. BODY.DISCUSSION: This randomized and active-controlled phase IIb study was designed to be hypothesis generating. It had the main goals to determine the optimal dose of trabedersen for use in further clinical development in patients with high-grade glioma and to compare the efficacy and safety of trabedersen to standard chemotherapy. The primary endpoint tumor control rate at 6 months did not show statistically significant differences among the treatment groups. Endpoints based on tumor assessments early after treatment start are recognized surrogate endpoints for OS, the "gold standard" endpoint for oncology trials.21,22 However, it has turned out in recent years that this may not be appropriate for therapies that have a completely different mode of action from fast-acting cytotoxic or cytoreductive agents.22,23 This particularly applies to immuno-based therapies, such as trabedersen, which rely on building an immune response over time.23,24 Indeed, the results of this study show that the clinically relevant beneficial effect of trabedersen increases over time, which was visible especially in the 10 μM trabedersen group and was most evident at 14 months regarding tumor response (Table 2). An increase in tumor response over time for trabedersen had already been observed in phase I/II studies,12 where 2 patients had long-lasting complete tumor remissions appearing several months after initiation of treatment. One patient from these studies is still alive and in complete remission more than 8 years after start of trabedersen treatment. The other patient was in complete remission and died of an unrelated cause (myocardial infarction) more than 2 years after start of trabedersen treatment. Taken together, the full clinically relevant beneficial effects of trabedersen can be accurately assessed only at later time points (eg, after 14 months for response assessment and after 24 months for survival assessment), and this finding will be incorporated into the design of subsequent, confirmatory studies (see also below). Despite the fact that the primary endpoint did not reach statistical significance, the results indicate that the optimal dose is 10 μM trabedersen, as both efficacy (tumor control rate, tumor progression rate, and survival rate) and safety results tended to be superior for the 10 μM dose compared with the 80 μM dose, despite the inferior prognosis (higher age and tumor volume) of the 10 μM group at baseline. Although higher efficacy of the lower dose may seem counterintuitive, in vitro assays in human GBM cells have shown that 10 μM trabedersen has a stronger inhibitory effect upon TGF-β2 secretion when compared with 80 μM (manuscript in preparation), although the mechanism for this is not fully understood. There was an advantage in long-term survival for the 10 μM trabedersen group, especially when compared with standard chemotherapy. In the entire study population, the 2-year survival rate for 10 μM trabedersen was 39% vs 22% for standard chemotherapy. The beneficial effect of 10 μM trabedersen concerning the 2-year survival rate was even more pronounced in patients with GBM aged ≤55 years with KPS >80% (40% vs 13%) and patients with AA (83% vs 42%). Remarkably, the median survival of patients with AA treated with 10 μM trabedersen exceeded that of standard chemotherapy by 17.4 months (39.1 vs 21.7 months). However, it has to be taken into consideration that the sample sizes of the AA subgroup and the group with GBM patients aged ≤55 years with KPS >80% were small and, therefore, restrict generalization of the results. Despite the clinical relevance of the treatment effects observed with trabedersen and the consistency with findings from previous phase I/II studies,12 the hypotheses generated in this phase II study need to be confirmed in prospective clinical studies. To this end, a randomized and controlled phase III study in patients with AA has been initiated (see below). In this study, the proportion of patients with AEs considered related or possibly related to the study drug was highest in the standard chemotherapy group and lowest in the 10 μM trabedersen group. The patients treated with trabedersen had a higher rate of NCI-CTC grade 3/4 AEs than patients in the standard chemotherapy group, mainly regarding neurological events. The exact reason(s) for this has not been fully clarified to date and will be closely examined in current and upcoming studies. Part of the reason for the higher frequency of AEs in the trabedersen groups may be that there were more scheduled study visits for the patients (up to 23 visits) compared with those with standard chemotherapy (up to 9 visits), thus allowing more chances for the observation of AEs. Moreover, all patients of the trabedersen groups had biopsies taken before start of therapy and 20 patients at 3 months after start of therapy, while no biopsies were taken from patients of the control group. Generally, the frequency and nature of the reported events were typical for patients with primary brain tumors who usually have a large number of accompanying neurological disorders.25,26 One reason for more neurological AEs observed in the trabedersen groups may be the mode of administration of trabedersen through CED (ie, the infusion process through an implanted catheter).12 Although events related to the implantation and the procedure of drug administration cannot be completely avoided, those observed during this study were manageable, and their incidence can be further reduced by intensified training of the investigators. The finding that in the 10 μM trabedersen group more patients were assessed with CR or PR at month 14 than at month 6 (Table 2) supports the hypothesis from preclinical and phase I/II data that trabedersen acts mainly by inducing an immune response against the tumor. This is further supported by the fact that these responses continue to increase long after discontinuation of trabedersen—for instance, from SD to partial and even CRs without further antitumor treatment (see Supplementary Material, Figures S1 and S2). The suppression of immune reactions is commonly observed in patients with high-grade glioma.27 A major factor contributing to the suppression of the immune system is TGF-β2, as it has multiple effects on immunoregulatory functions, including the inhibition of lymphokine-activated killer cells and T-lymphocyte development and interleukin-2–dependent growth of T cells.5,28–31 Thus, the transient reduction in TGF-β2 levels via the antisense compound trabedersen may reverse TGF-β2–mediated immunosuppression. This may lead to the induction of a comprehensive, adaptive anticancer immune response that eliminates even residual cancer cells and has the potential to translate into a beneficial long-term outcome for patients. An immuno-based anticancer response is also in line with the observation in a previous phase I/II study where 1 AA patient had several localizations of the tumor in both brain hemispheres. All these tumor lesions receded completely after local therapy of only 1 tumor lesion with trabedersen.12 This phenomenon was also observed in patients of this phase IIb study. Pseudoprogression, a transient initial increase of the enhancing lesion,32 was seen in about 10% of trabedersen-treated patients in this study. In these patients, pseudoprogression was associated with a favorable survival (manuscript in preparation), as has been reported previously in high-grade glioma patients treated with radiotherapy and TMZ.33 In conclusion, 10 μM trabedersen represents the optimal dose for further clinical development of trabedersen. The 6-month intratumoral CED technique was found to be safe and well tolerated. On the basis of the promising results of this study, the randomized, active-controlled phase III study SAPPHIRE (Efficacy and Safety of AP 12009 in Adult Patients with Recurrent or Refractory Anaplastic Astrocytoma [WHO grade III] as Compared to Standard Treatment with Temozolomide or BCNU: A Randomized, Actively Controlled, Open-label Clinical Phase III Study) in patients with recurrent or refractory AA has been initiated to compare the 14-­month progression and 24-month survival rates and median OS of 10 μM trabedersen with standard chemotherapy. Additional survival and tumor response parameters as well as quality of life and safety will also be assessed in the study. BODY.SUPPLEMENTARY MATERIAL: Supplementary Material is available at Neuro-Oncology online.

TITLE: Pain perception during debridement of hypersensitive teeth elicited by two ultrasonic scalers ABSTRACT.OBJECTIVES: The ultrasonic NO PAIN technology (Electro Medical Systems, Nyon, CH) promises minimal pain during debridement due to linear oscillating action combined with a sinusoidal power output and feedback control. The aim of the present study was to measure pain perception on a visual analogue scale (VAS) during supportive periodontal therapy including debridement of hypersensitive teeth. Two ultrasonic scalers were used, one with and one without NO PAIN technology. ABSTRACT.MATERIAL AND METHODS: In a randomized-controlled clinical study with split-mouth design, 100 hypersensitive teeth matched for air blast hypersensitivity were either treated with the ultrasonic device Piezon Master 700 or the Mini Piezon (both EMS, Nyon, CH). Pain perception during debridement was assessed by a VAS (range 0–10). ABSTRACT.RESULTS: The average VAS for the test device Piezon Master 700 with NO PAIN technology was 3.16 ± 2.10, and for the control device Mini Piezon without NO PAIN technology 3.40 ± 2.59 (p = 0.490). Placing an arbitrary threshold at the VAS score of 3 for significant pain experience, 60 % of the subjects experienced no significant pain with either instrument. ABSTRACT.CONCLUSION: No statistically significant difference in perceived pain between the instruments used was found. ABSTRACT.CLINICAL RELEVANCE: Both ultrasonic devices showed very small pain intensities during debridement of highly hypersensitive teeth and can therefore be recommended for supportive periodontal therapy. BODY.BACKGROUND: The Consensus report of the 11th European Workshop on Periodontology on effective prevention of periodontal and peri-implant diseases reinforced the need to enrol patients treated for periodontitis in a supportive periodontal therapy regimen [1]. Supportive periodontal therapy aims at preventing the recurrence of periodontal disease in terms of tooth loss and additional attachment loss through periodic preventive interventions [2, 3]. Such regimen includes routine assessments of disease and oral hygiene status, behaviour modification and professional mechanical plaque and calculus removal (PMPR) [2]. The importance and effectiveness of supportive periodontal therapy in the secondary prevention of periodontal disease have been well established [1]. The authors of the Consensus report of the 11th European Workshop on Periodontology concluded that patients treated for periodontitis can maintain their dentition with limited variations in periodontal parameters when regularly complying with a supportive periodontal therapy regimen based on routine PMPR [1]. Additionally, patients irregularly complying with the planned supportive periodontal therapy regimen showed greater tooth loss and disease progression when compared to patients who comply regularly [1, 4]. Pain during PMPR was recently reported to be a significant factor influencing clinical compliance to periodontal therapy [5]. The healing of periodontal tissues after active periodontal therapy often results in gingival recession, and in addition, root debridement leads to loss of cementum [6, 7]. The short, sharp pain arising from exposed dentin in response to thermal, tactile, osmotic or chemical stimuli has been defined as dentin hypersensitivity [8]. Levels of dentin hypersensitivity may increase after surgical as well as non-surgical periodontal treatment [9–11]. Among periodontal patients, the occurrence of root sensitivity has been reported to reach up to 98 % [12]. PMPR often induces dentin hypersensitivity due to thermal or tactile stimuli. The ability to deliver dental care with a minimum of patient discomfort should be an essential part of a clinician's skills to avoid a decline of compliance with supportive periodontal therapy [13]. The EMS Piezon® NO PAIN technology (Electro Medical Systems (EMS), Nyon, CH) promises minimal pain during PMPR and no injury of the gingiva, due to controlled linear oscillating instrument movements parallel to the tooth surface, combined with a sinusoidal power output and feedback control [14]. The aim of the present randomized-controlled clinical study was to compare subjective pain intensities during PMPR of hypersensitive teeth with two piezoelectric ultrasonic devices, one including NO PAIN technology (Piezon Master 700, EMS, Nyon, CH), and one without (Mini Piezon, EMS, Nyon, CH). BODY.MATERIAL AND METHODS.ETHICAL CONSIDERATIONS: The Ethics Committee of the Medical University of Innsbruck, Austria, approved the study. The study was conducted in accordance with the 1964 Helsinki declaration and its later amendments. All subjects signed an informed written consent prior to the study enrolment. BODY.MATERIAL AND METHODS.STUDY SUBJECTS: For the study, 53 patients of the dental clinic of the Medical University of Innsbruck who were known for generalized and severe dentin hypersensitivity were recruited (Fig. 1). Subjects had to exhibit a minimum of two hypersensitive teeth in two different quadrants. For the respective teeth, an air blast stimuli score of 2 or 3 (Schiff Cold Air Sensitivity Scale - SCASS) had to be present at the baseline examination. Both teeth of the subject had to feature the same air blast stimuli score. Additionally, test and control teeth had to be from the same tooth category (category 1: incisive, category 2: canines and premolars, category 3: molars). Any teeth with cracked enamel, enamel defects, caries, or extensive/defective restorations, clinically diagnosed pulpitis, and teeth with orthodontic appliances were excluded. Additional exclusion criteria encompassed subjects with gross oral pathology, PMPR or orthodontic treatment within the last 3 months, subjects with eating disorders, as well as pregnant or lactating women, and psychiatric disorders. Current users of anticonvulsants, antihistamines, antidepressants, sedatives, tranquillisers, anti-inflammatory drugs or daily analgesics were also excluded.Fig. 1Patient flow chart. Patients were preselected by their dentist/student regarding inclusion and exclusion criteria. Only patients known for generalized and severe dentin hypersensitivity were recruited for the clinical trial BODY.MATERIAL AND METHODS.CLINICAL INTERVENTION: At baseline, one investigator (HH) measured tactile and air blast hypersensitivity and selected two hypersensitive teeth in two different quadrants with SCASS 2 or 3. For SCASS, the test and control teeth were isolated from the adjacent teeth by the placement of red boxing wax. Air was delivered from a standard dental unit air syringe at maximal pressure (45 psi) and at an environmental temperature of 19–24°. Air was applied for 1 s at a distance of 1 cm perpendicular to the buccal surface of the tooth. The SCASS was used to assess the subject's response to the stimulus. The scale is graduated into four units: 0 = subject does not respond to the stimulus; 1 = subject does not respond to the stimulus, but considers stimulus to be painful; 2 = subject responds to air stimulus, but does not move away from the stimulus; 3 = subject responds significantly to air stimulus, moves away from the stimulus, and requests immediate termination of the stimulus [15]. Patients were informed before testing about the different units of the scale. Tactile hypersensitivity was assessed by scratching on the dentinal surface with a sharp-tipped probe and a maximum pressure of 70 g. Pressure of 70 g was calibrated with a letter balance before each investigation. The subjects graded pain intensity on a visual analogue scale (VAS) (0 = no pain and 10 = extreme, unbearable pain). Patients were instructed to point at the VAS. Probing pocket depths recorded at six sites per tooth were available from all patients. Recession depth was measured on the buccal aspect of the tooth. Both teeth were matched for air blast hypersensitivity and were randomly assigned by the second investigator (MS) to one of two treatment groups by rolling a dice: (1) supragingival debridement for 30 s using an ultrasonic scaler with the specific NO PAIN technology (Piezon Master 700, EMS, Nyon, CH), or (2) supragingival debridement for 30 s using an ultrasonic scaler without the NO PAIN technology (Mini Piezon, EMS, Nyon, CH). For both devices, the same tip was used (EMS Instrument PS). According to oral advice of the manufacturer, the power of both devices was set to 50 %. Temperature of physiological saline solution and water for cooling was adjusted to 24 °C. During debridement, the lower end of the tip was applied from coronal to apical with minimal pressure using brushing strokes parallel to the tooth surface. The blinded investigator HH performed the follow-up examinations: Intervention blinded patients were asked to protocol their perception of the instrumentation immediately after the treatment (main outcome measure) on an interval scale (VAS) ranging from 0, representing no pain or discomfort, to 10, representing maximum pain and discomfort. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSES: Standard descriptive methods were used to summarize the parameters studied. The Wilcoxon signed-rank test was used to evaluate differences between values for pain perception during PMPR (VAS, main outcome). Differences of baseline hypersensitivity levels between treatment groups were evaluated with the chi-square test (SCASS) or Wilcoxon signed-rank test (tactile hypersensitivity, VAS). All statistical tests of the hypotheses were two-sided, and a level of significance of alpha = 0.05 was employed. BODY.RESULTS.BASELINE DATA: One hundred teeth in 50 subjects (31 females, 19 males) were enrolled in the study. All participants were Caucasians, aged 20–79 years (mean age ± SD = 44.84 ± 14.06). Fifty-six percent of the subjects had never smoked before, and 44 % were smokers. Thirty-six subjects showed baseline SCASS 2 on matched test and control teeth, and 14 subjects exhibited with SCASS 3 on matched test and control teeth. Six subjects with tooth category 1, 27 subjects with tooth category 2, and 17 subjects with tooth category 3 were included. There was no statistical significant difference between test and control teeth in baseline tactile hypersensitivity (VAS baseline 1.60 ± 2.09 and 1.62 ± 2.19, respectively; p = 0.617) (Table 1).Table 1Dentin hypersensitivity at baseline and pain perception during debridement of test and control teethPiezon Master 700(n = 50)Mini Piezon(n = 50)Tooth categories Category 1: incisives, n 66 Category 2: canines and premolars, n 2727 Category 3: molars, n 1717Periodontal parameters Probing pocket depth, m ± sd 3.43 ± 1.443.40 ± 1.47 Recession depth, m ± sd 2.03 ± 1.272.30 ± 1.26Airblast sensitivity scale SCASS 2, n 3636 SCASS 3, n 1414Tactile sensitivity scale, baseline Visual analogue scale, m ± sd 1.60 ± 2.091.62 ± 2.19Pain perception during debridement Visual analogue scale, m ± sd 3.16 ± 2.103.40 ± 2.59One hundred teeth in 50 subjects (31 females, 19 males; mean age ± SD = 44.84 ± 14.06) were enrolled in the study. Fifty-six percent of the subjects had never smoked before, and 44 % were smokers. Two teeth in two different quadrants were matched for SCASS and tooth category n number, SCASS Schiff Cold Air Sensitivity Scale, m mean, sd standard deviation No statistically significant difference in perceived pain between the instruments used was found. For the test device Piezon Master 700 with NO PAIN technology, the average VAS value during debridement was 3.16 ± 2.10, and for the control device Mini Piezon without NO PAIN technology, this was 3.40 ± 2.59 (p = 0.490). The median was 3 for both instruments (range 1–10) (Fig. 1). Placing an arbitrary threshold for significant pain experience at the VAS score of 3 [16], 60 % (n = 30) of the subjects experienced no significant pain (VAS 0 to 3) with either instrument. Further assuming another arbitrary limit at 7 [16], two subjects perceived great pain (VAS 7 to 10) during treatment with the Piezon Master 700, and seven patients perceived great pain during treatment with the Mini Piezon (Fig. 2).Fig. 2Box plot for the pain perception during debridement (median, outliers, 10, 25, 75, and 90 % percentiles). Pain perception of the instrumentation was assessed immediately after the treatment on an interval scale (visual analogue scale, VAS) ranging from 0, representing no pain or discomfort, to 10, representing maximum pain and discomfort. With a median of 3, pain perception was low for both devices BODY.DISCUSSION: Many studies have highlighted the importance of regular supportive periodontal therapy including PMPR. Among the most well-known studies are Hirschfield and Wasserman [17], McFall [18], Lindhe and Nyman [19], Goldman et al. [20], and Axelsson and Lindhe [21]. Indeed, there is a significant increase in tooth loss in non-compliers or irregular compliers compared to compliers [22]. Incidence of new sites with probing depth of > or =5 mm varied between 3.2 % for the compliant and 5.8 % for the non-compliant patients (mean delay from the scheduled recall sessions: compliant within 1–6 weeks; and not compliant >6 weeks) [23]. A painless treatment increases patient comfort during PMPR, and might therefore increase patient compliance [5]. This in turn may provide a better long-term prognosis for periodontal therapy. In the present randomized-controlled and double blind trial, 100 teeth with dentin hypersensitivity were enrolled. Two teeth in each subject were matched according to air blast hypersensitivity and tooth category and were randomly assigned to debridement with an ultrasonic device with or without NO PAIN technology. Debridement of hypersensitive dentin was restricted on supragingival areas to avoid pain by gingival injury, which would have falsified the result. Pain perception of the instrumentation was recorded instantly with a VAS. Verbal reports are known to be shaped by a variety of psychosocial variables. Additionally, pain is not a simple sensory state but is influenced by cultural learning, the meaning of the situation, attention and other psychological variables [24]. Therefore, to overcome inter-individual differences between test and control patients, we investigated both devices in a split-mouth clinical trial. To overcome intra-individual differences in pain perception between different teeth, tooth categories and air blast hypersensitivity were matched between test and control teeth. Pain perception during instrumentation was low for both ultrasonic devices (VAS median 3), and there was no statistically significant difference between the two treatment modalities. More subjects (n = 7) perceived great pain (VAS 7–10) with the Mini Piezon compared to subjects exposed to the Piezon Master 700 (n = 2) (Fisher Exact Probability Test: p = 0.159). Our results are in line with previous studies on pain perception during debridement with piezoceramic ultrasonic devices. Braun et al. compared pain intensities during debridement with hand instruments (Gracey-curettes, Hu-Friedy, Leimen, Germany), a piezo ultrasonic instrument (SirosonTMS, instrument N°3, Siemens, Bensheim, Germany) or the VectorTM-system (Duerr Dental, Bietigheim-Bissingen, Germany); mean VAS values for pain perception during therapy was 3.7 ± 1.8 for the piezo ultrasonic device [25]. Kocher et al. compared pain intensities during debridement with a sonic (Sonicflex2000, KaVo, Biberach, Germany) and a piezoceramic ultrasonic scaler (PiezonMaster 400, EMS, Nyon, CH) in a split-mouth design; the median VAS was 3 for both instruments [16]. Kocher et al. concluded that the motion of the instrument's tip might be redundant with respect to perceived pain [16]. The EMS Piezon® NO PAIN technology (Electro Medical Systems (EMS), Nyon, CH) promises minimal pain during PMPR and no injury of the gingiva, due to controlled linear oscillating instrument movements parallel to the tooth surface, combined with a sinusoidal power output and feedback control [14]. Emmelmann studied in his thesis the motion of instrument tips in an unloaded and loaded mode with a high speed camera with 7500 frames-per-second and provided first evidence that the instrument's tip (EMS® Instrument P) of the Piezon Master 700 has a slight elliptic motion [26]. Possibly, the motion of the instrument's tip with the Piezon Master 700 is very similar to the Mini Piezon, which is also equipped with the Piezon® technology (Fig. 3). The feedback control does not seem to have a significant effect on pain reduction in supportive periodontal therapy where little calculus is present.Fig. 3Frequency distribution of VAS scores for the treatment with a the Piezon Master 700 and b the Mini Piezon (both EMS, Nyon, CH). Pain perception of the instrumentation was assessed immediately after the treatment on an interval scale (visual analogue scale, VAS) ranging from 0, representing no pain or discomfort, to 10, representing maximum pain and discomfort. Placing an arbitrary threshold at the VAS score of 3, 60 % (n = 30) of the subjects experienced no significant pain with either instrument. Further assuming another arbitrary limit at 7, two subjects perceived great pain during treatment with the Piezon Master 700, and seven patients perceived great pain during treatment with the Mini Piezon In conclusion, both ultrasonic devices investigated in the present study showed very small pain intensities during debridement of highly hypersensitive teeth and can therefore be recommended for supportive periodontal therapy.

TITLE: Canagliflozin Lowers Postprandial Glucose and Insulin by Delaying Intestinal Glucose Absorption in Addition to Increasing Urinary Glucose Excretion ABSTRACT.OBJECTIVE: Canagliflozin, a sodium glucose cotransporter (SGLT) 2 inhibitor, is also a low-potency SGLT1 inhibitor. This study tested the hypothesis that intestinal canagliflozin levels postdose are sufficiently high to transiently inhibit intestinal SGLT1, thereby delaying intestinal glucose absorption. ABSTRACT.RESEARCH DESIGN AND METHODS: This two-period, crossover study evaluated effects of canagliflozin on intestinal glucose absorption in 20 healthy subjects using a dual-tracer method. Placebo or canagliflozin 300 mg was given 20 min before a 600-kcal mixed-meal tolerance test. Plasma glucose, 3H-glucose, 14C-glucose, and insulin were measured frequently for 6 h to calculate rates of appearance of oral glucose (RaO) in plasma, endogenous glucose production, and glucose disposal. ABSTRACT.RESULTS: Compared with placebo, canagliflozin treatment reduced postprandial plasma glucose and insulin excursions (incremental 0- to 2-h area under the curve [AUC0–2h] reductions of 35% and 43%, respectively; P < 0.001 for both), increased 0- to 6-h urinary glucose excretion (UGE0–6h, 18.2 ± 5.6 vs. <0.2 g; P < 0.001), and delayed RaO. Canagliflozin reduced AUC RaO by 31% over 0 to 1 h (geometric means, 264 vs. 381 mg/kg; P < 0.001) and by 20% over 0 to 2 h (576 vs. 723 mg/kg; P = 0.002). Over 2 to 6 h, canagliflozin increased RaO such that total AUC RaO over 0 to 6 h was <6% lower versus placebo (960 vs. 1,018 mg/kg; P = 0.003). A modest (∼10%) reduction in acetaminophen absorption was observed over the first 2 h, but this difference was not sufficient to explain the reduction in RaO. Total glucose disposal over 0 to 6 h was similar across groups. ABSTRACT.CONCLUSIONS: Canagliflozin reduces postprandial plasma glucose and insulin by increasing UGE (via renal SGLT2 inhibition) and delaying RaO, likely due to intestinal SGLT1 inhibition. BODY: The sodium glucose cotransporter (SGLT) 2 is the major transporter responsible for reabsorption of glucose filtered through the renal glomerulus (1). SGLT2 is a high-capacity, low-affinity transporter expressed primarily at the luminal membrane of the early segments of the proximal renal tubules (1). SGLT1 is a low-capacity, high-affinity transporter expressed in the distal segment of the proximal tubule (1), in the intestinal mucosa of the small intestine (2), and in other tissues to a lesser extent (3). Although SGLT1 plays a smaller role in renal glucose absorption than SGLT2, SGLT1 is the primary pathway involved in intestinal glucose and galactose absorption (2,4,5). Pharmacologic inhibition of SGLT2 is a novel approach to lowering plasma glucose in hyperglycemic individuals by blocking renal glucose reabsorption, lowering the renal threshold for glucose (RTG), and thereby markedly increasing urinary glucose excretion (UGE). Canagliflozin, an SGLT2 inhibitor in development for the treatment of patients with type 2 diabetes (6–10), is also a low-potency SGLT1 inhibitor. In vitro, canagliflozin inhibited sodium-dependent 14C-α-methylglucoside uptake in cells expressing human SGLT2 or SGLT1 with half-maximal inhibitory concentrations (IC50) of 4.4 ± 1.2 and 684 ± 159 nmol/L, respectively (8). Because the maximum plasma concentrations of unbound canagliflozin in subjects treated with canagliflozin 300 mg once daily are ∼100 nmol/L (maximum plasma concentrations are ∼10 μmol/L [11] and protein binding is ∼99% [unpublished data]), only minimal systemic inhibition of SGLT1 is expected in subjects treated with canagliflozin 300 mg. In clinical studies in healthy subjects and subjects with type 2 diabetes, treatment with canagliflozin provided dose-dependent increases in UGE compared with placebo (7,9). In healthy subjects treated with escalating doses of canagliflozin given 10 min before a mixed meal, doses of canagliflozin higher than 200 mg reduced postprandial plasma glucose and insulin concentrations to a greater extent than lower doses of canagliflozin, even when compared with doses that provided similar UGE during the postprandial period (7). These pronounced reductions in postprandial glucose and insulin excursions observed with canagliflozin doses higher than 200 mg were only observed for the first meal after dosing; similar reductions beyond that expected on the basis of increased UGE were not observed after later meals (lunch and dinner) given on the same day (7). On the basis of these observations, it was hypothesized that after dosing and during drug absorption, canagliflozin concentrations within the lumen of the intestinal tract could be sufficiently high to provide transient inhibition of intestinal SGLT1-mediated glucose absorption, thereby lowering postprandial plasma glucose and insulin concentrations. The current study investigated the effects of a single 300-mg oral dose of canagliflozin on intestinal glucose absorption and metabolism in healthy subjects (ClinicalTrials.gov Identifier: NCT01173549). This study used a dual-tracer method to test the hypothesis that canagliflozin 300 mg slows the rate of systemic appearance of orally administered glucose (RaO) during a mixed-meal tolerance test (MMTT) compared with placebo. BODY.RESEARCH DESIGN AND METHODS.STUDY POPULATION: This study was conducted from 22 November 2010 to 29 September 2011 at a single center in San Diego, California. The study protocol and all amendments were reviewed and approved by the University of California, San Diego Institutional Review Board. This study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and that are consistent with good clinical practices and applicable regulatory requirements. All subjects provided written informed consent to participate in the study after having been informed about the nature and purpose of the study, participation/termination conditions, and the possible risks and benefits of treatment. This study enrolled healthy men aged 18–45 years with a BMI of ≥20 and ≤27 kg/m2, stable body weight of ≥50 kg (<5% change during the 3 months before screening), and fasting plasma glucose (FPG) of <6.1 mmol/L. Further eligibility criteria are described at ClinicalTrials.gov (NCT01173549). BODY.RESEARCH DESIGN AND METHODS.SAFETY ANALYSES: Vital sign measurements, 12-lead electrocardiograms, physical examinations, and clinical laboratory tests were performed at predefined time points throughout the study. Adverse events (AEs) were monitored from the signing of informed consent until completion of the last study-related procedure. BODY.RESEARCH DESIGN AND METHODS.STUDY DESIGN: This was a randomized, double-blind, placebo-controlled, two-period crossover study consisting of a screening phase, a 25-day double-blind treatment phase (including two 1-day treatment periods and a washout period of 7–21 days between periods 1 and 2), and a follow-up phase of up to 10 days after period 2. Subjects were randomized to one of two treatment sequences: canagliflozin 300 mg in period 1, followed by matching placebo in period 2, or vice versa. On days –3 and –2 of each period, subjects were counseled to adhere to a specified diet (∼55% carbohydrate, 30% fat, 15% protein, and total caloric intake of ∼30 kcal/kg body weight). On the morning of day –1 of each study period, subjects were admitted to the clinical research unit in a fasting state for safety analyses, followed by standardized meals. On the morning of day 1 of each study period, after an overnight fast of at least 8 h, subjects received a primed (25 μCi), continuous intravenous infusion of 3H-glucose (0.25 μCi/min) for approximately 9 h. Three hours after starting the intravenous infusion and 20 min after administration of study drug, subjects received a standard 600-kcal MMTT (55% carbohydrate, 30% fat, 15% protein). The liquid component of the MMTT consisted of an oral solution of 75 g glucose, which was mixed with 75 μCi 14C-glucose, and an acetaminophen solution (960 mg in 30 mL); acetaminophen absorption was used as an indirect measure of gastric emptying (12,13). Subjects returned for a final follow-up visit for safety analyses 7 to 10 days after discharge on day 1 of period 2. BODY.RESEARCH DESIGN AND METHODS.CLINICAL EVALUATIONS: On day 1, subjects emptied their bladder before and after the 3-h 3H-glucose isotope equilibration period (from t = –3 h to t = 0 [the start of the meal]), and urine was collected over the intervals of 0 to 2 h and 2 to 6 h for determination of urinary glucose and creatinine concentrations. Blood samples were collected at t = –20, –10, 0, 15, 30, 40, 50, 60, 75, 90, 105, 120, 150, 180, 210, 240, 270, 300, and 360 min for measurements of plasma glucose (labeled and unlabeled) and insulin. Additional blood samples were drawn at predefined time points for determination of canagliflozin and acetaminophen concentrations and for analysis of other pharmacodynamic markers, including concentrations of glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), and peptide YY (PYY). BODY.RESEARCH DESIGN AND METHODS.BIOANALYTICAL ANALYSES: Plasma and urine glucose concentrations were measured using a hexokinase enzymatic assay, and plasma insulin concentration was determined using an electrochemiluminescent sandwich immunoassay (Roche Diagnostics, Indianapolis, IN). 3H-glucose and 14C-glucose specific activities were determined using the assays described by Mudaliar et al. (14) and Kreisberg et al. (15), respectively. Recycling of 14C-glucose over the course of the procedure was negligible, with recycled 14C generally below the limit of detection, similar to the observations of others (16,17). Active and total plasma GLP-1 levels were measured using an electrochemiluminescent sandwich immunoassay (Meso Scale Discovery, Gaithersburg, MD). Total plasma GIP was measured using an enzyme-linked immunosorbent assay, and total plasma PYY was measured using a radioimmunoassay (Millipore, Billerica, MA). Plasma acetaminophen concentration was determined using a validated high-performance liquid chromatography (HPLC) method with ultraviolet detection at PRA International, Assen, the Netherlands (calibration range = 0.500–50 μg/mL). BODY.RESEARCH DESIGN AND METHODS.GLUCOSE FLUX ANALYSIS: RaO, the rate of endogenous glucose production (EGP), and the rate of total glucose disposal (Rd) were determined from the measured plasma glucose, 3H-glucose, and 14C-glucose profiles using a circulatory model of glucose kinetics (18–20). The infused 3H-glucose profile was used to determine glucose clearance over time, and the 14C-glucose and plasma glucose profiles were used to determine the relative amounts of ingested and endogenous glucose in the circulation. The resulting rates of appearance of endogenous and oral glucose were determined by fitting the model of glucose kinetics to the oral and endogenous glucose profiles. All calculations were performed using the GLUTRAN toolbox (licensed from Mari and colleagues at the Institute of Biomedical Engineering, National Research Council, Padova, Italy) in Matlab 7.10 software (18). To separate total Rd into tissue glucose disposal (tissue Rd) and UGE, the rate of UGE at each time point was estimated from the UGE collections over the 0- to 2-h and 2- to 6-h intervals. This was done by calculating RTG over these intervals, as previously described (7,9), and then estimating UGE at each interval using the following equation:where the glomerular filtration rate (GFR) was estimated using the Modification of Diet in Renal Disease equation and PG is plasma glucose. Tissue Rd was then calculated as total Rd − UGE. BODY.RESEARCH DESIGN AND METHODS.PHARMACODYNAMIC PARAMETERS: The total amount of glucose appearance and disappearance over the intervals from 0 to 1 h, 0 to 2 h, 2 to 6 h, and 0 to 6 h was determined by calculating the area under the curve (AUC) of the associated rates of glucose appearance and disappearance time profiles over each time interval. Incremental AUCs (denoted as ΔAUC) for plasma glucose, insulin, and gut peptides were defined as the positive area above the premeal value. BODY.RESEARCH DESIGN AND METHODS.STATISTICAL ANALYSIS: For the primary pharmacodynamic parameters of RaO AUC0–1h and RaO AUC0–2h, data were log-transformed for analysis. Mixed-effects models were fitted with the logarithm of the parameter of interest as the dependent variable; sequence, period, and treatment as fixed effects; and subject as a random effect. The null hypothesis that the mean RaO AUCs (on the log-scale) are equal for canagliflozin 300 mg and placebo was tested using a one-sided (left-sided) α level of 5%. Using the estimated least squares (LS) means and intrasubject SD from the mixed-effects model, 90% CIs were constructed for the difference in means on the log scale between canagliflozin 300 mg and placebo. The CI limits for the difference in mean AUCs were exponentiated to yield the 90% CI for the ratio of geometric mean RaO AUCs of canagliflozin to placebo. All secondary pharmacodynamic variables were summarized with descriptive statistics for each treatment. Mean (95% CIs) differences between canagliflozin 300 mg and placebo were determined for all pharmacodynamic variables. Total Rd was analyzed using a mixed-effects linear model based on log-transformed data. The model included the logarithm of total Rd as the dependent variable; sequence group, period, and treatment as fixed effects; and subject as a random effect. Acetaminophen pharmacokinetic parameters (Cmax and AUCs) were compared between canagliflozin and placebo groups by constructing a 90% CI for the ratio of geometric means using mixed-effects modeling of the data with the logarithm of the pharmacokinetic parameters as the dependent variable; sequence, period, and treatment as fixed effects; and subject as a random effect. The relationship between RaO AUC and AUC acetaminophen was assessed by linear regression analysis. Comparison of the regression lines for canagliflozin versus placebo was performed using an ANCOVA model in Prism 5.01 software (GraphPad Software, Inc., La Jolla, CA). For all subjects, based on individual plasma concentration-time profiles, the total and incremental AUCs for glucose, insulin, PYY, GIP, and GLP-1 from 0 to 1 h, 0 to 2 h, and 0 to 6 h (glucose and insulin only) were calculated using the trapezoid rule using WinNonlin 5.2.1 software (Pharsight Corporation, Mountain View, CA). Unless otherwise specified, all results shown are mean ± SD. BODY.RESULTS: This study enrolled 20 subjects, and 19 completed both treatment periods (1 subject was withdrawn for noncompliance with study prohibitions). Subject baseline and demographic characteristics are summarized in Table 1. Table 1 Subject baseline and demographic characteristics BODY.RESULTS.POSTPRANDIAL PLASMA GLUCOSE AND INSULIN RESPONSES: After ingestion of the standardized meal, the postprandial plasma glucose and insulin excursions were reduced with canagliflozin 300 mg compared with placebo (Fig. 1A and B). Mean postprandial plasma glucose ΔAUC values were ∼44%, 35%, and 26% lower during the 0- to 1-h, 0- to 2-h, and 0- to 6-h postmeal intervals, respectively, after administration of canagliflozin 300 mg (arithmetic mean ± SD ΔAUC of 1.88 ± 0.77, 3.67 ± 1.41, and 4.80 ± 1.90 [mmol/L] ⋅ h, respectively) compared with placebo (3.34 ± 1.43, 5.61 ± 2.62, and 6.44 ± 2.57 [mmol/L] ⋅ h, respectively). Similarly, postprandial plasma insulin ΔAUC values were ∼43%, 43%, and 33% lower during the 0- to 1-h, 0- to 2-h, and 0- to 6-h postmeal intervals, respectively, for canagliflozin (arithmetic mean ± SD ΔAUC of 271 ± 232, 514 ± 273, and 738 ± 335 [pmol/L] ⋅ h, respectively) compared with placebo (474 ± 263, 906 ± 521, and 1,104 ± 624 [pmol/L] ⋅ h, respectively). Figure 1Mean ± SEM concentration-time profiles from predose to 6 h after the standard meal for plasma glucose (A), insulin (B), oral 14C-glucose tracer (C), and infused 3H-glucose tracer (D); rate of oral glucose appearance (E); and glucose absorption as a function of time after the standard meal (F). CANA, canagliflozin. The oral 14C-glucose tracer concentration was reduced with canagliflozin compared with placebo over the first 2 h after the MMTT (Fig. 1C). The infused 3H-glucose tracer concentration was similar between treatments for the first 3 h after the MMTT; between 3 and 6 h after the MMTT, the plasma concentration of 3H-glucose was lower with canagliflozin treatment than with placebo (Fig. 1D). BODY.RESULTS.R: Canagliflozin treatment blunted and delayed the postprandial increase in RaO compared with placebo (Fig. 1E). Treatment with canagliflozin reduced the amount of oral glucose absorption (AUC RaO) compared with placebo by 31% over the 0- to 1-h interval (ratio of LS geometric means [90% CI] of 0.69 [0.60–0.80]; P < 0.001) and by 20% over the 0- to 2-h interval (0.80 [0.71–0.89]; P < 0.01; Fig. 1F). However, this decrease in AUC RaO over the first 2 h with canagliflozin was nearly matched by a 34% increase in AUC RaO in the 2- to 6-h interval for canagliflozin compared with placebo (1.34 [1.19–1.51]), such that the AUC RaO over 0 to 6 h was only ∼6% lower for canagliflozin compared with placebo (0.94 [0.91–0.98]; P = 0.003; Fig. 1F). Administration of canagliflozin also reduced the maximum RaO by approximately 24% compared with placebo (LS geometric means of 7.16 and 9.38 mg/kg/min, respectively). BODY.RESULTS.UGE AND RT: Canagliflozin treatment increased UGE (Fig. 2A). Mean UGE after treatment with canagliflozin was 5.9 and 12.2 g during the 0- to 2-h and 2- to 6-h intervals, respectively, compared with less than 0.15 g in each interval with placebo. In canagliflozin-treated subjects, mean RTG was 4.1 ± 1.8 mmol/L over the 0- to 2-h interval and 2.4 ± 0.8 mmol/L over the 2- to 6-h interval. RTG values could not be determined in placebo-treated subjects because most had only minimal (<200 mg) UGE, which is consistent with expectations based on the plasma glucose profiles shown in Fig. 1 and with the commonly reported RTG values of ∼10.0 to 11.1 mmol/L in untreated, healthy subjects (21,22). Figure 2Effects of canagliflozin (CANA) treatment on UGE rate (A), EGP rate (B), total Rd (C), and tissue Rd (D) over 6 h after the standard meal and on total glucose turnover from 0 to 2 h (E) and 0 to 6 h (F) postmeal. The calculated mean total amount of oral glucose absorption over 0 to 6 h was 79 g with placebo and 75 g with canagliflozin. The mean value in the placebo group is slightly higher than the 75-g oral glucose load that was ingested, giving a calculated bioavailability of glucose that was slightly >100%. This slight overestimation may be due partly to recycling of the 14C glucose tracer through the Cori cycle that could not be accounted for due to limitations of assay sensitivity not allowing accurate detection of the recycled tracer amounts (see research design and methods). Values shown are mean ± SEM in A–D and mean in E and F. BODY.RESULTS.RATES OF EGP AND R: Before the MMTT, the rate of EGP was ∼2 mg/kg/min in both treatment groups, consistent with values reported in other studies in healthy subjects (23,24). EGP was rapidly suppressed after the MMTT, with similar suppression of EGP observed with canagliflozin or placebo treatments (Fig. 2B). From 3 to 6 h postmeal (when plasma glucose concentrations were generally back to premeal values), the rate of EGP was modestly higher with canagliflozin than with placebo (Fig. 2B), and the calculated total amount of EGP over 2 to 6 h was ∼20% higher with canagliflozin than placebo (geometric mean ratio = 1.20, P = 0.005). Although canagliflozin treatment dramatically increased UGE compared with placebo (Fig. 2A), total and tissue Rd were both lower with canagliflozin treatment than with placebo for ∼2 h after the MMTT (Fig. 2C and D), with AUC0–1h and AUC0–2h for total Rd reduced by ∼18% and 19% with canagliflozin compared with placebo (P = 0.026 and P = 0.009, respectively). Thus, despite the increase in UGE with canagliflozin treatment, the reduction in postprandial plasma glucose and insulin over the first 2 h is not explained by increased glucose disposal because Rd over that time interval is lower with canagliflozin than with placebo. Total Rd increased by ∼25% over the 2- to 6-h interval with canagliflozin compared with placebo (P < 0.001) so that the total amount of glucose disposal over the 6-h period (total Rd AUC0–6h) was essentially identical with canagliflozin and placebo (P = 0.78). BODY.RESULTS.TOTAL GLUCOSE TURNOVER: The total amounts of glucose appearance and disappearance over the 0- to 2-h and 2- to 6-h intervals are shown in Fig. 2. Over the 0- to 2-h interval, treatment with canagliflozin reduced mean RaO AUC (i.e., the amount of oral glucose absorbed) by 11 g, which was almost twice as large as the increase in UGE during this interval (6 g; Fig. 2E). This suggests that most of the observed reductions in plasma glucose and insulin excursions with canagliflozin treatment are due to decreased intestinal glucose absorption over the first 2 h after the meal. However, the total amounts of glucose appearance and disappearance over the 0- to 6-h interval were essentially identical between treatments (Fig. 2F). BODY.RESULTS.GASTRIC EMPTYING AND POSTPRANDIAL PLASMA GIP, PYY, AND GLP-1 RESPONSES: Mean plasma acetaminophen concentrations were ∼10% lower over the first 2 h after the meal with canagliflozin compared with placebo (LS geometric mean AUC0–2h of 9.21 and 10.29 μg ⋅ h/mL, respectively, giving a LS geometric mean ratio of 0.90 (90% CI 0.84–0.95; P = 0.004; Supplementary Fig. 1A). However, this reduction in the gastric emptying rate is insufficient to explain the decrease in RaO observed with canagliflozin compared with placebo, because the relationship between acetaminophen absorption and glucose absorption was altered by canagliflozin treatment (Supplementary Fig.1B). Over both the 0- to 1-h and 0- to 2-h intervals, the relationship between RaO and acetaminophen absorption was shifted downward with canagliflozin treatment, demonstrating that the reduction in RaO could not be explained solely by reductions in gastric emptying. Changes in gut peptide concentration during the 0- to 2-h postprandial period were consistent with delayed intestinal glucose absorption (Fig. 3). Incremental postprandial GIP was reduced by ∼50% with canagliflozin compared with placebo (arithmetic mean ± SD ΔAUC0–2h of 30.1 ± 11.1 and 63.5 ± 20.2 pmol ⋅ h/L, respectively). The incremental postprandial PYY concentration was ∼60% higher from 0 to 2 h with canagliflozin than with placebo (21.9 ± 13.5 and 13.6 ± 10.8, respectively), and total GLP-1 was ∼35% higher with canagliflozin than with placebo (13.7 ± 6.88 and 10.1 ± 5.38, respectively). Figure 3Mean ± SEM plasma concentration-time profiles of GIP (A), PYY (B), total GLP-1 (C), and active GLP-1 (D). CANA, canagliflozin. BODY.RESULTS.SAFETY: Canagliflozin was well tolerated, with no discontinuations due to AEs. No subjects reported symptoms suggestive of glucose malabsorption or gastrointestinal adverse effects. BODY.CONCLUSIONS: Results of this study show that in healthy subjects, a single 300-mg dose of canagliflozin administered before a meal reduced postprandial plasma glucose excursions by two mechanisms: increased UGE due to renal SGLT2 inhibition and delayed absorption of ingested glucose. The delay in oral glucose absorption is likely due to local and transient intestinal SGLT1 inhibition resulting from high intestinal canagliflozin concentrations within the intestinal lumen during the period of drug absorption. Although canagliflozin reduced intestinal glucose absorption by ∼31% over the first hour and by ∼20% over the first 2 h after a meal, this initial reduction was almost entirely compensated for by an increase in glucose absorption from 2 to 6 h after the meal. As a result, there was only a small difference (<6%) in the total appearance of orally ingested glucose in plasma over the full 6-h period after the meal between canagliflozin and placebo treatments. These findings demonstrate that canagliflozin treatment is not associated with any meaningful glucose malabsorption, and consistent with this, no symptoms of malabsorption were reported in this study. No increase in glucose malabsorption (assessed using a hydrogen breath test) was observed in subjects with type 2 diabetes treated with canagliflozin 300 mg twice daily for 4 weeks (9). This is in contrast to serious malabsorption symptoms observed in individuals with inactivating genetic mutations in SGLT1 (3). This lack of glucose malabsorption in subjects treated with canagliflozin is likely due to the rapid absorption of canagliflozin, such that intestinal canagliflozin concentrations are only transiently sufficiently high to inhibit SGLT1. The changes in gut peptide secretion observed with canagliflozin treatment are generally consistent with expectations based on intestinal SGLT1 inhibition leading to delayed glucose absorption. GIP-secreting K cells are primarily found in the proximal small intestine, and the observed reduction in plasma GIP concentrations with canagliflozin treatment is consistent with recent data showing that SGLT1-mediated glucose uptake is pivotal for GIP secretion (5). Similarly, the increase in plasma PYY and total GLP-1 observed after t = 30 min suggests increased glucose absorption in the more distal intestine where the GLP-1–secreting L cells are primarily found. However, it remains uncertain why similarly rapid increases in GLP-1 and PYY were observed in the first 30 min in both treatment groups. The early increments in GLP-1 and PYY (occurring before ingested nutrients have reached the more distal intestine) have been commonly reported in other studies (25), and the source of this early GLP-1 and PYY secretion remains uncertain, with possibilities including 1) a neural signal from the proximal small intestine that reaches L cells in the more distal intestine (26), 2) release from the small number of L cells in the proximal small intestine (27), and/or 3) release by a subset of enteroendocrine cells that cosecrete GIP and GLP-1 (28). Because SGLT1 is also reported to be essential for GLP-1 secretion (5) and no reduction in early GLP-1 secretion was observed with canagliflozin, it is not clear that the early increment in GLP-1 secretion can be explained by GLP-1 secretion from cells in the proximal small intestine. The observed reduction in serum insulin is likely due to decreased plasma glucose, because the relationship between plasma glucose and the insulin secretion rate was unchanged by canagliflozin treatment (data not shown). Although a slight delay in gastric emptying (estimated by plasma acetaminophen concentrations) was observed with canagliflozin treatment compared with placebo, the reductions in RaO observed with canagliflozin are greater than can be accounted for by the modest delay in gastric emptying. The observed effects of canagliflozin on RaO are consistent with the hypothesis that canagliflozin 300 mg transiently inhibits intestinal SGLT1-mediated glucose absorption, with perhaps a small contribution of delayed gastric emptying, possibly related to the increased GLP-1 levels. Endogenous glucose production was suppressed to a similar extent with both treatments after the meal, but EGP was higher with canagliflozin than with placebo over the 3- to 6-h period after the meal (Fig. 2). The elevated EGP is likely a compensatory response that enables normal plasma glucose concentrations to be maintained in the presence of sustained UGE, but the mechanism(s) leading to the increased EGP is not known. Although this study demonstrated that a single 300-mg dose of canagliflozin delays intestinal glucose absorption in healthy subjects, there are some important limitations. Because only one dose strength of canagliflozin was tested, the dose-response relationship for the effect of canagliflozin on intestinal glucose absorption cannot be established from this study. Results from an earlier study suggested that doses >200 mg of canagliflozin were required to see pronounced reductions in postprandial glucose excursions (7), but it is not known whether greater delays in intestinal glucose absorption could be achieved with higher doses of canagliflozin or whether malabsorption would be observed with higher doses. In addition, because this mechanistic study only characterized the response to a single dose in healthy subjects, further studies will be required to characterize the effects of sustained canagliflozin treatment on intestinal glucose absorption in subjects with type 2 diabetes. In conclusion, canagliflozin 300 mg reduces postprandial plasma glucose and insulin concentration in healthy subjects by two distinct mechanisms: 1) increasing UGE due to renal SGLT2 inhibition and 2) delaying RaO, which is likely due to transient intestinal SGLT1 inhibition.

TITLE: Effectiveness of Caregiver Training in Mindfulness-Based Positive Behavior Support (MBPBS) vs. Training-as-Usual (TAU): A Randomized Controlled Trial ABSTRACT: Caregivers of individuals with intellectual and developmental disabilities (IDD) often end up having their medical and psychological well-being compromised due to the stressful nature of caregiving, especially when those in their care engage in aggressive behavior. In this study, we provided caregivers with mindfulness-based training to enable them to better manage their psychological well-being and, through this, to also enhance specific indices of quality of life of the individuals in their care. Thus, the aim of the present study was to evaluate in a randomized controlled trial (RCT) the comparative effectiveness of Mindfulness-Based Positive Behavior Support (MBPBS) and Training-as-Usual (TAU) for caregivers in a congregate care facility for individuals with severe and profound IDD. The comparative effects of the two training conditions were assessed in terms of caregiver variables care recipient variable (number of aggressive events), and agency variables Results showed that MBPBS was significantly more effective than TAU in enabling the caregivers to manage their perceived psychological stress, and to reduce the use of physical restraints and stat medications for aggressive behavior of the individuals in their care. In addition, there were significant reductions in aggressive events by the individuals in their care, 1:1 staffing of individuals with aggressive behavior, and staff turnover. Furthermore, the MBPBS training was significantly more cost-effective than the TAU training. If replicated in future RCT studies, MBPBS may provide an effective means of enhancing socially acceptable bidirectional engagement of caregivers and care recipients within a person-centered context. BODY.INTRODUCTION: Caregivers, regardless of whether they are unpaid family members or paid staff, often end up having their psychological well-being compromised due to the stressful nature of caregiving (McIntyre et al., 2002; Hastings et al., 2006; Herring et al., 2006). Many change jobs because of psychological stress (Hastings and Beck, 2004) and burnout (Chung and Harding, 2009), or require therapy if they continue in their role of caregiving. In this context, psychological stress results from emotional and physiological reactions to job-related demands that a caregiver is unable to cope with, and burnout results when prolonged stress exhausts the physical and emotional strength of the caregiver. Recent research suggests that caregivers can enhance the self-management of their psychological stress by engaging in a disciplined practice of meditation (Singh, 2014). There are several mechanisms that may come into play when a caregiver regularly practices meditation, especially mindfulness (Anālayo, 2016). For example, enhanced mindfulness may provide caregivers better emotional self-regulation during periods of acute stress. It may also increase cognitive flexibility, when their responses are informed by awareness of what is unfolding in the present moment without the distortions of their own emotions and perceptions of the events. Every time caregivers are able to view each unfolding event with a beginner's mind, perceiving each event as if it is occurring for the first time, then "right action" emerges (Suzuki, 1970). Caregivers of individuals with intellectual and developmental disabilities (IDD) face additional stress from the severe challenging behaviors of individuals in their care (Hensel et al., 2012; Didden et al., 2016). For example, individuals with IDD evince aggression that is often of low frequency but high intensity, and are likely to physically hurt their caregivers as well as their peers. The prevalence rate of aggression in this population varies considerably, ranging from about 7% (Emerson et al., 2001) to over 50% (Tenneij et al., 2009; Jahoda et al., 2013). Current research suggests that when caregivers are stressed due to the aggressive behavior of the individuals in their care, they tend to develop a negative attitude toward the individuals, eventually leading to negatively interacting with them or avoiding them (Jahoda et al., 2013). Indeed, caregiver stress may lead them to recommend that individuals in their care who are aggressive be treated with restrictive procedures such as psychotropic medications, emergency medications, and physical restraints (Singh et al., 2011a; Deveau and McGill, 2014). In response, agencies have typically taken one of three general approaches to assist caregivers in delivering services to individuals with IDD. In the most widely used approach, caregivers are provided additional training in managing the challenging behaviors of individuals in their care. This could take many forms. For example, caregivers often receive new employee and in-service training in behavior management procedures, typically involving the principles and practice of positive behavior support (PBS; MacDonald, 2016; Morris and Horner, 2016). When implemented with fidelity, PBS has been shown to be immensely successful in caregiver management of the challenging behaviors of individuals with IDD. However, PBS procedures may not be used with fidelity in actual practice because of staff shortages, the inability of staff to use the procedures with more than one person at a time, caregiver stress, and the intensity of effort required (Allen et al., 2005; Didden et al., 2016). While assisting caregivers to better manage the aggressive behavior of the individuals is a viable and logical approach, it does not help the caregivers to manage their own stress and burnout. A second approach involves providing training that enables caregivers to better manage their work-related psychological distress. For example, Noone and Hastings (2009, 2010) showed that when caregivers participate in Promotion of Acceptance in Carers and Teachers training, it enables them to significantly decrease their psychological distress even when faced with a slight increase in occupational stress. This training included three key components of Acceptance and Commitment Therapy (Hayes et al., 1999)—acceptance, cognitive mindfulness, and values clarification. In a similar approach, Brooker et al. (2013) showed that when caregivers participate in occupational mindfulness (OM) training, they are able to decrease their stress, enhance psychological well-being, and increase job satisfaction. The OM training includes mindfulness practices, aspects of positive psychology (e.g., signature strengths; Seligman, 2002), and various cognitive therapy exercises. A third approach is to enhance the ability of the caregivers to skillfully use behavior management strategies and to learn ways of reducing their own occupational stress. For example, Singh et al. (2009) added a mindfulness-based training to the existing training in behavior management principles for caregivers, and demonstrated positive changes in the behaviors of both the caregivers and the individuals in their care. Brooker et al. (2014) essentially replicated these results using a different set of mindfulness-based training procedures. In a small multiple-baseline design study, Singh et al. (2015) evaluated the effects of an integrated mindfulness-based training with PBS training (i.e., Mindfulness-Based Positive Behavior Support [MBPBS]; Singh et al., 2016a). The results suggested that the MBPBS training enabled the caregivers to greatly reduce their psychological stress, eliminate staff turnover, and substantially reduce and then eliminate the use of physical restraints with individuals who evinced aggressive behavior. In a proof-of-concept quasi-experimental design study, Singh et al. (2016b) further evaluated the effectiveness of MBPBS training for caregivers. The results corroborated earlier findings of lowered caregiver psychological stress and staff turnover, and significantly less use of physical restraints. Furthermore, in both studies, a benefit-cost analysis showed substantial financial savings for the agency due to their staff participating in the MBPBS training (Singh et al., 2015, 2016b). These studies strongly suggest that caregivers can learn to regulate their emotions more effectively through MBPBS training than with standard agency in-service training. Furthermore, enhanced caregiver emotional regulation appears to have benefits for the caregivers (e.g., reduced psychological stress and staff turnover), as well as for those in their care (e.g., reduced engagement in aggressive behavior). These studies focused on caregivers who provided services to individuals with IDD who functioned at mild to moderate levels but did not include those who functioned at severe and profound levels. In addition, these studies did not use control groups to compare the effects of the different training using a robust experimental design. Thus, the aim of the present study was to evaluate the comparative effectiveness of MBPBS and Training-as-Usual (TAU) for caregivers in a congregate care facility for individuals with severe and profound IDD, using a randomized controlled trial (RCT). The comparative effects of the two training conditions were assessed in terms of caregiver variables (i.e., use of physical restraints, use of stat [emergency] medications, perceived psychological stress), a care recipient variable (i.e., number of aggressive events), and agency variables (i.e., 1:1 staffing of individuals with aggressive behavior, staff turnover, benefit-cost of the two trainings). BODY.MATERIALS AND METHODS.SETTING: The study was conducted at a large congregate care facility for individuals with IDD. The agency served individuals who were at the severe and profound levels of functioning and exhibited varying levels of challenging behaviors (e.g., physical aggression, property destruction, pica, rumination, stereotypy). The individuals resided in six homes, with each home accommodating between 6 and 10 individuals (mean = 8 per home). In total, there were 48 long-term beds, all of which were filled except when an individual needed short-term admission at a local hospital for acute medical care. All individuals exhibited challenging behaviors, but only 34 of the 48 individuals exhibited these behaviors at a level of severity and/or frequency that required the caregivers to implement formal behavior management plans. Of the 34 with formal behavior management plans, up to 10 of them required 1:1 staffing on any given day for aggressive behavior toward staff and/or peers. A 1:1 staffing level was used for the safety of the individual, staff and peers. BODY.MATERIALS AND METHODS.PARTICIPANTS: As a part of the facility's consumer engagement plan for enhancing the health and wellness of individuals with IDD, the entire caregiver staff was required to receive additional in-service training. All 107 caregivers were enrolled, of which 30 did not meet the inclusion criteria (i.e., full-time employment, consent to participate in the training, and availability during the training). Using a random number generator, the remaining 77 caregivers were randomized into MBPBS or TAU conditions. Of the 39 caregivers randomized to the MBPBS group, 2 dropped out before the study due to personal reasons (i.e., one due to change of job and the other due to late stage pregnancy). None of the 38 caregivers randomized to the TAU group dropped out. Figure 1 presents a CONSORT participant flow diagram. FIGURE 1Participant flow through the trial (CONSORT diagram). The sociodemographic data for both the caregivers and the individuals with IDD in their care are presented in Table 1. Table 1 Socio-demographic characteristics of the caregivers and individuals with IDD in their care for the Mindfulness-Based Positive Behavior Supports (MBPBS) and Training-as-Usual (TAU) conditions. MBPBS TAU Caregivers Individuals with IDD Caregivers Individuals with IDD Number of participants 37 24 38 24 Mean age in years (SD) 43.05 (10.39) 39.21 (7.61) 45.08 (7.87) 42.33 (9.22) Age range (years) 23–62 27–54 25–59 24–57 Gender: males 14 (37.83%) 16 (66.67%) 10 (26.32%) 16 (66.67%) Level of functioning     Severe na 9 (37.5%) na 7 (29.17%)     Profound na 15 (62.5%) na 17 (70.83%) Number of individuals on psychotropic medications na 20 (83.33%) na 19 (79.16%) Number of individuals with mental illness na 20 (83.33%) na 19 (79.16%) Number of individuals with behavior plans for aggressive behavior na 18 (75%) na 16 (67%) na, not applicable. BODY.MATERIALS AND METHODS.ETHICS STATEMENT: All training procedures in the study were in accord with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from all caregivers who participated in the study. The provider agency, caregivers, and facility review committee approved the MBPBS and TAU training and data collection. BODY.MATERIALS AND METHODS.PROCEDURE.EXPERIMENTAL DESIGN: A RCT was conducted to evaluate the comparative effectiveness of MBPBS vs. TAU on caregiver, individuals with IDD, and agency variables. Unless otherwise stated, standard agency data collection procedures were followed as part of the study protocol. BODY.MATERIALS AND METHODS.PROCEDURE.INTERVENTIONS.MINDFULNESS-BASED POSITIVE BEHAVIOR SUPPORTS: The standard 7-day MBPBS protocol, as reported by Singh et al. (2015, 2016b), was used. The training was presented in three parts, spread over a 10-week period. Part I lasted one 8-h day, Part II was five 8-h days (i.e., 40 h), and Part III was one 8-h day. All 37 caregivers in this condition received all the MBPBS training in a group format. Table 2 presents the MBPBS program and a brief outline of each day's training. Table 2 Outline of the 7-day MBPBS program. PART I Day 1 (First 1-day training) Samatha meditation Kinhin meditation Vipassanā meditation Five hindrances—sensory desire, ill will, sloth and torpor, restlessness and remorse, and doubt Daily logs and journaling PART II Day 2 Review of meditation practice (First day of 5-day intensive training) Introduction to the Four Immeasurables ( Brahmavihara : metta —lovingkindness; karuna —compassion; mudita —empathetic joy; upekkha —equanimity) Equanimity meditation Beginner’s mind Applications to PBS practice Day 3 Review of day 2 instructions and practices Further instructions on the Four Immeasurables Equanimity meditation Lovingkindness meditation Being in the present moment Applications to PBS practice Day 4 Review of days 2 and 3 instructions and practices Further instructions on the Four Immeasurables Equanimity meditation Lovingkindness meditation Compassion meditation The three poisons—attachment, anger, and ignorance Applications to PBS practice Day 5 Review of days 2 to 4 instructions and practices Further instructions on the Four Immeasurables Equanimity meditation Lovingkindness meditation Compassion meditation Joy meditation Attachment and anger—shenpa and compassionate abiding meditations Applications to PBS practice Day 6 Review of days 2 to 5 instructions and practices Review and practice Samatha, Kinhin, and Vipassanā meditations Review of the Four Immeasurables Practice equanimity, lovingkindness, compassion, and joy meditations Attachment and anger—meditation on the soles of the feet Review of applications to PBS practice Review of the MBPBS training program PART III Day 7 (Second 1-day training) Review of the meditation instructions and practices (daily logs) Review and practice Samatha, Kinhin, and Vipassanā meditations Review of the Four Immeasurables Practice equanimity, lovingkindness, compassion, and joy meditations Emotion regulation and anger—meditation on the soles of the feet Instructions for practicing three ethical precepts—refrain from (a) harming living creatures, (b) taking that which is not given, and (c) incorrect speech Applications to PBS practice Review of the 7-day MBPBS training program Part I was on the first day of the first week of training, during which the caregivers received instructions in and practiced three foundational meditations: Samatha, Kinhin, and Vipassanā. The caregivers received in-depth instructions and practiced the fundamentals of meditation posture. They were instructed to sit comfortably with a straight spine, without slouching or stretching the shoulders, with their head tilted slightly forward, eyes slightly open or closed, with the tip of their tongue lightly touching the upper palate, the right hand resting over the left hand on the lap, with thumbs just touching, and breathing evenly (Buksbazen, 2002). They were taught to focus on their breathing, without deliberately changing the length of each breath. They learned to count an inhalation and exhalation as one breath until they reached 10 breaths, before restarting the counting cycle. They were taught to simply observe their discursive thoughts and emotions, without interacting with them or trying to suppress them. That is, they were required to focus their awareness on whatever took place in their mind without judgment or engagement. Samatha meditation is the foundational meditation that provides the practitioner with the stability of mind on which to build all other meditation practices. In addition, they were taught Kinhin (walking) meditation and Vipassanā (insight) meditation (McDonald, 2005). Kinhin is a walking meditation that enables a person to be in the present moment while walking slowly and mindfully. Vipassanā meditation is used to gain insight into the true nature of reality through mindfulness of breathing, thoughts, feelings, and actions (Shonin et al., 2015). Toward the end of the first day of training, all caregivers were instructed to develop a personal meditation practice, beginning with a few minutes each day and incrementally increasing it until they reached between 20 and 30 min of daily practice. Finally, they were required to log their daily meditation practice. The caregivers were required to practice the three meditations daily until training in Part II that was scheduled for the fifth week. During Part II (days 2 to 6 of training), the caregivers received instructions on the nature of the Four Immeasurables (equanimity, lovingkindness, compassion, and empathetic joy), and meditation practices in equanimity, lovingkindness, compassion, and empathetic joy (Kyabgon, 2004). They also received instructions on the concepts and application of the beginner's mind (Suzuki, 1970), being in the present moment, the three poisons—attachment, anger and ignorance (Kyabgon, 2004), shenpa and compassionate abiding (Chödrön, 2007, 2010; Kongtrül, 2008), meditation on the Soles of the Feet (Singh et al., 2011b), and the general concept of emotion regulation and application of the various meditations in the caregivers' work and private life. Part III was scheduled on the first day of the 10th week (i.e., seventh full day of training), for follow-up, wrap-up, and follow-through meditation practices. This involved further meditation practice, review of the caregivers meditation practices and experiences, questions and answers from the group, and how the caregivers would continue their practice till the end of week 40—when formal aspects of the study concluded—and beyond. For the PBS component of the MBPBS training, Part I was devoted to ascertaining the current knowledge of the caregivers in the principles and practice of PBS, and collaboratively developing a training program in PBS within the context of mindfulness-based practices. The PBS training program was informed by current literature on PBS (Morris and Horner, 2016) and staff training in PBS (MacDonald, 2016), the caregivers' lived experience of working with individuals with IDD who periodically engaged in high-intensity but low frequency aggressive behavior, and the seamless interface with the caregivers' personal practice of mindfulness. During Part II (i.e., the 5-day training), the caregivers were instructed in the following five components of standard PBS plans: setting event strategies, preventive strategies, teaching strategies, consequence strategies, and quality of life outcomes (Lucyshyn et al., 2015). In terms of interfacing with their mindfulness practices, they were given instructions on mindful observation of the individual's behavior, mindful communication (with a focus on mindful prompting and feedback), mindful pause between requests and prompts, and mindful use of reinforcement contingencies that focused on the rate, quality, magnitude, delay, and specificity of the reinforcement delivered contingently and non-contingently to the individuals with IDD in their care (Singh et al., 2016a). Part III (i.e., the seventh full day of training), involved a review of the mindfulness-based PBS practice, discussion of the need for formal PBS programs, questions and answers regarding the MBPBS practice, and follow-through PBS practices. BODY.MATERIALS AND METHODS.PROCEDURE.INTERVENTIONS.TRAINING-AS-USUAL: The TAU in-service training followed the same three-Part, 7-day training timeline as in the MBPBS training. The training staff of the provider agency provided this training. It covered the following general areas of applied behavior analysis: definitions and characteristics; principles, processes and concepts; behavioral assessment; evaluation of outcomes; development and implementation of behavior management plans; and ethical considerations in using a behavioral approach to interventions (Cooper et al., 2007; Mayer et al., 2012). The caregivers were given instructions in reading, understanding, and implementing behavior management plans, observing the implementation of behavior support plans by expert staff, role playing implementation of PBS plans, and getting feedback on their implementation efforts. Further, they discussed their current behavior support plans, their effectiveness, fidelity of implementation, data collection, graphing the data, and revisions based on the data. BODY.MATERIALS AND METHODS.PROCEDURE.TRAINING ADHERENCE: For both training conditions, the caregivers' attendance at the 7-day training program was documented. In addition, caregivers in both training conditions were requested to record the time they spent in daily meditation during the 40 weeks of the study. All caregivers in both training conditions attended and fully participated in the 7 days of training. The daily logs showed that all 37 caregivers in the MBPBS training condition began their practice of meditation following Part I training (i.e., first 1-day training) and continued throughout the 40 weeks of the study. The duration of meditation gradually increased from a few minutes during Part I and averaged 15 min by the end of training in Part II (i.e., the 5-day training). There was a further increase following the 5-day training, and this averaged 33 min by the end of training in Part III. Thereafter, the caregivers averaged between 25 and 40 min of daily meditation, with occasional meditation holidays. Overall, on average, the caregivers in the MBPBS condition meditated for 89% (range: 0–96%) of the days. Two caregivers from the TAU training condition had a personal meditation practice prior to the study and both continued their meditation practice during the 40 weeks of the study. On average they meditated between 20 and 30 min daily, with occasional breaks from meditation. BODY.MATERIALS AND METHODS.PROCEDURE.TRAINERS: The MBPBS trainer was an experienced behavior analyst at the BCBA-D level, with over 35 years of hands-on experience in developing and implementing behavior support plans. In addition, the trainer had a 40-year personal meditation practice and experience in the mindful delivery of services in behavioral health. Segments of training in Parts I, II, and III were videotaped and 10 randomly selected segments of 10–15 min from each day of the 7-day training (i.e., 70 training segments) were rated for fidelity of training by another qualified meditation trainer who was also an expert in PBS. The fidelity of the MBPBS training was rated at 100% for both the meditation instructions and the training in PBS. The TAU trainer was an experienced behavior analyst at the BCBA level, with over 20 years of training experience in behavior management. This trainer did not have a personal meditation practice. As with the MBPBS training, segments of training in Parts I, II, and III were videotaped and 10 randomly selected segments of 10–15 min from each day of the 7-day training (i.e., 70 training segments) were rated for fidelity of training by another qualified trainer in behavior management. The fidelity of the behavioral training was also 100%. BODY.MATERIALS AND METHODS.PROCEDURE.MEASURES.AGGRESSIVE EVENTS: An aggressive event was defined as an individual hitting, biting, scratching, punching, kicking, slapping, or destroying property. Staff recorded each instance of an aggressive event on an incident reporting form at the point of occurrence and this was later entered in the facility's incident management database. By policy, each incident was double-checked by the home supervisor for occurrence and accuracy of reporting. The reliability of reporting and logging the occurrence of aggressive events was 98% (range: 94–100%). BODY.MATERIALS AND METHODS.PROCEDURE.MEASURES.PHYSICAL RESTRAINTS: A physical restraint was defined as a brief physical hold of an aggressive individual by a caregiver when there was imminent danger of physical harm to the individual, peers or staff, and the behavior could not be controlled with verbal redirection. Staff recorded each instance of the use of a physical restraint at the point of occurrence and this was later entered in the facility's risk management database. By policy, each use of physical restraint was double-checked by the home supervisor for occurrence and accuracy of reporting. The reliability of reporting and logging the occurrence of physical restraints was 100%. BODY.MATERIALS AND METHODS.PROCEDURE.MEASURES.STAT MEDICINE: Stat medicine is prescribed during medical, psychiatric or behavioral emergencies for the immediate safety of the individual. Stat medication was defined as an emergency medication prescribed for the immediate calming of an individual who was aggressive and could not be managed by other means, including physical restraints. Each prescription was counted as one event as recorded by a registered nurse in the individual's Medication Administration Record (MAR). Only those prescriptions that were prescribed specifically as emergency medication for aggressive behavior were counted. BODY.MATERIALS AND METHODS.PROCEDURE.MEASURES.ONE-TO-ONE STAFFING: A 1:1 staffing is the level of supervision used when an individual needs close attention for a specific reason and it is designed to ensure the safety of the individual, peers or staff. For the purpose of this study, 1:1 staffing was defined as the level of enhanced observation ordered by a physician or psychologist for an individual with IDD who evinces aggressive behavior. Each individual's treatment team determined the need for level of supervision, the nursing administration assigned the staff, and the home manager ensured the provision of level of supervision on a shift-by-shift basis. Level of supervision staff was recorded as being present for the assigned duties 100% of the time. BODY.MATERIALS AND METHODS.PROCEDURE.MEASURES.STAFF STRESS: Caregivers in both training conditions completed the Perceived Stress Scale-10 (PSS-10; Cohen et al., 1983) as a measure of perceived stress at two time points: on the first and last day of the 40-week study. The PSS-10 provides an index of the degree to which people perceive their lives as stressful and indicates how often they have found their lives to be unpredictable, uncontrollable, and overloaded in the last month. This rating scale includes items such as, "In the last month, how often have you found that you could not cope with all the things that you had to do?" The caregivers responded to 10 questions on a five-point scale, ranging from 0 (never) to 4 (very often). Their responses were summed to create a psychological stress score, with higher scores indicating greater psychological stress. The PSS-10 has adequate psychometric characteristics (Cohen and Williamson, 1988). In the present study, Cronbach's alpha was 0.82, indicating good reliability. BODY.MATERIALS AND METHODS.PROCEDURE.MEASURES.STAFF TURNOVER: The facility's Human Resources Department provided the staff turnover data, which included all instances of any staff member leaving the employment of the agency due to staff injury on the worksite during the 40 weeks of the study period. Data were extracted only for the fulltime staff involved in the MBPBS and TAU training conditions. BODY.MATERIALS AND METHODS.PROCEDURE.MEASURES.COST EFFECTIVENESS DATA: The facility's Finance Department provided cost data on (1) work days lost due to staff injury, (2) instances of 1:1 staffing, (3) staff needing medical and physical rehabilitation therapy due to injury, (4) staff resigning due to staff injury who were replaced, (5) staff required for MBPBS and TAU training, and (6) temporary staff required during MBPBS or TAU training. All costs were included, regardless of whether the costs were borne by the agency or by workers' compensation. BODY.MATERIALS AND METHODS.DATA ANALYSES: The effectiveness of the MBPBS and TAU conditions were examined in several ways. For several variables, the unit of analysis was a count variable for an entire condition, not for individuals within a condition. These examples included the number of aggressive events per week, the number of uses of physical restraints per week, the number of emergency stat medicine prescriptions per week, and the number of additional 1:1 staffing needed per week. As these variables are not at the individual level, they do not lend themselves to traditional analyses for RCTs. Therefore, we employed two strategies to examine change across time within conditions and differences across groups. First, we were able to examine change across time within each condition by treating each group as an n of 1. In doing so, we plotted the count of each variable for each condition across all weeks of the study. Second, we averaged counts per week across the 10-week Training phase and the 30-week post-training phase, respectively, for each condition. The resulting M's (with SD's) could then be compared across groups by phase with independent samples t-test (and corresponding Cohen's d values as effect size measures). Using paired samples t-test, we compared change across phases within each condition. Note that alternative approaches, such as use of a mixed-model ANOVA, were not possible because the unit of analysis was not individuals. The data on perceived stress were unique to individuals. Therefore, we used a mixed-model ANOVA to compare main effects of condition, time, and their interaction. Effect sizes reported include η2 for an overall effect size, and Cohen's d's for direct comparisons for a specific phase across conditions, or for a specific condition across time. BODY.RESULTS.DEMOGRAPHIC VARIABLES: We ran a series of Chi-Square and Independent Samples t-test to compare demographic characteristics of participants in the MBPBS and TAU conditions. There were no statistical differences between the groups (all p's > 0.05). BODY.RESULTS.CAREGIVER VARIABLES.PERCEIVED STRESS: Figure 2 shows there was a decrease in the perceived stress score from the first day of Training (Time 1) to the last day of post-training (Time 2) of 36.15% in the MBPBS condition and 9.02% in the TAU condition. Differences across time and between conditions on PSS were examined with a 2 (condition: MBPBS vs. TAU) × 2 (timepoint: pre vs. post) mixed model ANOVA. The results revealed a significant interaction, F(1,73) = 73.70, p < 0.001 (η2 = 0.50). Figure 2 shows there was no significant difference between groups on the first day of Training (pre) phase (Cohen's d = 0.36), but there was a significant difference, with a large effect size, on the last day of the post-training phase (Cohen's d = 2.78). The within-groups effect sizes from pre to post for the MBPBS and TAU conditions were as follows: Cohen's d = 2.60 and 0.70, respectively. FIGURE 2Ratings of caregiver perceived stress on PSS-10 on the first day of training and the last day of post-training in the two conditions, Mindfulness-Based Positive Behavior Supports (MBPBS) and Training-as-Usual (TAU). Note that higher scores indicate greater psychological stress. Error bars report standard error of the mean. BODY.RESULTS.CAREGIVER VARIABLES.PHYSICAL RESTRAINTS: As evident in Figure 3, the weekly use of physical restraints decreased substantially from the Training to the post-training phase for the MBPBS condition, while there was no such decrease in the TAU condition. On average, the use of physical restraints in the MBPBS condition was 8.00 (range = 2–13) per week during the Training phase and 0.53 (range = 0–3) in the post-training phase. Similarly, on average, the use of physical restraints in the TAU condition was 13.60 (range = 9–17) per week during the Training phase and 10.77 (range = 5–16) in the post-training phase. During the Training phase, there was a statistically significant difference in physical restraint use per week across the MBPBS (M = 8.00, SD = 4.06) and TAU conditions (M = 13.60, SD = 2.72), t(18) = 3.62, p = 0.002 (Cohen's d = 1.62). During the post-treatment phase, use of physical restraints was lower in the MBPBS condition (M = 0.53, SD = 0.90) than in the TAU condition (M = 10.77, SD = 3.21), t(58) = 1.73, p = 0.10 (Cohen's d = 4.34). FIGURE 3Mean number of physical restraints per week used by caregivers contingent on aggressive behavior of the individuals in the MBPBS and TAU conditions. Error bars report standard error of the mean. BODY.RESULTS.CAREGIVER VARIABLES.STAT MEDICATION: As shown in Figure 4, the weekly use of stat medications decreased substantially from the Training to post-training phase for the MBPBS condition, while there was no such decrease in the TAU condition. On average, the use of stat medication in the MBPBS condition was 6.40 (range = 2–11) per week during the Training phase and 0.23 (range = 0–3) in the post-training phase. Similarly, on average, the use of stat medication in the TAU condition was 8.60 (range = 5–12) per week during the Training phase and 7.17 (range = 2–14) in the post-training phase. During the Training phase, there was no statistically significant difference in mean use of stat medications per week between the MBPBS (M = 6.40, SD = 3.24) and TAU conditions (M = 8.60, SD = 2.37), t(18) = 1.73, p = 0.10 (Cohen's d = 0.77). During the post-training phase, the use of stat medications in the MBPBS condition was significantly lower (M = 0.23, SD = 0.68) than in the TAU condition (M = 7.17, SD = 3.30), t(58) = 11.28, p < 0.001 (Cohen's d = 2.91). FIGURE 4Mean number of stat medicines per week used by caregivers contingent on aggressive behavior of the individuals in the MBPBS and TAU conditions. Error bars report standard error of the mean. BODY.RESULTS.CARE RECIPIENT VARIABLE.AGGRESSIVE EVENTS: Figure 5 shows the number of aggressive events by each training condition. On average, in the MBPBS condition there were 21.75 (range = 19–27) aggressive events per week during the Training phase and 5.91 (range = 0–21) in the post-training phase. Similarly, on average, in the TAU condition there were 22.25 (range = 17–26) aggressive events per week during the Training phase and 17.75 (range = 10–24) in the post-training phase. There was no significant difference between the MBPBS (M = 21.40, SD = 2.76) and TAU conditions (M = 21.70, SD = 3.71), t(18) = 0.21, p = 0.84 (Cohen's d = 0.09) during the Training phase. However, the difference between conditions was significant in the post-training phase, t(58) = 11.01, p < 0.001 (Cohen's d = 2.84), with the MBPBS condition (M = 4.97, SD = 4.86) demonstrating fewer aggressive events than the TAU condition (M = 17.63, SD = 4.01). FIGURE 5Mean number of aggressive events per week exhibited by the individuals in the MBPBS and TAU conditions. Error bars report standard error of the mean. BODY.RESULTS.AGENCY VARIABLES.1:1 STAFFING: Figure 6 shows the average 1:1 staffing (in addition to regular staffing) required for the care, safety and protection of staff and peers from individuals with IDD who were periodically aggressive. In the MBPBS condition, on average, 4.40 (SD = 1.78; range = 2–7) additional staff was required each week during Training and 0.13 (SD = 0.35; range = 0–1) during post-training. In the TAU condition, on average, 4.90 (SD = 1.45; range = 3–7) additional staff was required each week during Training and 4.53 (SD = 1.59; range = 3–9) during post-training. The difference between the two groups during the Training phase was not statistically significant, t(18) = 0.69, p = 0.50, but the difference between MBPBS vs. TAU post-training phases was statistically significant, t(58) = 14.80, p = 0.0001. FIGURE 6Mean number of additional caregivers used for 1:1 staffing in the MBPBS and TAU conditions. Error bars report standard error of the mean. BODY.RESULTS.AGENCY VARIABLES.STAFF TURNOVER: In terms of staff turnover, no caregiver in the MBPBS condition resigned due to injury and stress during the Training or post-training phases. Three caregivers resigned (all due to injury) during the Training phase in the TAU condition and 11 caregivers resigned (seven due to injury, four due to stress and injury) during the post-training phase. The difference between the MBPBS (M = 0.00, SD = 0.00) and TAU (M = 0.30, SD = 0.48) Training phases was not statistically significant, t(18) = 1.98, p = 0.06. During the post-training phase, the difference between the MBPBS (M = 0.00, SD = 0.00) and the TAU (M = 0.37, SD = 0.67) conditions was statistically significant, t(58) = 3.02, p = 0.004. BODY.RESULTS.AGENCY VARIABLES.COST EFFECTIVENESS: Table 3 presents the cost effectiveness data for the MBPBS and TAU conditions. When compared to the TAU condition, the number of lost days of work due to staff injury was reduced by about 92% in the MBPBS condition, with a savings of $82,992.00. Commensurate with this savings, there was additional savings of $72,128.00 during the 40 weeks of the study in terms of additional costs of 1:1 staffing. The cost of medical and physical rehabilitation therapy services for the two injured staff during the MBPBS condition was $39,000.00 compared to $351,000.00 for the 18 staff injured during the TAU condition. While no additional costs were incurred in the MBPBS condition due to any staff turnover during the 40-week study period, the cost of training 14 new staff in the TAU condition was $17,680.00. The cost of providing alternate (temporary) staff for those who were in the MBPBS training was $41,440.00 compared to $42,560.00 in the TAU condition. Finally, the cost of providing the MBPBS training was $30,000.00 compared to $2,000.00 for TAU training. Overall, when compared to the TAU condition, there was a savings of $457,920.00 for an equivalent period during the MBPBS condition; that is, a savings of over 78%. Table 3 Comparative costs for 40 weeks of MBPBS training condition ( n = 37) compared to 40 weeks of TAU training condition ( n = 38) for the human services variables. Cost Variables Cost MBPBS TAU MBPBS TAU Lost days of work and cost due to staff injury 42 536 $7,056.00 $90,048.00 Number of staff-days and cost of 1:1 staffing 96 740 $10,752.00 $82,880.00 Number and cost of staff needing medical and physical rehabilitation therapy 2 18 $39,000.00 $351,000.00 Number of staff resigned due to staff injury and training costs for new hires 0 14 $0.00 $17,680.00 Number of training days and cost of training 10 10 $30,000.00 $2,000.00 Cost of temporary staff during training 37 38 $41,440.00 $42,560.00 Total additional costs for the two time periods $128,248.00 $586,168.00 Total overall savings $457,920.00 BODY.DISCUSSION: Caregivers in mental health provide services for individuals with diverse abilities and challenging behaviors that often sap their mental and physical resources, and lead to stress and burnout. The traditional solution has been to teach caregivers specialized techniques, such as behavior analytic skills, that will help them to be more effective in managing the challenging behaviors of the individuals in their care (MacDonald, 2016; McIntyre and Neece, 2016). While this approach has been found effective, caregivers often work in situations where such skills alone may not be enough to curb their stress and burnout, because of the intensity and frequency of the challenging behaviors they face, multiple individuals engaging in challenging behaviors, the shortage of well-trained staff when the need arises, and the emotional toll of such work (Hastings, 2002; Allen et al., 2005; Crawford et al., 2010; Deveau and McGill, 2014). An emerging approach involves not only enhancing the management skills of the caregivers in terms of the needs of the individuals they serve, but also teaching them self-management skills that enhance the caregivers' psychological well-being, thereby making them more resilient in their work situation (Noone and Hastings, 2009, 2010; Brooker et al., 2013, 2014). For example, a series of exploratory proof-of-concept studies reported that adding mindfulness-based skills to caregivers previously or concurrently trained in PBS reduces their stress and burnout, as well as the challenging behaviors of those in their care (Singh et al., 2009, 2015, 2016b). Related studies have supported the notion that caregivers and their clients with disabilities mutually benefit when the caregivers are given training in mindfulness-based approaches (Brooker et al., 2013, 2014). The present study strengthens the evidence-base for this approach. In a RCT, the current study demonstrated multiple beneficial effects of training caregivers in MBPBS compared to TAU in a congregate care long-term care facility for individuals with IDD. First, with regard to caregiver variables, the effects of the MBPBS training were evident in terms of statistically significant reductions in perceived psychological stress and job resignations due to stress and work-related injury. In addition, following training in MBPBS, staff greatly reduced using physical restraints and stat medicines contingent on the individuals' aggressive behaviors. These findings confirm earlier reports of significant reductions in the use of physical restraints and emergency medications for individuals with aggressive behavior following training of caregivers in MBPBS (Singh et al., 2015, 2016b). Furthermore, Brooker et al. (2014) reported similar findings in terms of reduced staff use of PRN ("as needed") medicines for behavior control, seclusions, and emergency chemical restraints following training in a mindfulness-based training program. Any reduction or elimination of the use of restrictive procedures indicates an enlightened approach to the care of people with diverse abilities, particularly those with IDD (Singh, 2016). Second, with regard to the care recipient variable, reductions in aggressive incidents were significantly greater with the individuals receiving care from caregivers trained in MBPBS, than with those receiving care from caregivers trained in TAU. Caregivers in the MBPBS training condition did not receive instructions on how to manage the behavior of specific individuals in their care who engaged in aggressive behavior. In addition, existing behavior management plans for aggressive behavior were not reviewed or revised as a component of the MBPBS training condition. Given that the only difference between the MBPBS and TAU conditions was the nature of the training the caregivers received, the significantly reduced frequency of aggressive behavior evident in the MBPBS condition could be attributed to the personal change in the caregivers due to training in MBPBS. Similar reductions in incidents of verbal and physical aggression were reported in previous studies in which staff was trained in MBPBS (Singh et al., 2009, 2015, 2016b). All these findings suggest that training in MBPBS may change the very nature of the reciprocal interactions between the caregivers and the individuals in their care, moving them from a negative to a positive trajectory (Sameroff, 1995). Third, with regard to agency variables, there was a significant reduction in the assignment of 1:1 staffing of individuals with aggressive behavior in the MBPBS condition when compared to the TAU condition. Indeed, when compared to the TAU condition, 1:1 staffing was rarely used following caregiver training in MBPBS. There was no staff turnover in the MBPBS condition compared to 14 in the TAU condition. Finally, the cost-effectiveness data showed substantial savings in the MBPBS condition when compared to the TAU condition. Costs were estimated for several standard variables (i.e., lost days of work due to staff injury, 1:1 staffing, treatment related costs for work-related staff injury, hiring and training of new staff, MBPBS and additional TAU training costs, temporary staff during training days) during the study period. There was an overall savings of 78.12% with the MBPBS training condition compared to the TAU training condition. Except for the costs of MBPBS training, which was much higher than for TAU, cost savings were realized on all other key variables in the MBPBS condition. Although the overall cost savings were somewhat less in the present study, they still aligned well with those from previous studies that reported savings 87.75% (Singh et al., 2015) and 89% (Singh et al., 2016b). Coupled with the enhanced psychological -being of the caregivers and reduced aggressive behavior of the care recipients, these cost savings suggest that MBPBS may be a clinically useful and financially viable training for caregivers. The results of this RCT indicate that the traditional training provided in large facilities and community group homes for individuals with IDD may not be very effective in ameliorating the stress and burnout of the caregivers. While such training may enable caregivers to provide the required services detailed in each individual's Individual Support Plan (ISP), the training is not very effective in assisting caregivers to effectively manage the behavior of individuals who engage in severe challenging behaviors, such as aggression, property destruction, and self-injury (Didden et al., 2016; Hoch et al., 2016). Furthermore, traditional training falls short in another respect—it does not teach caregivers how to successfully respond to workplace stress, compassion fatigue, and burnout. It is inevitable that caregivers will be stressed in large congregate care facilities for people with disabilities because of inherent demands in the job. Thus training should encompass strategies that enable caregivers to change their relationship to the job demands. Fortunately, mindfulness-based training does just that by teaching caregivers how to respond differently to the same daily work stresses, because they are typically not in position to change the nature of the job requirements (Hwang and Singh, 2016). The MBPBS training enabled staff to respond in a calm and mindful way rather than to react negatively to the challenging behaviors of the individuals (Singh et al., 2016a). We suspect that disciplined meditation practice enables the caregivers to gradually change their relationship to their perceived mental and emotional experiences that arise when providing care to the individuals. This ability to step back and observe their thoughts and emotions as they occur results in cognitive, emotional and behavioral flexibility which helps them to respond more adaptively to difficult situations, thus reducing psychological stress and burnout (Shapiro et al., 2006). Indeed, it is likely that this metacognitive awareness enables caregivers to distance themselves from their reactive thoughts and emotions, and reperceive difficult situations as transient mental events (Safran and Segal, 1990). The Samatha and Kinhin meditations in the MBPBS training enable the caregivers to pay attention to the individuals with non-judgmental awareness, supporting them to work through their ISPs rather than by controlling their challenging behaviors. The meditations on the Four Immeasurables enable the caregivers to develop more equanimity in the face of daily work hassles and stresses, view the individuals and other staff with lovingkindness and compassion, and to demonstrate empathic joy as events unfold. Their training in adopting a beginner's mind enables them to see more possibilities in terms of how to provide care to the individuals with challenging behaviors, buttressed by their training in seeing each individual and each event as if for the first time, without the baggage of history and emotional biases. For example, this mindset enables them to avoid reacting to the challenging behaviors of the individuals based on their premature cognitive commitment to control aggressive behavior through physical restraints and stat medications. While the actual mechanisms of the observed change due to training in mindfulness are yet to be explicated, it is increasingly evident that training staff in mindfulness produces behavioral changes in the staff as well as the individuals in their care (Eliassen et al., 2016). In addition to its strengths as a RCT, this study has limitations as well. An important one is the issue of equivalence of the training in each of the two conditions of the study. It can be argued that the MBPBS condition included more training components than the TAU condition and, thus, should produce better outcomes. A standard control group design could not be used because congregate care facilities are required by policy to provide in-service training, thus restricting the comparison to the TAU condition. To address this possible limitation, future studies could evaluate the differential effects of MBPBS against standard behavioral training, the current gold standard of training in the care of individuals with challenging behaviors. Also, this study was executed in a congregate care facility for individuals who functioned at the severe to profound levels of IDD. Whether similar outcomes would be expected in other settings is open to speculation. Previous studies were executed in community group homes, and with individuals who had higher levels of intellectual functioning, factors that could likely produce similar or better results. In addition, it would be instructive to ask the caregivers in the MBPBS condition their views on what and how changes occurred as a consequence of their participation in the meditation practices. Furthermore, future research should investigate the importance of the mindfulness trainer's personal meditation practice and the authenticity of the teachings as factors that may impact outcomes (Carmody and Baer, 2009; McCown et al., 2010). Mindfulness-Based Positive Behavior Supports provides a paradigm shift in terms of how caregivers and care recipients can be mutually engaged in enhancing the quality of their lives. This model is based on multiple theoretical underpinnings, including Sameroff's (1995) bidirectional transactions, mindfulness (Shonin et al., 2015), PBS (Morris and Horner, 2016), and patient engagement (Institute of Medicine, 2011; Barello et al., 2014; Graffigna et al., 2016), which is known as person-centered planning in the field of IDD (Ratti et al., 2016). In their lexicographic literature review, Barello et al. (2014) noted that the concept of patient engagement has changed over time, and still remains an elusive concept in clinical practice and health care policy. Coulter (2011, p. 10) has suggested that when patients and caregivers work together on patient engagement, they "promote and support active patient and public involvement in health and healthcare and... strengthen their influence on healthcare decisions, at both the individual and collective levels." The MBPBS model is grounded in mutual engagement of both the caregiver and care recipient, and by changing the behavior of one of the pair, the behavior of the other changes because of the bidirectional transactions that occur (Sameroff, 1995; Singh and Singh, 2001). Training caregivers in MBPBS not only elicits and enhances the individual's activation—his or her ability and willingness to self-manage challenging behaviors—but also promotes engagement in positive behavior, as evidenced in the present and previous studies (Singh et al., 2015, 2016b). There are several implications of this study. First, these findings need further validation in terms of the effects of MBPBS against other proven techniques, such as behavior analytic strategies, as well as replications by other research teams. Second, the effects of MBPBS need to be assessed in different care settings, such as institutions, group homes, family homes, and within the larger community settings. Third, the effects of MBPBS need to be further evaluated with parents and teachers of individuals with IDD, caregivers of other populations, such as the elderly and individuals with neurodevelopmental disorders. These kinds of studies will help refine the MBPBS model and delineate the boundary conditions for its effectiveness. Furthermore, future research should also focus on dissemination, especially given the finding that few evidence-based practices are actually implemented in real-world settings (Katz, 2010). The challenge is to make MBPBS not too intensive or effortful, but still produces replicable and long-lasting effects at a reasonable cost (Glasgow et al., 2003; Singh, 2016). In sum, this RCT provides further evidence that MBPBS may be a viable approach to caregiver training to improve the psychological well-being of both caregivers and the care recipients. This study showed that, when provided training in MBPBS, caregivers could significantly reduce their own stress levels and the use of restrictive procedures (e.g., physical restraints and emergency medications) when confronted with challenging behaviors by individuals with intellectual disabilities. When caregivers changed the nature of their care, the individuals reduced their aggressive behaviors and obviated the need for 1:1 staffing. Finally, the cost effectiveness data suggest that agencies may want to implement MBPBS training not only because of the benefits for the caregivers and care recipients, but also for financial savings. BODY.AUTHOR CONTRIBUTIONS: NS: designed and executed the study, assisted with the data analyses, and wrote the paper. GL: collaborated with the design and writing of the study. BK: analyzed the data and wrote part of the results. JC: collaborated with the design and writing of the study. AW: collaborated in the writing and editing of the final manuscript. BODY.CONFLICT OF INTEREST STATEMENT: NS is the developer of the MBPBS program. All the other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

TITLE: Comparison of Two Protocols in the Management of Glucocorticoid-induced Hyperglycemia among Hospitalized Patients ABSTRACT.CONTEXT:: There is limited literature focusing on the management of glucocorticoid-induced hyperglycemia (GCIH). ABSTRACT.AIMS:: The primary objective was to compare the mean blood glucose between the experimental group (new protocol) and the control group (standard protocol) in the management of GCIH. The secondary objective was to compare other parameters of glycemic efficacy, variability, and safety parameters. ABSTRACT.METHODS:: This was a randomized, open-labeled, parallel arm trial. Adult patients who were given glucocorticoid (minimum dose equivalent to prednisolone 10 mg) in the past 24 h and had 2 h postmeal plasma glucose ≥200 mg/dl were included in the study. Patients randomized to control group received standard basal-bolus insulin. In the experimental group, a "correctional insulin" matching the glycemic profile of the glucocorticoid administered was provided with or without "background" basal-bolus insulin. The parameters of glycemic efficacy, variability, and safety were compared. P < 0.05 was considered statistically significant. ABSTRACT.RESULTS:: Data of 67 patients included in the study were analyzed, of which 33 patients were in the experimental group and 34 patients in the control group. The mean blood glucose in the experimental and the control group was 170.32 ± 33.46 mg/dl and 221.05 ± 49.72, respectively (P = 0.0001). The parameters for glycemic variability were all significantly lower in patients in the experimental group. The hypoglycemia event rate was low in both the groups. ABSTRACT.CONCLUSION:: When compared to the standard basal-bolus insulin protocol, the new protocol showed lower mean blood glucose and lower glycemic variability. BODY.I: Glucocorticoids are popular therapeutic agents in clinical medicine. One of the major side effects of glucocorticoids is to cause an increase of blood glucose (hyperglycemia). The effect of glucocorticoids on glucose metabolism was first described by Ingle[1] in 1941. Management of glucocorticoid-induced hyperglycemia (GCIH) among hospitalized patients is a challenge. A study revealed that 64% of indoor patients who received glucocorticoids equivalent to prednisolone ≥40 mg developed hyperglycemia.[2] Hyperglycemia induced by glucocorticoids has an impact on mortality and morbidity. Studies in patients with hematological malignancies who develop GCIH have shown increased hospital stay, mortality, sepsis, and shorter periods of remission.[3] Based on our review of literature and understanding of the pharmacokinetics and pharmacodynamics of glucocorticoids, we developed a new protocol for the management of GCIH in hospitalized patients. The new protocol included the use of a "correctional insulin" which matches the glycemic profile of the glucocorticoid administered with or without "background" basal-bolus insulin. The dose of correctional insulin was based on the dose of the administered glucocorticoid and weight of the patient. The primary aim of the study was to compare the mean blood glucose between the experimental group (new protocol) and the control group (standard basal-bolus insulin protocol) in the management of GCIH in hospitalized patients. The secondary objective was to compare other parameters of glycemic efficacy between the two groups, glycemic variability. and safety between the two groups. BODY.M: This study was conducted at a single tertiary care institution in New Delhi. This was a randomized, open-labeled, parallel arm trial. Permission for the study was granted by the Institutional Ethics Committee on August 4, 2014. The study was conducted from August 2014 to April 2016. We used the data from the pilot study conducted by Seggelke et al.[4] for calculating our sample size. The calculated sample size was 23 for each group (total of 46). However, to improve the power of the study, we had decided to enroll 92 patients (double the calculated sample size). This gave our study a power of 99% with an alpha error of 0.05. Patients screened for eligibility to be included in the study were nonpregnant patients of ages 18 years and above, with or without diabetes mellitus (Type 1, Type 2, or other forms of diabetes mellitus), who were admitted to the nonintensive care unit of our hospital from August 2014 to April 2016. A screening was conducted to find the patients who were given systemic glucocorticoid (oral or parenteral) for any indication (minimum dose equivalent to prednisolone dose of 10 mg) in the past 24 h. Plasma glucose was checked 2 h after lunch and 2 h after dinner within 24–48 h of the patients receiving the glucocorticoid. The patients included in the study were those who had 2 h postlunch and/or 2 h postdinner plasma glucose ≥200 mg/dl. The detailed inclusion and exclusion criteria are given in Table 1. Table 1 Inclusion and exclusion criteria for the study BODY.M.STUDY PROCEDURE: Patients were recruited in the study once they signed the consent form. On recruitment, the patients were randomized to either the control group or the experimental group using computer-generated random numbers. Baseline demographic and clinical data were collected from the hospital records for all patients included in the study. All oral antidiabetics, premixed insulin, and noninsulin injectable antidiabetics were stopped in both the groups. Patient already taking basal-bolus insulin at home was allowed to continue the same with necessary adjustments as per the group allotted to them. All patients received fixed carbohydrate meals three times a day. Unless there was a specific reason, all patients received their meals at fixed times of the day. In all cases, capillary blood glucose (CBG) monitoring was carried out four times a day (three times before meals and at bedtime) using a glucometer. Our hospital uses glucometers from the same manufacturer in all the wards and it is regularly calibrated. All glucometer readings were informed to a member of the endocrinology team in real-time and also noted on the nursing charts. Data were obtained from these nursing charts by study investigators the next day. Adjustments were made to insulin regimen depending on the protocol assigned to the patient. Additional CBG readings were taken if the patient complained of symptoms suggestive of hypoglycemia and/or the treating endocrinologist ordered additional readings. If the additional blood glucose reading was suggestive of hypoglycemia (blood glucose <70 mg/dl) or severe hyperglycemia (blood glucose >300 mg/dl), the readings were counted in the event rate of hypoglycemia or severe hyperglycemia, respectively; however, the readings were not taken to calculate the other glycemic parameters. Any cases of hypoglycemia (blood glucose <70 mg/dl) were managed according to the existing protocol developed by the endocrinology department. If patient was given any correction for hypoglycemia, then the blood glucose readings for the rest of the day were discarded from the study. If the patient developed any episode of blood glucose >250 mg/dl, urinary ketones were checked. If any patient developed diabetic ketoacidosis (defined as blood glucose >250 mg/dl, urine ketone positive, pH <7.3, and serum bicarbonate <18 mEq/l), it was appropriately managed and that patient was excluded from the study. However, if the patient did not have diabetic ketoacidosis, the hyperglycemic excursion was managed as per the protocol assigned to the patient. If the patient developed blood glucose >400 mg/dl without ketoacidosis, the patient was given additional corrective dose of insulin as per the hospital protocol, irrespective of the group he was randomized to, and blood glucose readings for the rest of the day were discarded from the study. The event was however counted in the even rate for severe hyperglycemia. BODY.M.CONTROL GROUP: Treatment protocol for the control group was standard basal-bolus insulin regimen as detailed in the Endocrine Society guidelines.[5] BODY.M.EXPERIMENTAL GROUP: Patients randomized to the experimental group were managed according to the new protocol developed by us. The basis of this protocol is that giving an additional "correctional insulin" which matches the glycemic profile of the glucocorticoid administered would negate the glycemic effect of the glucocorticoid. The first step in the experimental group was to classify the patient according to whether they had established diabetes mellitus or developed hyperglycemia secondary to administration of glucocorticoids. They were divided into two groups, namely Group 1 and Group 2. Group 1 was the group of patients having established diabetes mellitus. Patients included in this group were those who fulfilled any of the following criteria: (a) patient having a history of diabetes mellitus as per their medical records, (b) patients on oral/injectable antidiabetic medications or insulin, and (c) glycated hemoglobin (HbA1c) ≥6.5%. The second group patients (Group 2) were those who did not have established diabetes mellitus but developed hyperglycemia after administration of glucocorticoids. Patient who did not fulfill any of the criteria enlist above were categorized as Group 2. The heart of our study was the administration of a "correctional insulin" to be given along with the glucocorticoid depending on the dose and type of glucocorticoid administered. The insulin given as correctional insulin would match the glycemic profile of the glucocorticoid administered. This is presented in Tables 2 and 3. Table 2 Type of correctional insulin to be used along with the glucocorticoid administered in the experimental group Table 3 Dose of the correctional insulin given along with the respective glucocorticoids in the experimental group Apart from the correctional dose of insulin, patients with established diabetes mellitus (Group 1) were given additional background basal-bolus insulin while those in Group 2 were not given any additional background insulin. The indication for background insulin is detailed in Table 4. Table 4 Summary of protocol used in the experimental group Those patients who were assigned to receive additional background insulin (Group 1) were given basal-bolus insulin. The total starting dose of insulin was same as that in the control group [Table 5]. The total starting dose was divided as 50% basal and 50% bolus insulin. Basal insulin used was insulin glargine, and short-acting bolus insulin used was insulin lispro. The basal insulin glargine was given at bedtime and the dose titrated as shown in Table 6. Table 5 Total starting dose of insulin for patients in the experimental and control group Table 6 Adjustment of basal insulin dose in the experimental and control groups Bolus insulin was administered 15 min before each meal. A supplemental dose of short-acting insulin lispro was given depending on premeal CBG readings. The premeal blood glucose readings were informed to the treating endocrinologist on the phone and the supplemental dose of insulin as administered as per the scale shown in Table 7a. This scale is based on the "usual" column of the supplemental insulin scale described in the Endocrine Society guidelines.[5] The patients in the control group were given "higher" dose of supplemental correctional insulin based on the "insulin-resistant" column of the supplemental insulin scale described in the Endocrine Society guidelines [Table 7b]. This was an additional dose over and above the scheduled bolus dose of insulin in both control and experimental groups. Table 7a Supplemental dose of premeal insulin for experimental protocol Table 7b Supplemental dose of premeal insulin in the control group Those who were basal-bolus insulin before admission were allowed to continue the same in the experimental group. The basal insulin and bolus insulin were changed to glargine and lispro, respectively, for the purpose of the study. In addition, "correctional insulin" was administered depending on the dose and type of glucocorticoid administered. Patients were followed up for the entire length of hospital stay till discharge. The discharge advice for the diabetes management was determined by the treating endocrinologist in consultation with the treating physician. BODY.M.MEASUREMENT OF THE GLYCEMIC PARAMETERS: CBG monitoring was carried out four times a day (fasting, prelunch, predinner, and bedtime) using a glucometer. Data from day 1 of randomization were not used to calculate any of the parameters mentioned below because the patient would be still in a titration phase on day 1. Hence, the blood glucose readings taken to calculate the mean blood glucose and all the other parameters excluded the readings from day 1 in both the control and the experimental groups. BODY.M.MEASUREMENT OF THE GLYCEMIC PARAMETERS.COMPARISON OF OTHER PARAMETERS OF GLYCEMIC EFFICACY: Apart from the mean blood glucose, the mean fasting blood glucose, mean prelunch, mean predinner, mean bedtime, and mean premeal blood glucose were calculated from the blood glucose data. The percentages of blood glucose readings in the target range were calculated. The target range for fasting, prelunch, predinner, and bedtime blood glucose values was determined as 100–140 mg/dl. The target range for bedtime blood glucose was 140–180 mg/dl. BODY.M.MEASUREMENT OF THE GLYCEMIC PARAMETERS.COMPARISON OF OTHER PARAMETERS OF GLYCEMIC VARIABILITY: We compared the parameters for glycemic variability between the two groups. Standard deviation (SD) of blood glucose was calculated using standard mathematical equation. Mean daily SD is the mean of the SD of blood glucose of each individual day. Mean daily delta blood glucose is the mean of the daily maximum minus daily minimum blood glucose.[6] Mean amplitude of glycemic excursion (MAGE) is the mean of the absolute values of the increases or decreases of blood glucose from the nadir or the peak (the increase or decrease should be more than one SD of that day to qualify). Service et al.[7] have described in detail the procedure for calculating MAGE from CBG readings. BODY.M.MEASUREMENT OF THE GLYCEMIC PARAMETERS.COMPARISON OF PARAMETERS OF SAFETY: Percentage of readings showing hypoglycemia and severe hyperglycemia were calculated for assessment of the safety of the two protocols. Hypoglycemia was defined as blood glucose value <70 mg/dl, and severe hyperglycemia was defined as blood glucose value of >300 mg/dl. BODY.M.LABORATORY METHODS: Plasma glucose concentration was measured with glucose oxidase method on Beckman Coulter Unicel DXC-800 analyzer. HbA1c was measured using ion-exchange high-performance liquid chromatography (Bio-Rad D-10 analyzer). The reference range of the test was from 3.8% to 18.5%. Contour® Next glucometer was used for CBG measurements (manufactured by Bayer HealthCare LLC). Fingertips were used for CBG sample using standard lancing device provided by the manufacturer. The test strips used flavin adenine dinucleotide-glucose dehydrogenase method for blood glucose testing.[8] The results of the glucometer are referenced to plasma/serum blood glucose values. The measuring range of the glucometer is 10–600 mg/dl. BODY.M.STATISTICAL ANALYSIS: All parametric variables were expressed as mean ± SD, and nonparametric variables were expressed as median ± interquartile range. Unpaired t-test was used to compare the means of parametric data and Mann–Whitney U-test was used to compare nonparametric data. Categorical variables were compared using Chi-square test. P <0.05 was considered statistically significant. IBM SPSS Statistics version 20.0 software (IBM Co., Armonk, NY, USA) was used for carrying out statistical analysis. BODY.R.PARTICIPANT FLOW AND PATIENT NUMBER: A total of 3110 adult, nonpregnant patients admitted to the nonintensive care unit of our hospital from August 2014 to April 2016 were screened for eligibility. Of these, 234 patients received glucocorticoids for any indication with dose equivalent to prednisolone 10 mg or above. Plasma glucose was checked 2 h after lunch and 2 h after dinner within 24–48 h of the patient receiving the glucocorticoid. A total of 103 patients developed 2 h postlunch and/or 2 h postdinner plasma glucose ≥200 mg/dl. Five of these patients either declined or were unable to give consent. A total of six patients were excluded because they met one or more of the exclusion criteria. Two of these refused to take insulin and four of the patients were given enteral or total parenteral nutrition. The remaining 92 patients were randomized to either experimental or control group. All patients received the allocated intervention. On follow-up during the hospital stay, 10 patients in the experimental group and 11 patients in the control group were excluded. The reasons for exclusions are detailed in Figure 1. Figure 1Participant flow diagram (based on consort 2010 flow diagram) As per our study procedure, CBG readings from day 1 of randomization were excluded in the final analysis. For those patients who developed hypoglycemia which was corrected or severe hyperglycemia which was corrected, the CBG data for the rest of the day were excluded from the analysis. If after exclusion of these CBG readings, the remaining number of CBG was ≤8, the patient was excluded from analysis. There were three such patients in the experimental group and one such patient in the control group who was excluded from the final analysis. After all the exclusions, data of 34 patients in the control group and 33 patients in the experimental group were analyzed. The mean age of the patients in the study was 54 years. 71% of the study patients recruited were males. The mean weight and body mass index of the patients were 71 kg and 25.6 kg/m2, respectively. The mean HbA1c was 6.8% and mean creatinine was 1.2 mg/dl. There was no statistically significant difference in the baseline demographic data of the two groups [Table 8]. Table 8 Baseline demographic and clinical characteristics of the two groups Patients with established diabetes mellitus who received glucocorticoids as well as nondiabetics who developed hyperglycemia after administration of glucocorticoids were included in the study. Forty-two percent of patients in the experimental group and 64% patients in the control group had preexisting diabetes mellitus. Although the proportion of patients with preexisting diabetes in the experimental and control group was different, the difference was not statistically significant (P = 0.08). Oral prednisolone was the most frequently used glucocorticoid in both the groups. Besides this, oral or parenteral hydrocortisone, methylprednisolone, and dexamethasone were also used. The difference in the proportions of various glucocorticoids used in the two groups was different. However, the proportion of patients receiving prednisolone versus glucocorticoid other than prednisolone was not statistically significant between the two groups (P = 0.22). There was no significant difference among the doses of the prednisolone between the two groups. The dose of all glucocorticoids was converted to the equivalent dose of prednisolone and compared between the two groups. The dose all glucocorticoid when converted to the equivalent dose of prednisolone showed no significant difference between the two groups [Table 9]. Oral prednisolone, when it was used, was most frequently prescribed in the morning time (between 8 and 9 a.m.) in both groups. Table 9 Mean dose of glucocorticoid used in the two groups BODY.R.COMPARISON OF PARAMETERS OF GLYCEMIC EFFICACY BETWEEN EXPERIMENTAL AND CONTROL GROUPS: The glycemic parameters were compared between experimental and control groups. The mean blood glucose and all the other blood glucose readings including fasting, prelunch, predinner, bedtime, and premeal overall were all significantly lower in experimental group [Table 10 and Figure 2]. The mean blood glucose in the experimental group was 170.32 mg/dl while that in the control group was 214.99 mg/dl. The experimental group had better glycemic control as compared to the control group. Figure 2Difference in mean blood glucose readings at various time points in the experimental and the control groups (y axis shows blood glucose in mg/dl) Table 10 Comparison of glycemic parameters in experimental group versus control group The parameters which assessed the blood glucose in target ranges were higher in the experimental group. However, except for the percentage of premeal blood glucose in target range, the differences were not statistically significant. The parameters for glycemic variability (SD of blood glucose, SD of premeal blood glucose, mean delta blood glucose, mean daily SD, and MAGE) were all significantly lower in patients in the experimental group [Table 11]. The experimental group had lower glycemic variability as compared to the control group. Table 11 Comparison of glycemic variability parameters in experimental group versus control group The hypoglycemia event rates (hypoglycemia defined by blood glucose <70 mg/dl) were low in both the groups [Table 12]. None of the patients in our study in either of the groups developed severe hypoglycemia (defined as blood glucose <50 mg/dl and/or associated with come or seizure). No mortality was reported in either of the two groups during the study period. Table 12 Comparison of parameters of safety between experimental and control groups The event rate for severe hyperglycemia (defined as blood glucose >300 mg/dl) was significantly lower in the experimental group [Table 12]. No patients in either of the two groups developed diabetic ketoacidosis during the study. BODY.D: There are a number of mechanisms by which glucocorticoids cause an increase of blood glucose. Glucocorticoids enhance the hepatic gluconeogenesis, and they cause an increase in the peripheral insulin resistance. They also cause degradation of proteins and lipids providing the substrate for gluconeogenesis.[910] The inhibition of peripheral glucose uptake by skeletal muscles is seen early after administration of glucocorticoids. This explains the reason that glucocorticoids cause initial postprandial hyperglycemia.[11] Existing literature on the topic of management of GCIH focuses on two major ideas. Studies on the glycemic effects of glucocorticoids show that in the initial period, glucocorticoids have a predominant effect on postprandial blood glucose.[12] The first school of thought, to tackle GCIH, is increasing the premeal bolus insulin to counteract the postprandial surge of blood glucose. This is also the basis of the current guidelines proposed by the Endocrine Society.[5] The use of this basal-bolus insulin approach for GCIH is well established, and there is evidence that it is superior to sliding-scale insulin which was used in the past.[13] An alternative approach was first proposed by Clore and Thurby-Hay.[14] In a review article by Perez et al.,[15] a modified version of the above approach has been proposed for the management of GCIH. The basis of the second school of thought is that if we can give a specific insulin simultaneously with the glucocorticoid administration, we would be able to negate the hyperglycemic effect of the glucocorticoid.[15] The authors have proposed that the type of insulin we would use for neutralizing the glucocorticoid should have the same peaks and duration of action as the hyperglycemic effect of the glucocorticoid.[14] A similar approach is followed by the Australian Diabetes Society guidelines for the management of hyperglycemia in indoor patients.[16] However, the evidence for the second approach is limited. Clore and Thurby-Hay[14] first recommended an alternative approach to the management of GCIH. They acknowledged the fact the glucocorticoids primarily increased postprandial blood glucose. However, they differed in the explanation to this phenomenon. It is often believed that the postprandial hyperglycemia seen in GCIH is due to the effect of morning glucocorticoids on the peripheral insulin sensitivity. However, Clore and Thurby-Hay[14] proposed that the reason for the phenomenon was probably the effect of morning glucocorticoid in causing insulin resistance throughout the day, rather than specific a postprandial defect. They gave an analogy of managing diabetes in pregnancy to explain their theory. Pregnant diabetics initially develop postprandial surge just as it is seen with glucocorticoids. However, as the pregnancy advances and the insulin resistance increases, the requirement of basal insulin progressively increases; however, the bolus ratio for meals (insulin: carb ratio) does not change.[17] The study done by Burt et al.[12] later in 2011 provides evidence for their theory. The nondiabetic cohort in the study by Burt et al., who received glucocorticoids, developed a hyperglycemic peak after 6–8 h despite not having a significant postprandial glycemic surge. Their major recommendation which helped us shape our protocol was the use of insulin neutral protamine Hagedorn (NPH) along with prednisolone. They also recommended that the dose of the insulin NPH should be based on the dose of the prednisolone administered and weight of the patient. A number of guidelines recommend the use of NPH along with morning prednisolone. The guidelines by Australian Diabetes Society[16] and a recent guideline by the Joint British Diabetes societies[18] endorse similar protocols. Expert reviews on the topic also support the theory.[14151920] The Endocrine Society, however, does not endorse this protocol because of lack of evidence.[5] As far as the glycemic profile of methylprednisolone is concerned, there is little reason to believe that it is any different from that of prednisolone. Seggelke et al.[4] demonstrated the glycemic profile of morning dose of methylprednisolone in patients with cystic fibrosis. The pattern of glycemia was similar to what we have seen in the studies done for prednisolone. One of the unique aspects of our protocol is the use of insulin glargine along with insulin dexamethasone and use of regular human insulin along with hydrocortisone. Our recommendation comes from the studies which have shown the glycemic profile of dexamethasone and hydrocortisone as well as its pharmacokinetics. It is well known that dexamethasone has a longer half-life than other glucocorticoids.[21] Sethi et al.[22] demonstrated that the glycemic effect of dexamethasone starts in about 3 h. Eberhart et al.[23] and Lukins and Manninen[24] showed that glycemic peak due to dexamethasone is at around 9–10 h after administration. The glycemic effect of dexamethasone may last as long as 48 h.[20] Insulin glargine has a longer half-life and a longer duration of effect, and hence, it is a more suitable insulin to administer with the dexamethasone. This was the basis of our recommendation. Hydrocortisone has a much shorter half-life as compared to other glucocorticoids. Vila et al.[25] showed that hydrocortisone produced a glycemic peak 2 h after administration in healthy volunteers. Since regular human insulin has peak after 2 h, it is perfect insulin to be administered along with hydrocortisone. There are a few published studies which are similar to our study, which have studied and compared various protocols for the management of GCIH. Grommesh et al.[26] published a study for the management of GCIH in hospitalized patients. In the study by Grommesh et al., NPH was used irrespective of the type of glucocorticoid used, while in our study we used the "corrective insulin" depending on the type of glucocorticoid which was used. Ruiz de Adana et al.[27] published a clinical trial comparing insulin glargine versus insulin NPH as basal insulin in patients with GCIH. The protocol used in the experimental group differs from our study. In our study, we used insulin NPH along with prednisolone over and above the basal-bolus insulin (which included insulin glargine). However, in the study published by Ruiz de Adana et al.,[27] they have used insulin NPH three times a day before each meal along with the bolus insulin. Based on the results of our study, we suggest several recommendations for clinical practice. We recommend that all patients who receive glucocorticoids must have multiple blood glucose measurements for diagnosis of GCIH. We recommend the use of insulin NPH along with prednisolone and methylprednisolone, regular human insulin along with hydrocortisone, and insulin glargine along with dexamethasone as "correctional insulin." The dose of correctional insulin should be based on the dose of the administered glucocorticoid and weight of the patient. For patients having preexisting diabetes mellitus given glucocorticoids, we recommend using correctional insulin over and above the background basal-bolus insulin regimen. For patients not having preexisting diabetes and developing GCIH, just the correctional insulin dose along with the administered glucocorticoid would suffice. Our study had several limitations. We proposed using specific insulin as "correctional insulin" for the management of hyperglycemia induced by dexamethasone and hydrocortisone, which was a unique part of our study. However, the number of patients enrolled in the study who received dexamethasone and hydrocortisone was too less to draw any conclusion based on a subgroup analysis. We did not collect data for the dose of insulin used in the experimental and control group. These data would have helped us understand whether there was a difference in insulin requirement between the two groups. Our study could be a primer for the future research on the field of GCIH. We propose dedicated scientific trials on the management of hyperglycemia secondary to dexamethasone, hydrocortisone, and other glucocorticoids. We also recommend that more studies should be conducted on the dysglycemic effects of systemic as well as other forms of glucocorticoids in both normal volunteers and hospitalized patients to understand the risk factors for GCIH and the glycemic patterns of the administered glucocorticoid. BODY.C: We developed a new protocol for the management of GCIH among hospitalized patients. The new protocol incorporated the use of "correctional insulin" matching the glycemic profile of the glucocorticoid administered to negate the effect of the glucocorticoid. When compared to the standard basal-bolus insulin protocol, the new protocol showed lower mean blood glucose and lower glycemic variability. The new protocol had lesser episodes of severe hyperglycemia, and the risk of hypoglycemia was negligible. BODY.C.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.C.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Characteristics Associated With Maintenance of Mean A1C <6.5% in People With Dysglycemia in the ORIGIN Trial ABSTRACT.OBJECTIVE: To assess the success and baseline predictors of maintaining glycemic control for up to 5 years of therapy using basal insulin glargine or standard glycemic care in people with dysglycemia treated with zero or one oral glucose-lowering agents. ABSTRACT.RESEARCH DESIGN AND METHODS: Data from 12,537 participants in the Outcome Reduction with Initial Glargine Intervention (ORIGIN) trial were examined by baseline glycemic status (with or without type 2 diabetes) and by therapeutic approach (titrated insulin glargine or standard therapy) using an intention-to-treat analysis. Median values for fasting plasma glucose (FPG) and A1C and percentages with A1C <6.5% (48 mmol/mol) during randomized treatment were calculated. Factors independently associated with maintaining updated mean A1C <6.5% were analyzed with linear regression models. ABSTRACT.RESULTS: Median A1C in the whole population was 6.4% at baseline; at 5 years, it was 6.2% with glargine treatment and 6.5% with standard care. Of those with diabetes at baseline, 60% using glargine and 45% using standard care had A1C <6.5% at 5 years. Lack of diabetes and lower baseline A1C were independently associated with 5-year mean A1C <6.5%. Maintaining mean A1C <6.5% was more likely with glargine (odds ratio [OR] 2.98 [95% CI 2.67–3.32], P < 0.001) than standard care after adjustment for other independent predictors. ABSTRACT.CONCLUSIONS: Systematic intervention with basal insulin glargine or standard care early in the natural history of dysglycemia can maintain glycemic control near baseline levels for at least 5 years, whether diabetes is present at baseline or not. Keeping mean A1C <6.5% is more likely in people with lower baseline A1C and with the glargine-based regimen. BODY: There is a strong epidemiologic relationship between hyperglycemia and long-term complications of type 2 diabetes (1–4), and intensive treatment studies have verified that improving glycemic control can delay or prevent some of these complications (5–8). However, interventional studies have shown that diabetes is a progressive disorder, and the treatments used often do not prevent a gradual increase of hyperglycemia over time (9–11). The Outcome Reduction with Initial Glargine Intervention (ORIGIN; NCT00069784) trial compared the medical outcomes of two different glycemic treatment approaches for people with dysglycemia either with or without a diagnosis of diabetes who were taking no more than one oral glucose-lowering drug (12). The 12,537 participants were randomized to treatment with either basal insulin glargine, which was systematically titrated to maintain fasting plasma glucose (FPG) ≤5.3 mmol/L (≤95 mg/dL), or to standard care based on oral therapy. Cardiovascular and other medical outcomes of ORIGIN and the glycemic control results for the whole population have been reported previously (13,14). Here we report the glycemic control results separately for the subgroups with and without diabetes at enrollment and assess the independent associations of glycemic status and other baseline characteristics, and the treatment regimen used, with maintenance of updated mean A1C below <6.5% for up to 5 years of follow-up. BODY.RESEARCH DESIGN AND METHODS.PARTICIPANTS: The rationale and design of the ORIGIN trial were reported previously (9). In brief, it was a multinational randomized trial with a 2 × 2 factorial design that tested two pairs of interventions. Titrated basal insulin glargine was compared with standard stepwise oral therapy, and an omega-3 fatty acid supplement with placebo. Participants were required to have a prior cardiovascular (CV) event or other evidence of high CV risk together with documented dysglycemia, defined as either impaired fasting glucose, impaired glucose tolerance (or the two together), or newly detected or previously diagnosed type 2 diabetes. Participants with diabetes could be treated with lifestyle alone or accompanied by no more than a single oral glucose-lowering agent. The present analysis concerns the glycemic intervention, with use of omega-3 fatty acids included only as a covariate. Data from all 12,537 individuals in 40 countries were assessed. BODY.RESEARCH DESIGN AND METHODS.INTERVENTIONS: Participants assigned to standard care continued their prior glucose-lowering treatment and were managed according to the investigators' judgment and local guidelines for glycemic control and therapeutic approaches. Investigators were advised not to prescribe insulin for standard participants unless they were on full doses of two or more oral agents, and if insulin was added, glargine was not to be used. Participants assigned to basal insulin glargine who were taking a thiazolidinedione prior to randomization stopped this medication but continued to take other glucose-lowering agents. Insulin glargine (Lantus; Sanofi) was added to their regimen starting at 2–6 units daily, based on fasting glucose levels. Participants were advised to inject the glargine in the evening and to self-titrate the dosage using a simple algorithm supported by the site investigators. Self-measured, plasma-referenced fasting capillary blood glucose tests were done at least twice-weekly to guide titration, with the goal of achieving and maintaining fasting glucose at ≤5.3 mmol/L (≤95 mg/dL). Other oral agents could be continued, reduced, or discontinued as judged appropriate during treatment with insulin glargine. The only oral agent that could be added (if not previously used) was metformin, which the site investigator initiated for individual participants if judged necessary to limit the risk of hypoglycemia. The importance of lifestyle management was periodically reinforced in both treatment groups. BODY.RESEARCH DESIGN AND METHODS.MEASUREMENTS: In addition to self-measured glucose tests, venous blood for measurement of FPG and A1C at local laboratories was collected at intervals during treatment. Measurements of A1C were done at baseline, yearly thereafter, and at the end of treatment for all participants. All participants in the glargine treatment group and the nondiabetic participants in the standard care group had FPG levels measured at baseline, annually, and at the end of treatment. Participants with diabetes in the standard care group had FPG measurements at baseline, after 2 years, and at the end of treatment. Historical information about participants (such as smoking and alcohol habits, prior CV events, depression, and other medical problems) was systematically collected at baseline by participant self-report. BODY.RESEARCH DESIGN AND METHODS.STATISTICAL ANALYSIS: Summary statistics were computed for baseline characteristics of the whole population and for subgroups by glycemic treatment allocation and by glycemic status at enrollment (diabetes absent or present). Median FPG and A1C with interquartile ranges were computed for each subgroup for all time points. Percentages of participants in each subgroup having A1C <6.5 and <7.0% (two levels commonly identified as targets for glycemic control) (15,16) were calculated for each time point from the beginning to the end of the trial. To determine the relationships between baseline characteristics and glycemic outcomes, findings for all randomized participants during the first 5 years of treatment were analyzed. Maintenance of an A1C <6.5% during treatment was defined as having the mean of all available annual measurements, including the 1-year value, up to and including the 5-year value (i.e., the updated mean A1C) below those levels. Univariable analyses of the relationships between clinical characteristics and a maintained A1C <6.5% were performed using linear regression models. Characteristics with a P < 0.1 in univariate analyses were entered into multivariable models. Model 1 included all baseline characteristics meeting this criterion, including baseline glycemic status (diabetes vs. no diabetes), but not glycemic treatment allocation. The independent effect of allocation to basal insulin glargine versus standard care was assessed by adding this variable to model 2, which included all variables statistically significant at P < 0.05 in model 1. The unadjusted effect of allocation to insulin glargine within subgroups was estimated using logistic regression and statistical tests for interactions between allocation and these subgroups. The results of these analyses were displayed as a forest plot. BODY.RESULTS.BASELINE CHARACTERISTICS: The characteristics of the ORIGIN population at enrollment, divided by treatment assignment and glycemic status, are shown in Table 1. Of 12,537 subjects randomized, 6,264 were assigned to treatment with insulin glargine and 6,273 to standard care. For the whole population, the mean age was 63.5 years, median FPG 6.9 mmol/L (125 mg/dL), and median A1C 6.4% (46 mmol/mol). The two randomized treatment groups were alike in baseline characteristics. Eighty-eight percent of participants had either a prior diagnosis of diabetes (of mean duration of 5.4 years) or newly detected diabetes. The 12% without diabetes clearly differed from those with diabetes in FPG and A1C levels and also in other ways, including more frequent prior CV events, use of alcohol, depression, and use of statins and β-blockers. Table 1 Baseline characteristics of participants BODY.RESULTS.GLYCEMIC RESPONSES OF PARTICIPANTS WITH AND WITHOUT DIABETES.MEDIAN FPG.: The median period of follow-up on randomized treatment was 6.2 years. The effect of treatment allocation on the responses of FPG and A1C during treatment is shown in Fig. 1. For participants without diabetes, the median FPG (interquartile range) was 6.1 mmol/L (5.5–6.4) prior to randomized treatment (Fig. 1A). Standard care led to little change of FPG in this subgroup, but insulin glargine caused a sustained decrease to median values of 5.0 (4.5–5.5), 4.9 (4.4–5.5), 5.0 (4.5–5.7), and 5.1 mmol/L (4.5–5.8) at 1, 2, 5, and 7 years. For participants with diabetes, the median baseline FPG was 7.2 mmol/L (6.2–8.4). With standard care, the values at 2 years and the end of treatment were 6.8 (5.9–8.1) and 7.0 mmol/L (5.9–8.4) (Fig. 1B). Treatment with glargine reduced median FPG to 5.2 (4.6–5.9), 5.0 (4.4–5.8), 5.1 (4.5–6.1), and 5.3 mmol/L (4.5–6.4) after 1, 2, 5, and 7 years. Figure 1Median FPG values and median A1C values at baseline, yearly during randomized treatment, and at the end of treatment are shown separately for participants without diabetes (A for FPG, C for A1C) and with diabetes (B for FPG, D for A1C) at baseline. The group assigned to use basal insulin glargine is shown by solid lines and solid circles, and the group assigned to standard care by broken lines and open circles. The numbers at the bottom of each panel show the number of observations included at each point in time. BODY.RESULTS.GLYCEMIC RESPONSES OF PARTICIPANTS WITH AND WITHOUT DIABETES.MEDIAN A1C.: For participants without diabetes, A1C changed little from baseline with either regimen (Fig. 1C and Supplementary Table 1). With standard therapy, the median A1C was 5.7% (5.4–6.1) at baseline, 5.7% (5.4–6.1) at 1 year, and 6.0% (5.6–6.4) after 5 years. For glargine-treated participants, median A1C was 5.7% (5.4–6.0) at baseline, 5.6% (5.3–5.9) at 1 year, and 5.8% (5.5–6.1) at 5 years. For participants with diabetes, the median A1C at baseline was 6.6% (6.0–7.3) (Fig. 1D). During standard care, the median A1C values at 1, 2, 5, and 7 years were 6.3 (5.8–7.0), 6.4 (5.9–7.0), 6.6 (6.1–7.3), and 6.6% (6.1–7.3). Corresponding values during treatment with glargine declined to 6.0 (5.5–6.5), 6.0 (5.6–6.6), 6.3 (5.8–6.9), and 6.3% (5.8–6.9). For comparison with these median values, mean A1C during treatment is also provided in Supplementary Table 1. BODY.RESULTS.GLYCEMIC RESPONSES OF PARTICIPANTS WITH AND WITHOUT DIABETES.PERCENTAGES <7.0 AND 6.5% A1C.: Of participants without diabetes at entry, >90% sustained A1C levels <7.0% (53 mmol/mol), and >75% achieved an A1C <6.5% (48 mmol/mol) throughout randomized treatment with both regimens (Fig. 2A and C). Of participants with diabetes, 66% had an A1C <7.0% and 47% had A1C <6.5% before starting treatment (Fig. 2B and D). During glargine treatment, the percentage in the diabetic subgroup achieving an A1C <7.0% increased to 88% at 1 year and 77% at 5 years, and the percentage achieving an A1C <6.5% was 74% at 1 year and 60% after 5 years. With standard care, the percentages in the diabetic subgroup were 76% with A1C <7.0% at 1 year and 66% at 5 years, and 58% with A1C <6.5% at 1 year and 45% at 5 years. Figure 2Percentages of participants with A1C values <7.0 and <6.5% at baseline and yearly during randomized treatment are shown separately for those without diabetes (A for <7.0%, C for <6.5%) and with diabetes (B for <7.0%, D for <6.5%) at baseline. The group assigned to use basal insulin glargine is shown by solid lines and solid circles, and the group assigned to standard care by broken lines and open circles. BODY.RESULTS.PREDICTORS OF SUCCESS IN MAINTAINING MEAN A1C <6.5%: Associations of various clinical factors with maintaining a mean level of <6.5% for up to 5 years are shown in Table 2. Univariate associations with this outcome are shown for all baseline characteristics. Multivariable model 1, which included baseline characteristics with a univariate P < 0.1 for this association, displays several factors with independent associations with maintaining A1C <6.5%. These were greater age, reported use of alcohol, diagnosis of depression, lower A1C, lower waist-to-hip ratio, greater grip strength, lower albumin-to-creatinine ratio (ACR), lack of diabetes at baseline, and perhaps lack of statin use (P = 0.053). Model 2 included the baseline characteristics independently associated with mean A1C <6.5% in model 1 with a P value <0.05 as well as the effect of allocation to insulin glargine versus standard care. In model 2, greater age, use of alcohol, depression, lower A1C, lower waist-to-hip ratio, greater grip strength, lower ACR, and lack of diabetes were significant independent predictors. The adjusted odds ratio (OR) for success in maintaining A1C <6.5% when using glargine compared with standard care was 2.98 (P < 0.001). In a further multivariable analysis not reported in detail here, independent predictors of maintaining mean updated A1C <7.0% during 5 years of treatment were similar. Greater age, use of alcohol, lower A1C at baseline, and lack of diabetes at baseline were independent predictors, and the OR for glargine versus standard care was 2.41 (P < 0.001). Table 2 OR (95% CI) of maintaining a cumulative 5-year mean A1C <6.5% In an unadjusted subgroup analysis, the glargine-based regimen was more effective than standard care in all subgroups, including those with and without diabetes at baseline, with ORs close to 2 and no overlap of 95% CIs with unity (Supplementary Fig. 1). Three subgroup comparisons showed a nominally significant interaction with treatment assignment; glargine may have been modestly more effective in participants with higher waist-to-hip ratios (P = 0.011), greater grip strength (P < 0.001), and moderate use of alcohol (P = 0.029). BODY.RESULTS.GLUCOSE-LOWERING THERAPIES AND CONFIRMED HYPOGLYCEMIA: The usage of oral glucose-lowering agents prior to randomization is listed in Supplementary Table 2. Less than 2% of participants with dysglycemia not meeting the criteria of diabetes had used such agents prior to entry, and none at the time of oral glucose tolerance testing during screening. Of the participants with diabetes at enrollment, 33% were taking no oral therapy, 31% were taking metformin, and 33% were taking a sulfonylurea. Supplementary Table 3 displays usage of oral agents and insulin at the end of treatment. Of the participants without diabetes at entry, 69% of those assigned to glargine and 0.3% of those assigned to standard care were taking insulin at the end of the study. At the end of follow-up, 21% of those randomized to glargine and 29% of those randomized to standard care were taking one or more oral agents, most often metformin (18 and 24%; P < 0.003). Of the participants with diabetes at entry, insulin was used at the end by 82% of those who were assigned to glargine treatment and by 12% of those assigned to standard care (P < 0.001). Oral therapies were used by 71% of participants with diabetes assigned to glargine and 88% of those assigned to standard care (P < 0.001). Metformin was taken by 51 and 65% of subjects in the glargine and standard groups, respectively, and sulfonylureas were used by 28 and 52% (each <0.001). Two or more oral agents were taken by 14% of the glargine-treated group and 42% of the standard care group. The percentages of people with diabetes at enrollment having one or more hypoglycemic episodes confirmed by a glucose test of <3 mmol/L (<54 mg/dL) was 10.5 per 100 person-years with glargine and 3.0 per 100 person-years with standard treatment. The corresponding frequencies for those without diabetes at enrollment were 5.7 and 0.3 per 100 person-years. BODY.CONCLUSIONS: As previously reported, the methods of therapy used in ORIGIN maintained excellent control of both FPG and A1C for at least 5 years of follow-up in the whole study population with dysglycemia. The present analysis expands this demonstration by examining the subpopulations with and without diabetes at baseline separately. In the subgroup without diabetes at baseline, there was a small increase of median A1C from 5.7% (39 mmol/mol) initially to 6.0% (42 mmol/mol) with standard care and to 5.8% (40 mmol/mol) with glargine after 5 years of follow-up. In those with diabetes at entry, median A1C decreased slightly from the 6.5% (48 mmol/mol) baseline after initiation of either glargine or standard care, and then it slowly increased with both treatments. After 5 years, the median A1C was 6.6% (49 mmol/mol) with standard care and 6.3% (45 mmol/mol) with glargine. Relative stability of A1C levels over time was not unexpected for the subpopulation without diabetes, but the finding that both treatment regimens had sustained success in people with overt diabetes at entry is reassuring. In contrast, glycemic control steadily worsened over the course of 5–10 years in some other long-term studies of type 2 diabetes (9–11). Sustained glycemic control in ORIGIN presumably was related to the use in each treatment arm of "treat-to-target" schemes, which called for intensification of treatment in response to evidence of rising levels of glucose. The dosage of glargine was systematically adjusted, seeking FPG levels ≤5.3 mmol/L, and metformin could be added to mitigate the risk of hypoglycemia. Similarly, during standard therapy, oral medications were added and their dosage increased, with the aim of keeping A1C below either 6.5 or 7.0%, depending on locally accepted guidelines. At the end of treatment, 42% of those using the standard regimen were taking two or more oral agents, and 14% of participants assigned to glargine therapy were doing so. Treatment in the Belfast (9), UK Prospective Diabetes Study (UKPDS) (10), and A Diabetes Outcome Progression Trial (ADOPT) (11) studies, in contrast, was based on assignment to monotherapy regimens, including diet alone, metformin, sulfonylurea, thiazolidinedione, or basal insulin, with escalation of therapy only under certain conditions. The glycemic control attained in ORIGIN resembled that achieved in Action in Vascular Disease: Preterax and Diamocron Modified Release Controlled Evaluation (ADVANCE) (17), Action to Control Cardiovascular Risk in Diabetes (ACCORD) (18), and Veterans Affairs Diabetes Trial (VADT) (19), other trials in which glucose-lowering therapies in the intensive treatment groups were systematically adjusted seeking near-normal glycemic control. However, compared with the population in ORIGIN, participants in these studies had a longer duration of diabetes and in many cases established therapy with multiple glucose-lowering agents prior to enrollment. The A1C levels attained in ADVANCE (6.5% [48 mmol/mol]) and ACCORD (6.4% [46 mmol/mol]) were close to those at baseline in ORIGIN (6.5%), whereas those in VADT were slightly higher (6.9% [52 mmol/mol]). In all three studies, these values were achieved by strenuous efforts to improve control from higher A1C levels at baseline. Hence, maintenance of A1C at or below near-normal entry levels in ORIGIN contrasts with the other trials' efforts to restore previously inadequate glycemic control. Keeping glycemic control below a level associated with increasing risk of diabetes complications by advancing therapy as needed may be a more desirable approach than the historically common practice of allowing marked hyperglycemia to occur and then attempting to reduce levels to a lower target (20–22). This concept is in keeping with the recent adoption of A1C 6.5% as one option for timely diagnosis of diabetes to allow intervention to minimize the risk of complications (23,24). This analysis also identified baseline characteristics of ORIGIN participants that were predictive of maintaining an updated mean A1C from the first year of treatment to the end of treatment or 5 years of follow-up at <6.5%. This measure of treatment success was selected because it was close to the median value at entry to the trial and also reflects a level of control now endorsed as a threshold for diagnosing diabetes. Data after 5 years of treatment were not included because of the smaller numbers of participants who had been in the study long enough to complete 6 or 7 years. Individuals with diabetes at baseline were understandably less likely than those without diabetes to have this level of success in controlling A1C, having an adjusted OR of 0.309 (P < 0.0001). An association of long-term treatment success with lower baseline A1C, independent of the presence of overt diabetes, was also demonstrated, consistent with other studies. Other predictive factors included greater age, use of alcohol more than two times weekly, depression, lower A1C, and lower waist-to-hip ratio (all P < 0.001). Less clearly associated factors were greater grip strength (P = 0.038) and lower urinary albumin excretion (P = 0.035). Speculative explanations may be offered for these associations. Better success for individuals who were older at enrollment might reflect slower progression of the underlying defects of diabetes in those with later onset. Lower waist-to-hip ratio, greater grip strength (25), and lower albumin excretion might be markers for better physical fitness and a lower burden of microvascular complications of diabetes. Moderate use of alcohol may be associated with favorable physiological patterns and reduced risk of type 2 diabetes and mortality (26), and participants who reported depression might have been more willing to make changes to reduce depression or otherwise improve health-related behavior. Several of these associations support the assumption that various behavioral characteristics of participants influenced the success of these treatment regimens in maintaining glycemic control. Finally, allocation to basal insulin glargine with titration of dosage seeking normal FPG levels led to a nearly threefold increase in the likelihood of maintaining mean A1C <6.5% for 5 years. Moreover, this effect was observed in subgroups representing characteristics that were associated with success in maintaining A1C <6.5% for 5 years. This observation is not surprising given that an ambitious glycemic target for glargine titration, fasting glucose <5.3 mmol/L, was systematically sought. Experience in ORIGIN showed that early use of this basal insulin was able to both attain and maintain this level of control of FPG, and thereby keep A1C from rising above baseline levels (median 6.5% [48 mmol/mol]) for a majority of people with overt diabetes at baseline for up to 5 years (5-year median 6.3% [45 mmol/mol], 60% of participants <6.5%). However, the short- and long-term risks versus benefits of this method of treatment relative to standard care in ORIGIN are not yet well-defined. As previously reported (13,27), the use of systematically titrated glargine in ORIGIN was associated with 1.6-kg gain of weight and 0.7% increased incidence of severe hypoglycemia. These unwanted effects of seeking nearly normal glycemic control were less prominent in ORIGIN than in the trials in which participants had a longer duration of diabetes and more elevated A1C levels at baseline (18,19). For example, the mean gain of weight with the intensive treatment regimen in the VADT was 8.2 kg (19). Also, the annual incidence of severe hypoglycemia with intensive treatment in ACCORD was 3.1% (27), whereas it was 1.0% with basal insulin and 0.3% with standard therapy in ORIGIN (13). Although maintenance of nearly normal glycemic control for 5 years may be predicted to delay the development of complications of diabetes, the present analysis lacks information about the predictors and consequences of hypoglycemia and other unwanted effects of insulin glargine relative to standard care. Therefore, further examination of data from ORIGIN is needed to clarify the balance of risks to potential benefits from early intervention with insulin glargine. In conclusion, early intervention with basal insulin glargine or with standard care kept median A1C near the starting level for at least 5 years in both the subgroup without diabetes and the subgroup with diabetes at entry. Maintaining the mean of yearly A1C measurements at <6.5% (48 mmol/mol) was more often accomplished when the initial A1C was lower and with titrated basal insulin glargine than with standard care. However, the risks versus benefits of this approach remain to be determined by further analyses and additional follow-up of ORIGIN participants.

TITLE: Sugarsquare, a Web-Based Patient Portal for Parents of a Child With Type 1 Diabetes: Multicenter Randomized Controlled Feasibility Trial ABSTRACT.BACKGROUND: Raising a child with type 1 diabetes (T1D) means combining the demands of the disease management with everyday parenting, which is associated with increased levels of distress. A Web-based patient portal, Sugarsquare, was developed to support parents, by providing online parent-professional communication, online peer support and online disease information. ABSTRACT.OBJECTIVE: The first aim of this study was to assess the feasibility of conducting a multicenter, randomized controlled trial in Dutch parents of a child with T1D. The second aim was to assess the feasibility of implementing Sugarsquare in clinical practice. ABSTRACT.METHODS: The parents of 105 children (N=105) with T1D below the age of 13 participated in a 6-month multicenter randomized controlled feasibility trial. They were randomly assigned to an experimental (n=54, usual care and Sugarsquare) or a control group (n=51, usual care). Attrition rates and user statistics were gathered to evaluate feasibility of the trial and implementation. To determine potential efficacy, the parenting stress index (PSI-SF) was assessed at baseline (T0) and after 6 months (T1). ABSTRACT.RESULTS: Of a potential population of parents of 445 children, 189 were willing to participate (enrollment refusal=57.5%, n=256), 142 filled in the baseline questionnaire (baseline attrition rate=25%, n=47), and 105 also filled in the questionnaire at T1 (post randomization attrition rate during follow-up=26%, n=32). As such, 24% of the potential population participated. Analysis in the experimental group (n=54) revealed a total of 32 (59%) unique users, divided into 12 (38%) frequent users, 9 (28%) incidental users, and 11 (34%) low-frequent users. Of the total of 44 professionals, 34 (77%) logged in, and 32 (73%) logged in repeatedly. Analysis of the user statistics in the experimental group further showed high practicability and integration in all users, moderate acceptability and demand in parents, and high acceptability and demand in health care professionals. Baseline parenting stress index scores were related to the parents' frequency of logging on (ρ=.282, P=.03) and page-views (ρ=.304, P=.01). No significant differences in change in parenting stress between experimental and control group were found (F3,101=.49, P=.49). ABSTRACT.CONCLUSIONS: The trial can be considered feasible, considering the average enrollment refusal rate, baseline attrition rate and postrandomization attrition rate, compared to other eHealth studies, although lower than hypothesized. Implementing Sugarsquare in clinical practice was partly feasible, given moderate demand and acceptability in parent users and lack of potential efficacy. Parents who reported higher levels of parenting stress used Sugarsquare more often than other parents, although Sugarsquare did not reduce parenting stress. These results indicate that Web-based interventions are a suitable way of providing parents of children with T1D with additional support. Future studies should determine how Sugarsquare could reduce parenting stress, for instance by adding targeted interventions. Factors potentially contributing to successful implementation are suggested. ABSTRACT.TRIAL REGISTRATION: Nederlands Trial Register Number: NTR3643; http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=3643 (Archived by WebCite at http://www.webcitation.org/6qihOVCi6) BODY.INTRODUCTION.BACKGROUND: Type 1 diabetes (T1D) is a chronic metabolic disorder with a complex daily treatment regime, requiring patients to carry out a variety of health-related self-care behaviors, such as monitoring blood glucose levels, administering insulin, adhering to a diet, and exercising. In case of young children, parents are responsible for ensuring that these disease management tasks are performed. Having to combine these complex self-management tasks with regular parenting tasks in everyday life can have a profound impact on parents [1-10], indicated by elevated levels of stress and depressive symptoms in parents of a child with T1D [3,7,9,11], especially in those with young children and with children with a more recent diagnosis [2-7,12,13]. Family and parental functioning are related to well-being, self-care skills, and glycemic control in children, which makes it important that diabetes teams are aware of the impact of the disease and its treatment on parents [1,6,14-19]. Studies show that parents need easy access to their diabetes care team [8,20,21], local peer support [22-26], and tailored information about the disease and its management provided by their own diabetes team [8,27-30]. This positively affects their quality of life [8,23,26] and helps them adequately cope with the disease. New technologies such as the Internet can help diabetes teams in delivering these aspects [8,25,26,29,31-40]. Despite the great potential of the Internet and parents' positive attitude toward using Internet in care, there has been little research into the efficacy and feasibility of Internet interventions for the parents of chronically ill children, especially interventions that combine multiple aspects of care [38,39,41]. This is unfortunate, considering that chronically ill patients and their parents can benefit from using the Internet, because it facilitates the exchange of knowledge and information between patients and health care professionals. There are several challenges, when it comes to implementing and testing an Internet intervention in a clinical research context. eHealth studies are specifically subject to low retention rates (evaluation dropout), which are often the result of study-specific factors and low adherence rates (nonintervention usage) that are mostly intervention specific. These rates can lead to a loss of participants and thus to lack of statistical power [34,42-46]. Achieving successful recruitment is particularly problematic when multiple practices are involved, as practices often differ at an organizational level and local recruiters often have limited resources for recruitment [47,48]. BODY.INTRODUCTION.RANDOMIZED CONTROLLED TRIAL: To gain knowledge about the feasibility of conducting a randomized controlled trial (RCT) and implementing an Internet intervention in usual care for parents of a child with T1D, we developed a Web-based patient portal, called Sugarsquare [40]. Sugarsquare was specifically developed according to parents' needs and preferences [8,31] and is hypothesized to enable diabetes teams to improve their accessibility, facilitate local peer support, and provide tailored information [31]. An explorative, multicenter study was conducted to answer the following research questions: Is conducting an RCT concerning Sugarsquare feasible in a population of parents of a child with T1D in terms of:potential participants: what is the number of eligible parents?enrollment refusal rate: what is the proportion of parents who refuse participation?baseline attrition rate: what is the proportion of parents who drop out before baseline?follow-up attrition rate: what is the proportion of parents who drop out during the trial?Is implementation of Sugarsquare in daily clinical practice feasible in a population of parents of a child with T1D in terms of:practicability: are recipients able to use Sugarsquare?acceptability: do recipients use Sugarsquare?demand: do recipients continue to use Sugarsquare?integration: is Sugarsquare consistent with international guidelines for pediatric diabetes care?potential efficacy: is usage associated with change in parenting stress? BODY.METHODS.DESIGN AND SETTING OF THE STUDY: The participants for this study were recruited from 7 medical centers in the Netherlands, with a potential of 445 parents, from May 2012 to January 2013. Eligible participants were the parents of a child with T1D (one parent per child) younger than 13 years of age, had access to the Internet at home, and were able to comprehend the Dutch language. The children had to be treated in one of the participating centers during the entire course of the study. Participants were randomly assigned to one of two conditions: (1) an intervention condition and (2) a usual care control condition. Participants in the intervention condition had access to the intervention for 6 months in addition to care as usual. Participants in the control group received care as usual during that period. An extensive report of the offline recruitment of participants, the randomization and the procedure of the data collection is described in the Sugarsquare study protocol [31]. The study described in this study was part of a larger project [31], of which all procedures were approved by the Ethics Committees of Human Experimentation of the Radboud University Medical Center and the participating hospitals and are in accordance with the Declaration of Helsinki. BODY.METHODS.INTERVENTION: The final version of Sugarsquare consists of a Web-based patient portal providing online parent-professional communication, peer support, and disease information. Sugarsquare was developed at parents' explicit request and is based on a previous comparable intervention for adolescents with T1D [8,31,40]. Seven focus group interviews with parents [8,31] and a questionnaire for health care professionals affiliated to the cooperating centers were used to tailor the intervention to the preferences of both parents and health care professionals. In a series of pilots, involving parents and professionals participated, the intervention was further fine-tuned and facilitators and barriers were identified. The test phase ended when bugs were repaired and both parents and professionals felt the intervention was ready for use. In accordance with parents' preferences, the intervention was organized locally, so that each center for diabetes care has its own secured portal, which is only accessible to health care professionals of that particular center and to the parents of the children treated at that clinic. Sugarsquare is accessible through the Internet and has the following two main sections. BODY.METHODS.INTERVENTION.SECTION I: GENERAL: The first section provides online peer support and disease information and is accessible to all users (parents and health care professionals). Peer support is facilitated through a chat application, a forum application, and a blog application. Disease information is provided by means of downloadable documents and Web links. BODY.METHODS.INTERVENTION.SECTION II: PERSONAL: The second section is specific to individual patients and can only be accessed by the parents of that particular patient and their diabetes team. The section contains an overview of treatment goals and an application for easily accessible private contact between parents and health care professionals. This application is only used for nonurgent matters. The intervention has been described in the study protocol [31]. In the final version of Sugarsquare, disease information is incorporated in Section I, instead of Section II as described in the study protocol. Sugarsquare is secured by means of a 2-factor authentication, requiring a username-password combination and a personalized SMS code in the login procedure. Health care professionals of the local diabetes teams were appointed as coordinators for the local recruitment of participants and the local implementation of Sugarsquare. Screenshots of Sugarsquare for parents are displayed in Multimedia Appendix 1 [49]. BODY.METHODS.CARE AS USUAL: All children received care as usual, according to International Guidelines for Pediatric Diabetes Care [18,50], provided by a multidisciplinary team of pediatric diabetologists, diabetes nurse practitioners, dietitians, and psychologists. Parents and children were invited to visit the outpatient center for consultations with the pediatric diabetologist and nurse practitioner 4 times a year. Dieticians and psychologists were available on request by parents, children, or physicians. The diabetes care team could be contacted during business hours by telephone and email. An emergency telephone number could be accessed outside office hours to guarantee continuous access to care. Children of participants in both conditions (experimental and control) received care as usual during the entire study period. As such, Sugarsquare was used in addition to care as usual. During the study period, the parents in the experimental group could contact the diabetes care team via the portal instead of by telephone or email in case of nonurgent matters. The telephone number for emergencies was maintained. BODY.METHODS.MEASURES: Feasibility of the RCT was assessed in terms of the number of potential participants, the proportion of parents who refused participation, and the attrition rates. Demographic data of all the participants who were included in the final analyses were gathered at baseline. For assessment of feasibility of the intervention, expressed in terms of practicability, acceptability, and demand [40,51], individual user data of all participants in the experimental group, such as frequency of logins and number of messages posted on the forum, were logged digitally. For feasibility in terms of integration, we assessed whether Sugarsquare was of added value for working according to International ISPAD (International Society for Pediatric and Adolescent Diabetes) and or IDF (International Diabetes Federation) and ADA (American Diabetes Association) Guidelines for Diabetes Care [18,50], by checking 9 key-elements for diabetes care, derived from these guidelines. For feasibility in terms of potential efficacy, parenting stress was assessed by means of the Dutch version of the parenting stress index-short form (PSI-SF) [52] on T0, T1, and T2. The reliability and criterion validity of the Dutch PSI-SF are shown to be good [52]. The PSI-SF consists of 25 items answered on a 6-point Likert scale, ranging from "totally agree" to "totally disagree." An example of an item on the PSI-SF is "it is not always easy to accept my child the way he or she is." The sum score on the PSI-SF can be categorized into normal, subclinical, and clinical based on standardized cutoff scores described in the manual [52]. Parenting stress was assessed at the start of the study (T0=baseline), at 6 months after the start of the study (T1), and at 12 months after the start of the study (T2=follow-up). Also, at the end of the study we asked the local Sugarsquare coordinators, who were health care professionals and part of the local diabetes teams, to evaluate the study and identify facilitators and limitations for the implementation. Information about the child's glycemic control (HbA1c) and the number of hospital admissions (lasting over 24 h) for keto-acidosis or severe hypoglycemia were used to explore the potential efficacy of the portal. These data were taken from the child's medical files. Questionnaires for demographics and parenting stress were administered by means of a Web-based, secured survey program, called Radquest, which generates a closed survey system. The registered participants received an email with a Web link to the survey, which was paired with a unique user id. All items had to be answered and participants were able to change the answers until the participant submitted the completed survey. The data generated from the survey were stored on a secured server. Some participants preferred filling in a hardcopy questionnaire, which was sent to them by post. For an elaborate overview of all measures, see Table 1. BODY.METHODS.ANALYSES: Demographic data were analyzed descriptively, and differences at baseline between the experimental group and the control group were assessed using an analysis of variance (ANOVA). For feasibility, user data were analyzed by means of descriptive statistics. To compare differences in change in parenting stress between the experimental group and the control group, an analysis of covariance (ANCOVA) was performed on T1 data, using T0 data as covariate and the condition (experimental vs control) as fixed factor. A sensitivity analysis was conducted by means of a multiple imputation analysis (based on HbA1c scores at T1) to account for missing data. To test robustness of the results, a conservative analysis based on a Last Observation Carried Forward (LOCF) imputation was performed. Associations between user data and parenting stress at baseline were explored using Spearman ρ for nonparametric correlation due to high skewness of user data and a univariate ANOVA. Data on T2 were regarded as follow-up and were not analyzed in this study. BODY.METHODS.POWER CALCULATION: We calculated that the data of 180 parents would be needed for the final analysis in order to reach a medium effect size (d=0.50), with a Cronbach alpha of .05 (two-tailed test) and a beta of .10 [31]. On the basis of recent literature, a declination rate of 25% (n=80) and a dropout rate of 25% (n=60) was hypothesized [31,34]. As such, we would need to approach 320 parents in order to reach a minimum of 240 parents at the start of the study to have data for 180 participants in the final analysis [31]. Table 1 Variables used in the Sugarsquare study. Outcome Measures Demographics Age and gender of the child Age of onset and duration of diabetes Pen or pump treatment Age, gender, and educational level of the primary parent Social economic status of the parents Feasibility of the trial Potential population Total population of parents, N (%) Enrollment refusal Participants who consented (total population of parents), n (%) Baseline attrition Participants who completed T0 (participants enrolled), n (%) Postrandomization attrition (during follow-up) Participants who completed T1 (randomized participants), n (%) Feasibility of intervention Practicability (can they use it?) Inventory of difficulties logging in and downtime (inaccessibility) Acceptability (do they use it?) Percentage of users who logged in at least once and used all applications Demand or adherence (do they continue to use it?) Percentage of users who logged in repeatedly Integration (does it fit with the treatment?) Evaluation of international guidelines for diabetes care (ISPAD or IDF and ADA) when using Sugarsquare Potential efficacy (is usage associated with change in parenting stress?) Parenting stress index-short form (PSI-SF [ 44 ]) Exploration of change in medical parameters Medical parameters HbA1c Hospitals admissions due to glycemic disruptions BODY.RESULTS.FEASIBILITY OF THE RANDOMIZED CONTROLLED TRIAL: ENROLLMENT AND DROPOUT: All the parents of children with T1D, who were treated in 1 of the 7 cooperating centers for pediatric diabetes care, were invited by mail to participate in the study. The total population consisted of the parents of 445 children. A total of 189 parents of 189 children were willing to participate. The remaining 256 potential participants refused participation (enrollment refusal rate=57.5%). Frequently mentioned reasons for not participating were a lack of time, no interested in additional care and having to temporarily increase the focus on diabetes. A number of 142 parents filled in the baseline questionnaire. As such, 47 parents (baseline attrition rate=25%) dropped out before filling out the first questionnaire. Mentioned reasons for dropping out were a loss of interest and a lack of time. Subsequently, 105 parents also filled in the questionnaire at T1, meaning that 32 (postrandomization attrition rate during follow-up=26%) participants dropped out during the course of the study. Participants dropped out due to losing interest, a lack of time or because they changed from treatment center. As such, 23.6% (n=105) of the potential population successfully participated in the study (see also Figure 1). BODY.RESULTS.DEMOGRAPHICS: The demographic statistics of the 105 participants are displayed in Table 2. A one-way, between-group ANOVA revealed no significant differences in any of the variables at baseline between the centers. Figure 1Flowchart of inclusion of participants. Table 2 Demographics and baseline scores of the participants. Demographic variables Experimental group Control group Total group Parents (n) 54 51 105 Gender (male; female; filled in together) 49; 5 44; 5; 2 93; 10; 2 Educational level Lower secondary education, n (%) 2 (4) 4 (8) 6 (6) Middle secondary education, n (%) 3 (5) 4 (8) 7 (7) Higher secondary education, n (%) 24 (44) 19 (37) 43 (41) Middle tertiary education, n (%) 9 (17) 2 (4) 11 (11) Higher tertiary education, n (%) 9 (17) 19 (37) 28 (27) Academia, n (%) 7 (13) 3 (6) 10 (10) Child Age in years, mean (SD a ) 9,1 (2.9) 8,9 (2.5) 9 (2.7) Gender (female; male) 30; 24 27; 24 57; 48 HbA1c in mmol/mol, mean (SD) 64 (13.77) 62 (7.77) 63 (10.62) HbAc in %, mean (SD) 7,98 (1.17) 7,86 (0.71) 7,92 (0.97) Insulin therapy Injections, n (%) 10 (19) 15 (29) 25 (24) Pump, n (%) 44 (82) 36 (71) 80 (76) a SD: standard deviation. BODY.RESULTS.FEASIBILITY OF THE INTERVENTION: Data from the 54 participants in the experimental group and who therefore had access to Sugarsquare were used for the feasibility analysis and for the analysis relating user data and baseline scores on questionnaires. A proportion of 59% (n=32) of the parents who had access, used Sugarsquare during the trial (Table 3). Of the 32 unique parent users, 11 (34%) logged in repeatedly, at least once every 2 weeks and 9 (28%) logged in incidentally (3 times or more, but under once every 2 weeks), and 16 (41%) logged in once or twice during the study period. Table 3 also shows that 34 (77%) of 44 professionals who had received access at the start of the study, logged in and 32 (94%) logged in again. Thus, overall, 73% (n=32) of the professionals accessed Sugarsquare more than once. All users (parents and professionals) viewed all applications at least once when they logged in. The applications for forum (#page views=2838) and contact with the treatment team (#page views=2795) were viewed more often than the applications for information (#page views=415) and chat (#page views=683). Users reported no downtime, although 2 users reported that they sometimes could not access Sugarsquare, due to technical problems with the users' telecom providers. Some parents (n=8) said that the two-step security procedure as a hassle. Sugarsquare attributed to provision of care according to all 9 key elements, derived from the Global IDF or ISPAD and ADA Guidelines for Diabetes care in Childhood and Adolescence (see also Multimedia Appendix 2) [18,50]. According to the Sugarsquare coordinators, there were 3 factors that limited implementation. These factors were the two-step login procedure, the lack of customized instructions for health care professionals and the randomization on individual level. The local Sugarsquare coordinators and the multidisciplinary approach of the team were suggested as 2 factors that positively affected implementation. BODY.RESULTS.POTENTIAL EFFICACY: With regard to parenting stress, 82 (78%) parents (control and experimental condition) reported average or below average levels of parenting stress compared with Dutch healthy controls, 19 (18%) reported slightly elevated levels, and 4 (4%) reported very high levels of parenting stress (see also Table 4). The analysis revealed no significant differences in change in parenting stress over time between the two groups (F3,101=.49, P=.49), or between centers (F3,101=.31, P=.91), and nor was there an interaction between groups and centers (F3,101=1.16, P=.34). Similar results were obtained in an ANCOVA (Table 5) without the factor center and a sensitivity analysis, conducted by means of a multiple imputation analysis. Since no change was found, a conservative analysis using LOCF was not conducted. We also found no significant differences in change over time in HbA1c levels between the experimental group and the control group (F3,101=.040, P=.84). BODY.RESULTS.BASELINE PARENTING STRESS LEVELS AND PORTAL USAGE: The analysis revealed that parenting stress at baseline was significantly correlation with the frequency of logging in (ρ=.282, P=.03 Table 6) and the number of pages viewed (ρ=.304, P=.02). It seems that the greater stress parents experienced, the more parents logged in and the more pages they viewed. Table 3 Sugarsquare usage during the first phase (6 months) of the study period. User statistics Parents Professionals Parents and professionals Parents n (experimental group) 54 44 Unique visitors, n (%) 32 (59) 34 (77) Log-ins High frequent users, n (%) 12 (38) 12 (35) Moderate users, n (%) 9 (28) 20 (59) Low frequent users, n (%) 11 (34) 2 (6) #logins (n) 419 505 #logins, mean (SD a ) 7,8 (13) 11,5 (16) Page views #page views (n) 5690 8006 #mean page views, mean (SD) 105,4 (175) 182 (253) Information #Documents visits (n) 415 #Web links visits (n) 213 Patient-professional contact #Questions visits (n) 2795 #Questions input (n) 344 #Treatment visits (n) 674 #Treatment input (n) 29 Peer support #Forum visits (n) 2838 #Forum input (n) 147 #Chat visits (n) 683 #Chat input (n) 1653 a SD: standard deviation. Table 4 Distribution of parenting stress index (PSI) scores for the total group. PSI a -scores n (%) Normal stress scores 82 (78) Elevated stress scores 19 (18) High stress scores 4 (4) a PSI: parenting stress index. Table 5 Results of the analysis of covariance (ANCOVA) in parenting stress and HbA1c. Efficacy variables Experimental group Control group F T0 T1 T0 T1 Mean (SD a ) Mean (SD) Mean (SD) Mean (SD) PSI b 48.13 (19.46) 51.35 (22.32) 44.61 (17.60) 44.45 (17.89) .49 HbA1c 63.74 (12.77) 63.06 (8.98) 62.41 (7.77) 62.54 (8.64) .04 a SD: standard deviation. b PSI: parenting stress index. Table 6 Correlations of parenting stress at baseline and frequency of log-ins and page views. Efficacy variables #log-ins #page views Parenting stress (baseline) ρ =.282, P =.03 ρ =.304, P =.02 BODY.DISCUSSION.PRINCIPAL FINDINGS: This study investigated the feasibility of conducting a trial and implementing an Internet intervention in a population of parents of children with T1D, in daily clinical practice. It revealed that eHealth has the potential to create a platform for shared, daily disease management between professionals and parents. Sugarsquare seems to attract parents with relatively high stress levels. The participation rate and dropout rate in the RCT were average, compared with other trial studies and results indicated that conducting a trial concerning Sugarsquare was feasible. The implementation of Sugarsquare in clinical practice was partly feasible, given the high practicability in all users, moderate acceptability and demand in parent users, high acceptability and demand in professional users, high level of integration and lack of potential efficacy. It is interesting to note that parents reporting higher levels of parenting stress were more likely to use Sugarsquare compared with parents reporting lower levels. This is consistent with a recent study by Balkhi and colleagues [26], who reported that parents with higher stress levels more frequently visited diabetes-related online forums than did parents with lower stress levels. As no association between HbA1c and usage was found, it is assumed that general parenting stress is associated with usage and not stress related to medical condition of the child. However, it is quite possible that the parents who did not use Sugarsquare might do so if they have a temporary need for additional support or information, for instance if their child becomes ill, at onset of puberty or if they are planning a trip abroad. Our enrollment refusal rate (57.5%) and baseline attrition rate (25%) fell within the ranges described in the review by Karlson and Rapoff (2009), who found the refusal rates in eHealth studies to be ranging from 0% to 75% (mean 37%) and baseline attrition rates ranging from 0% to 35% (mean 4%) [53]. From this perspective, the rates in this study are reasonable. Still, we expected a lower enrollment refusal rate, since the intervention was requested by parents and fitted to their preferences by means of focus group interviews. It could be that the questionnaires, which had to be filled in by the parents on several occasions, discouraged potential participants [54]. It is also possible that, due to the research context, parents perceived this study as an externally driven project, which conflicted with their preference for a center-driven intervention [8] and might have negatively influenced their willingness to cooperate [55]. Our study was further confronted with an average postrandomization attrition rate during follow-up (26% vs 0-54%, mean 20% in Karlson and Rapoff) [34,53]. The eHealth studies are subject to low enrollment and high dropout rates. In order to resolve the issue of low enrollment, Lernmark and colleagues [56] suggested that clarity should be provided about what participants are expected to invest and about the potential added value of the study results for the individual participant, their clinic or care in general. Baxter and colleagues [57] suggested that interaction between researchers and participants is vital for keeping participants committed after they decide to participate. During the study, possibilities to improve the trial and implementation were identified. First of all, customized instructions for when and how to use Sugarsquare, would have helped them fit Sugarsquare into their daily workflow and encourage parents to use Sugarsquare [58-60]. Also, Sugarsquare was used in a research context and randomization took place on an individual level. As such, only a part of the population in each center participated in this study. This meant that health care professionals had to work using two procedures simultaneously, making their work very complex and intensive and complicating the integration of Sugarsquare in their workflow of everyday [61,62]. The research context also had a negative effect on the amount of interaction on Sugarsquare, since only a relatively small population of parents had access to the platform. Implementation would have been more successful if randomization was conducted on center level, which would have meant that a center would have used Sugarsquare for its entire population or not at all. Factors that might have contributed to the success of the trial and the implementation were also identified. The teams all appointed a team member dedicated to Sugarsquare, who coordinated local recruitment and implementation, and monitored Sugarsquare usage. This might have supported the teams in integrating the intervention in usual care, since studies in the past reported that this lead to increased awareness in the team for usage of innovative interventions [44,59,62]. Also, the multidisciplinary approach of the Diabetes teams in our study might have contributed to the implementation of Sugarsquare, since literature shows that members of multidisciplinary teams are used to working toward shared, organizational goals, which makes it easier to implement changes into their workflow [58,59]. Sugarsquare has a broad focus and consists of multiple, general, potentially feasible applications. These characteristics fit to the needs of the parents, as expressed in the focus groups [8]. However, because of this broad focus, it is difficult to establish which applications (information, peer contact, contact with staff) contributed to usage and to potential effect. As such, mechanisms of change could not be identified. Future studies could apply multiple study arms to adequately assess the value of single applications, which would increase the number of participants required. [63,64]. Another way of identifying potential working mechanisms and the value of single applications would be to collect qualitative data. This is expected to provide more insight into both and future researchers should consider collecting qualitative data in their study. In this study, we used a generic questionnaire to assess parenting stress, considering its broad use in pediatrics and the lack of a diabetes-specific one. Although generic parenting stress measures can be helpful for assessing stressors and distress, they might not be sensitive to issues specific to the parents of children with an illness or specific disease-related issues and, as such, failed to properly assess potential change in those domains [65]. Future studies could consider using an instrument designed for parents of a child with T1D or, in case this is lacking, an instrument for parents of pediatric patients, such as the Pediatric Inventory for Parents (PIP) or the recently validated pediatric parenting stress index (PPSI). The direct effect of the small sample size in this study is expected to be limited, since the sensitivity analyses did not show different outcomes compared with the completer analysis. However, indirectly, the limited number of participants in the local centers may have decreased the interaction on the local Sugarsquares and, with that, generalizability of the results. Future studies can avoid this by using randomization on center level. Sugarsquare can be considered as a promising tool for diabetes teams, virtually extending their diabetes center. It contributes to usual care, because it offers parents and professionals a secured, Web-based platform for parent-health care professional communication, moderated peer support, and tailored disease information. In addition, it especially attracts parents who experience higher parenting stress levels. Given the complications that arose when Sugarsquare was used together with conventional communication tools, it is recommended that Sugarsquare be used as the sole medium for regular communication between parents and diabetes team. Appointing a dedicated Sugarsquare manager and using adequate instructions for the involved professionals are also hypothesized to contribute to the integration of Sugarsquare in care as usual. In order to increase usage by parent users and to improve their acceptance of Sugarsquare in daily care, diabetes teams could continuously add new content to Sugarsquare. This is expected to keep Sugarsquare interesting and to invite parent users to post information as well. It is also important that all team members post information, which shows parent users that Sugarsquare is accepted by the whole team. This might lower the threshold for parent users to use and accept Sugarsquare. This has been found to be workable in 9 centers for diabetes care in the Netherlands, which have implemented Sugarsquare in usual care. In a recent study on the implementation of an eHealth intervention regarding online assessment of quality of life, it was noticed that successful implementation is affected by many factors acting on different aspects of implementing an intervention [66]. In general, they distinguish between factors on the level of the existing IT-structures (eg, usability, compatibility), organization (eg, support, expectations of management for usage), and the intervention itself (eg, easy to use, technical problems). As attrition rates as well as limited implementation are general challenges in eHealth, future studies should pay more attention to these factors. Another issue in the field of eHealth is that the financial costs of maintenance of interventions have yet to be included in systems for health care costs. The main problem that arises from this issue is the high number of interventions that are not implemented after a trail. When starting an intervention study, we advise researchers to start with a single center trial for exploration of feasibility and potential efficacy. When feasibility and potential efficacy are demonstrated, a multicenter implementation could be conducted, potentially combined with assessment of efficacy using a historic design. BODY.DISCUSSION.CONCLUSIONS: This study concerned a generic intervention, based on parents' preferences and needs, serving different aims, especially regarding shared disease management between parents and professionals. Our next step is to further develop the potential of Sugarsquare to serve as a platform for provision of more mechanism-focused interventions, targeted to reduce parenting stress, for instance, by providing online information or online cognitive behavior therapy. More generally, eHealth has possibilities to support monitoring of physical and psychosocial well-being, facilitate peer contact, interaction between patients and health care professionals and exchange of data. Sugarsquare can serve as central portal through which these applications or interventions can be accessed.

TITLE: Comparison between 5-day aprepitant and single-dose fosaprepitant meglumine for preventing nausea and vomiting induced by cisplatin-based chemotherapy ABSTRACT.PURPOSE: We aimed to compare the preventive effect of 5-day administration of aprepitant with single administration of fosaprepitant meglumine against nausea and vomiting symptoms due to highly emetogenic chemotherapy regimens comprising cisplatin (CDDP). ABSTRACT.METHODS: Subjects were inpatients who underwent chemotherapy for gastric cancer, esophageal cancer, lung cancer, or head and neck cancer with a regimen comprising 60 mg/m2 or higher dose of CDDP. In this randomised, open-label, controlled study, the subjects were assigned to a group given aprepitant for 5 days or a group given a single administration of fosaprepitant meglumine. The nausea and vomiting symptoms that emerged within 7 days after the first CDDP administration were investigated with a questionnaire form; the results were compared between the two groups. Risk factors affecting nausea and vomiting symptoms were also investigated. ABSTRACT.RESULTS: Of the 101 patients enrolled, 93 patients were included (48 in the 5-day aprepitant group and 45 in the single fosaprepitant meglumine group). No significant intergroup differences in the complete response rate or the complete control rate were found over the entire period. The nausea score tended to increase from day 3 in both groups, but no significant intergroup difference was observed. Furthermore, the investigation of risk factors affecting moderate or severe nausea symptoms indicated that the fosaprepitant meglumine administration was not a risk factor. ABSTRACT.CONCLUSIONS: Single administration of fosaprepitant meglumine was not inferior to 5-day administration of aprepitant for preventing acute and delayed nausea and vomiting symptoms occurring after administration of CDDP (60 mg/m2 or higher). BODY.INTRODUCTION: Chemotherapy-induced nausea and vomiting (CINV) necessitates modifications to the regimen in severe cases and is reportedly a factor affecting the prognosis of cancer treatment [1, 2]. CINV reduced the amount of food intake, resulting in malnutrition, body weight loss and reduced performance status (PS) [3]. The reduced PS leads to an increased incidence of haematotoxicity [4]. Therefore, it is critical to prevent CINV. The emetogenicity of anti-cancer agents has been categorised according to their risk levels in guidelines by the Multinational Association of Supportive Care in Cancer (MASCC) [5], the American Society of Clinical Oncology (ASCO) [6] and the National Comprehensive Cancer Network (NCCN) [7]. In all of these guidelines, cisplatin (CDDP) is classified as a highly emetogenic agent [8, 9]. Appropriate measures against CINV are essential in CDDP administration. In addition, CDDP is characterised by a biphasic emergence of CINV in the acute (within 24 h after starting the administration) and late phases (beyond 24 h after starting the administration) [10]. The development of antiemetic drugs has been remarkable; selective 5-HT3 receptor antagonists were developed in the 1990s. Ohmatsu et al. have reported that the granisetron + dexamethasone group fared better than the metoclopramide + dexamethasone group with respect to the prevention of CINV during CDDP administration [11]. Thereafter, it has been shown that the NK-1 receptor is closely involved in CINV [12], and substance P induces vomiting through binding to the NK-1 receptor in the central nervous system [13–15]. The NK-1 receptor has also been noted as a new target for antiemetic therapy [16]. NK-1 receptor antagonist, aprepitant, has been developed and was listed in the NCCN guidelines in 2004. Aprepitant is an anti-emetic agent with a novel mechanism of action. It inhibits the binding of substance P to the NK-1 receptor in the central nervous system [17, 18] and reduces the incidence of acute and delayed nausea and vomiting symptoms significantly when used in combination with the conventional anti-emetic therapy (concomitant use of ondansetron and dexamethasone) [19]. Aprepitant has also been listed in the drug price standard in Japan since December 2009 and accordingly included as an effective anti-emetic that is also used for delayed emesis in 'Guidelines for Proper Use of Anti-emetics Version 1', which was created by the Japan Society of Clinical Oncology in May 2010. Subsequently, fosaprepitant meglumine, a phosphorylated prodrug of aprepitant with improved water solubility, was developed. The non-inferiority of fosaprepitant meglumine to aprepitant in the anti-emetic effect in the acute and late phases has been demonstrated in a phase III study conducted in 27 countries to compare aprepitant administered for 3 days and fosaprepitant meglumine administered once in patients receiving the highly emetogenic anti-cancer agent CDDP (≥70 mg/m2) [20]. Based on this result, fosaprepitant meglumine has been listed in the NCCN guidelines since 2011 and also included in the Japanese drug price standard in November 2011. Thereafter, a phase III clinical study in Japanese patients treated with CDDP (≥70 mg/m2) [21] was conducted and demonstrated a superior effect in the fosaprepitant meglumine group compared to that of the placebo group. However, no studies have been conducted to directly compare the anti-emetic effect of aprepitant with fosaprepitant meglumine in Japanese patients. Moreover, although guidelines released by the Japan Society of Clinical Oncology have recommended 3 days of aprepitant administration, a domestic phase II clinical trial targeting Japanese subjects [22] has verified a 5-day administration of aprepitant. This is because aprepitant was developed in Japan as a drug that should be administered for 5 days. However, the overseas clinical trials [19, 23, 24] tested the 3-day regimen of aprepitant, and the NCCN guidelines [7] recommended the 3-day technique. Therefore, after carefully considering the domestic phase II clinical trial results, the Ministry of Health, Labour and Welfare in Japan decided that sufficient efficacy could be anticipated with 3-day aprepitant administration for Japanese patients and that up to 5 days of administration would be acceptable. In light of the above facts, we decided that it was necessary to compare the 5-day administration of aprepitant and a single administration of fosaprepitant meglumine to compare the efficacy of aprepitant and fosaprepitant meglumine in Japanese patients who had received CDDP, a highly-emetic anticancer agent. Moreover, a phase III trial [20] that compared the 3-day administration of aprepitant and the single administration of fosaprepitant meglumine compared antiemetic effects until 5 days after CDDP administration; however, this study did not examine the subsequent progress of the patients. Therefore, we evaluated the degree of nausea and vomiting symptoms until day 7 in Japanese patients who underwent chemotherapy with CDDP at Fujita Health University Hospital (the day of CDDP administration was day 1) to prospectively compare the effects on nausea and vomiting symptoms in a 5-day aprepitant administration group and in a single-dose fosaprepitant meglumine administration group. BODY.SUBJECTS AND METHODS.SUBJECTS: In this study, we enrolled Japanese patients who started to receive chemotherapy comprising CDDP (≥60 mg/m2) for lung cancer, gastric cancer, esophageal cancer, or head and neck cancer between January 2013 and March 2014 at the Fujita Health University Hospital. They provided consent to participate in this study after the intent of the study was explained to them. However, any patient who had nausea or vomiting within 24 h before starting administration of anti-neoplastic drugs, who could not receive a drug orally, who could not answer the questionnaire, who did not provide consent or who was considered unsuitable to this study was excluded. BODY.SUBJECTS AND METHODS.INVESTIGATIONS: An equal number of subjects were randomly assigned to the aprepitant treatment group (group A; aprepitant, a 5-HT3 receptor antagonist and dexamethasone) and the fosaprepitant meglumine treatment group (group B; fosaprepitant meglumine, a 5-HT3 receptor antagonist and dexamethasone). Anti-emetic agents were used in both groups in accordance with the dosage instruction on the package inserts. Specifically, in group A, aprepitant was administered at 125 mg on day 1 (the day administration of anti-neoplastic agents started) and at 80 mg daily on days 2–5, and a 5-HT3 receptor antagonist (palonosetron [0.75 mg], granisetron [3 mg] or azasetron [10 mg]) was administered, along with 6.6–9.9 mg of dexamethasone on day 1 and 3.3–6.6 mg daily on days 2–4. Group B subjects received fosaprepitant meglumine at 150 mg on day 1, 6.6–9.9 mg of dexamethasone on day 1 and 3.3–6.6 mg daily on days 2–4, and a 5-HT3 receptor antagonist (palonosetron [0.75 mg], granisetron [3 mg] or azasetron [10 mg]). BODY.SUBJECTS AND METHODS.ASSESSMENT: To survey the occurrence of nausea and vomiting, we prepared a nausea/vomiting recording form (Fig. 1) based on the MAT (questionnaire about nausea and vomiting), which was developed by the Multinational Association of Supportive Care in Cancer (MASCC). Investigators interviewed their patients daily about states of nausea and vomiting symptoms from day 1 (the day CDDP was administered) to day 7 and recorded the responses. Based on a report from Longo et al. [25], we conducted patient interviews only for the first course to minimise bias. Nausea symptoms were surveyed and recorded by a pharmacist according to the numeric rating scale (NRS; 11-point scale, in which 0 represents a condition without nausea and 10 represents a condition with the worst conceivable nausea). The results were then categorised according to the Likert scale into 'no symptom or mild' (NRS, 0–2), 'moderate' (3–6) and 'severe' (7–10). For vomiting symptoms, the number of times vomiting (including dry vomiting) occurred was surveyed every day and evaluated according to the National Cancer Institute-Common Terminology Criteria for Adverse Events Version 4.0 (NCI-CTCAE ver. 4.0). With regard to the effects of the drugs on nausea and vomiting, the proportion of the patients who did not experience vomiting or dry vomiting (the complete response [CR] rate) and the proportion of the patients who did not experience vomiting or dry vomiting and had 'no symptom or mild' nausea (the complete control [CC] rate) were determined and compared between the two groups. We also investigated age, sex, cancer type, administration of concomitant radiotherapy and use of opioid as potential confounding factors. We also examined susceptibility to motion sickness, history of alcohol consumption and history of pregnancy, which were proposed as risk factors for nausea and vomiting by Koeller et al. [26].Fig. 1Nausea/vomiting recording form BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSIS: Continuous variables in patient background data were expressed as mean ± SD, and intergroup comparison was performed with an unpaired t test. Patient background data composed of a nominal scale was expressed in %, and χ2 test was used for intergroup comparisons. χ2 test was used to compare the CR and CC rates between the two groups. The nausea score was expressed in a form of median (first quartile–third quartile), and changes in the score over time were compared with the Friedman test, followed by multiple comparisons with the Wilcoxon signed-rank test with Bonferroni correction. Intergroup comparisons were performed with the two-way repeated measures analysis of variance. To explore the risk factors for nausea and vomiting, univariate analysis was performed, and then multivariate logistic regression analysis was performed on items with a significance level less than 10 %. The analysis software used was SPSS Ver. 22.0 (IBM Corporation, Armonk, NY, USA); the significance level used was less than 5 %. BODY.SUBJECTS AND METHODS.STATEMENT OF ETHICS: This study was conducted in compliance with the 'Ethical Guidelines for Clinical Research' with an approval from the Ethical Review Board for Epidemiology/Clinical Research of our hospital. BODY.RESULTS.PATIENTS: Of 101 patients enrolled, 8 patients were excluded as per the exclusion criteria, and the remaining 93 patients were randomised: 48 in group A and 45 in group B. The patient inclusion flowchart is presented in Fig. 2. No differences were noted in the patient background between the two groups (Table 1).Fig. 2Study flow chartTable 1Patient backgroundGroup A (n = 48)Group B (n = 45) P valueAge (years)61.7 ± 11.765.4 ± 10.00.099Male, Female40,834,110.35CDDP (mg/m2)70 (60–80)70 (60–80)0.61Cancer type0.85 Lung cancer1412 Gastric cancer1510 Esophageal cancer79 Head and neck cancer1113 Unknown primary11RT concomitantly used23210.90Opioid concomitantly used330.93Alcohol consumption29260.80Susceptible to motion sickness360.25History of emesis due to pregnancy370.515-HT3 receptor antagonist0.93 Palonosetron2322 Other than palonosetron (granisetron or azasetron)2523 CDDP cisplatin, RT radiotherapy BODY.RESULTS.EFFICACY: The CR rates in the acute phase (days 1 and 2), the first stage of the late phase (days 3–5) and the second stage of the late phase (days 6 and 7) were separately compared between group A and group B. The CR rates in group A and group B were, respectively, 97.9 and 97.8 % for the acute phase (P = 0.96), 87.5 and 84.4 % for the first stage of the late phase (P = 0.67) and 89.6 and 90.0 % for the second stage of the late phase (P = 0.91), showing no significant differences between the two groups in all phases (Fig. 3a). The CR rate for the entire period was 85.4 % (41/48) in group A and 82.2 % (37/45) in group B, also showing no significant difference (P = 0.90). While comparing the CR rate between the acute and late phases in group A, no significant differences were found between the acute phase and the first stage (P = 0.36) or the second stage of the late phase (P = 0.63). The CR rate was similarly compared between the phases also in group B, but no significant differences were found between the acute phase and the first stage (P = 0.19) or the second stage of the late phase (P = 0.60).Fig. 3Comparison of the CR and CC rates between group A and group B The CC rate was also separately compared between the two groups. The CC rates in group A and group B were, respectively, 77.1 and 91.1 % for the acute phase (P = 0.066), 60.4 and 73.3 % for the first stage of the late phase (P = 0.19), and 66.7 and 71.1 % for the second stage of the late phase (P = 0.64). Although differences between the two groups were not of statistical significance in any phases, the CC rate in group A tended to be slightly lower in the acute phase (Fig. 3b). The CC rate for the entire period also did not differ significantly between group A (60.4 %, 29/48) and group B (64.4 %, 29/45) (P = 0.85). Comparisons of the CC rate between the acute and late phases in group A showed no significant differences between the acute phase and the first stage (P = 0.23) or the second stage of the late phase (P = 0.78). Similar phase-to-phase comparisons of the CC rate in group B also showed no significant differences between the acute phase and the first stage (P = 0.16) or the second stage of the late phase (P = 0.093). For day-to-day changes in the nausea score, while a significant consecutive increase was observed from day 3 to day 7 in group A, the score increased only on days 3 and 4 in group B. However, no significant differences were detected by the two-way repeated measures analysis of variance (Table 2).Table 2Daily changes in nausea score in group A and group BGroup AGroup BDay 10 (0–0)0 (0–0)Day 20 (0–1)0 (0–0)Day 30 (0–3)*0 (0–1)*Day 41 (0–3)**, ***0 (0–2)*Day 51 (0–3)**, ***0 (0–2)Day 60 (0–3)**0 (0–2)Day 70 (0–3)**0 (0–2) P value (Friedman test)0.00130.0013*P < 0.05; **P < 0.01 versus day 1; ***P < 0.05 versus day 2 (Wilcoxon's signed-rank test followed by Bonferroni correction), P = 0.658 (two-way repeated measures analysis of variance) BODY.RESULTS.RISK FACTORS: Risk factors for moderate or severe nausea symptoms were investigated. The two factors 'age' and 'susceptible to motion sickness' were of near statistical significance in the univariate analysis, but the multivariate analysis indicated that neither was a risk factor (Table 3).Table 3Risk factors for moderate or severe nausea symptomsUnivariate analysisMultivariate analysisOdds ratio (95 % CI) P valueOdds ratio (95 % CI) P valueAdministration of fosaprepitant0.840.69(0.36–1.95)Age (years)0.970.0750.970.21(0.93–1.00)(0.93–1.02)Female1.660.33(0.60–4.60)CDDP (mg/m2)1.020.24(0.98–1.06)Treatment with RT1.080.85(0.47–2.51)Concomitant use of opioid1.720.52(0.33–9.03)Alcohol consumption0.670.37(0.29–1.58)Susceptible to motion sickness3.790.0732.770.20(0.88–16.30)(0.59–12.96)Administration of palonosetron1.010.98(0.44–2.34) CDDP cisplatin, RT radiotheraphy BODY.DISCUSSION: For acute and delayed vomiting symptoms seen after CDDP-based chemotherapy, no difference in incidence was found between the 5-day aprepitant administration group and the single fosaprepitant meglumine administration group. Grunberg et al. compared the CR rate between the 3-day aprepitant administration group and the single fosaprepitant meglumine administration group of patients treated with at least 70 mg/m2 of CDDP and reported no significant intergroup differences in the acute phase (88.0 and 89.0 %, respectively) or the late phase (74.2 and 74.3 %). Although this result was in agreement with that of our study, Grunberg et al. used a higher CDDP dose and administered aprepitant for 3 days rather than 5 days [20]. These differences in conditions may underlie a lower CR rate observed in their study than that in our study. While a phase II study in Japanese patients treated with CDDP (≥70 mg/m2) showed that the CR rate in the 5-day aprepitant administration group was higher than that of the placebo group [22], the CR rate for the entire study period in the 5-day aprepitant administration group was 70.5 %, which was lower than the rate obtained in our study. This difference may be attributable to the difference in the CDDP dose between the two studies. In this study, we intentionally chose a CDDP dose of ≥60 mg/m2 for the following reason; in the Japanese gastric cancer guidelines, the first-line drug therapy options include combination therapy with CDDP (60 mg/m2) and S-1 (80 mg/m2) [27], and we thought that patients with gastric cancer, for which CDDP is a first choice, should be included in CDDP studies about Japanese subjects. In the present study, the CDDP dose did not differ between the two groups, and it is highly unlikely that the results were affected by the difference in the CDDP dose. Daily changes in the CC rate and nausea score were investigated, but neither showed significant differences between the two groups. Furthermore, the result from the logistic regression analysis of risk factors for nausea of moderate or higher severity indicated that fosaprepitant meglumine administration was not a risk factor. From these results, the nausea-suppressing effect of fosaprepitant meglumine administered once was found non-inferior to that of aprepitant administered for 5 days. In studies on the effect of aprepitant or fosaprepitant meglumine in CDDP-treated patients, the CR and CC rates have been used often, but the results have been based on the rate determined for a certain period [18–21] rather than the rate changes followed over the course of the study. While a visual analog scale [19–20] and other scoring systems [21] are generally used to evaluate the nausea symptom, we used the interview survey of patients with NRS for detailed, daily evaluation of the symptom. The demonstration of the absence of intergroup differences, even when changes in the nausea symptom were investigated in detail with NRS, provides a more solid support for the prophylactic equivalence of the two agents. In the study conducted by Grunberg et al. to compare aprepitant and fosaprepitant meglumine, the anti-emetic effect was not investigated beyond 5 days after the CDDP administration. In contrast, our present study suggested that the anti-CINV effect of fosaprepitant meglumine persisted over 7 days after the single administration. This is a new and very intriguing finding. Sekine et al. searched for risk factors that affected the incidence of CINV in multiple clinical studies conducted with chemotherapy-naïve patients in Japan and reported that the risk factors include female gender, less advanced age (less than 55 years) and no history of alcohol consumption. The risk of CINV emergence increases as the number of applicable risk factors increases [28]. However, our present investigation of risk factors for moderate or severe nausea indicated that no factors were influential. Moreover, the two groups were comparable in the number of risk factors: Patients with 1, 2 and 3 risk factors were, respectively, 20 (41.7 %), 7 (14.6 %) and 2 (4.2 %) in group A and 14 (31.1 %), 8 (17.8 %) and 2 (4.4 %) in group B. The risk factors reported by Sekine et al. were thus considered highly unlikely to have affected the results of the present study. In addition to aprepitant and fosaprepitant meglumine, the subjects in this study received another anti-emetic selected from granisetron, azasetron and palonosetron. While granisetron and azasetron reportedly are as potent as ondansetron in terms of the anti-emetic effect [29–31], Hashimoto et al. [32] reported that the CR rate in patients treated with a highly emetic regimen comprising CDDP at a dose of 50 mg/m2 or higher tended to be lower in the granisetron group than in the palonosetron group (59.1 versus 65.7 %; P = 0.0539). Based on this result, palonosetron administration was included as a variable in our risk factor analysis but was found uninfluential. This may be due to some differences in study conditions, but details remain unknown. In Japanese patients treated with CDDP + 5-FU (CDDP 80 mg/m2 on day 1 + 5-FU 800 mg/m2 on days 1–5) for head and neck cancer, Aoki et al. [33] investigated the nausea and vomiting symptoms over an observation period of 7 days from the start of chemotherapy (days 1–7) in the 3-day and the 5-day aprepitant administration group. They reported that the effect was maintained for 7 days in the 5-day group, while the effect declined from day 6 onwards in the 3-day group. In the present study, we compared the CR and CC rates between the acute phase and the first and second stages of the late phase but did not find any apparent decline of the effect in the single fosaprepitant meglumine administration group or the 5-day aprepitant administration group. Although conditions were not identical, results from our present study corroborated the results obtained by Aoki et al., suggesting that single administration of fosaprepitant meglumine was non-inferior to the 5-day administration of aprepitant in terms of persistence of the effect. BODY.CONCLUSIONS: The single administration of fosaprepitant meglumine was as effective as the 5-day administration of aprepitant for preventing acute and delayed nausea and vomiting symptoms occurring after the administration of CDDP (≥60 mg/m2).

TITLE: Comparison of low and standard pressure gas injection at abdominal cavity on postoperative nausea and vomiting in laparoscopic cholecystectomy ABSTRACT: Background and Objective: Postoperative nausea and vomiting (PONV) is the main concern for 40-70% of patients undergoing laparoscopic cholecystectomy. Our objective was to compare carbon dioxide gas at low pressure and standard pressure for the occurrence of PONV on patients undergoing laparoscopic cholecystectomy. Methods: This double- blind trial was conducted on 50 women patients aged between 18 to 60 years with acute cholecystectomy. The patients were divided into two groups: low pressure (LP) (received LP gas, 7-9 mmHg) and standard pressure (SP) (received SP gas, 14-15 mmHg). Nausea and vomiting in patients at hours 0-4, 4-8, 8-12, 12-24 after the surgery were recorded. Results: The frequency of PONV in the LP and SP groups did not demonstrate statistically significant different (P > 0.05). Nevertheless the frequency of shoulder pain after 4 hours at the LP group compared with SP group was significantly different (P < 0.023). Conclusions: The use of low pressure gas compared to standard pressure gas to create pneumoperitoneum could not reduce the PONV whereas the frequency of shoulder pain in LP group was reduced. Low pressure gas was associated with reduction of surgeon visibility and subsequently more prolonged surgery duration. BODY.INTRODUCTION: Laparoscopic cholecystectomy was introduced in 1992 as the gold standard intervention for gallstone operation.1 It can be associated with complications including dehydration, gastric aspiration and wound dehiscence.2 Almost 30 percent of all patients undergoing general anesthesia experience postoperative nausea and vomiting (PONV).3 PONV is the major distress of 40-70% of patients undergoing laparoscopic cholecystectomy.4 Although the precise mechanism of PONV is still unknown, we believe that high frequency of PONV in women undergoing laparoscopic operation may be due to pneumoperitoneum.5 Additionally, several cardiopulmonary changes occur during pneumoperitoneum, which is now introduced to allow laparoscopic cholecystectomy.6 Carbon dioxide (CO2) is noncombustible, quickly soluble in the blood, and relatively low-cost, so, it is the most common used gas for insufflation. Carbon dioxide is considered the major product of cellular metabolism and eliminated by a well-organized mechanism.7 Monitor of end tidal CO2 concentration is obligatory during the laparoscopy. It is suggested to employ the lowest intra-abdominal pressure allowing satisfactory exposure of the operative field, rather than using a routine pressure.8 The objective of this study was to find out the frequency of PONV in laparoscopic cholecystectomy at low pressure (8mm/Hg) vs. standard pressure (15mm/Hg) pneumoperitoneum. BODY.METHODS: Study design and population: This double-blind trial was performed in a hospital affiliated to pain research center in Jundishapur University located in Ahvaz, Southwest Iran, from Dec. 2012 to Sep. 2013. A total of 50 women patients aged between 18 to 60 years old with acute cholecystitis were enrolled into the study. Exclusion criteria was defined as the patients who had cholangitis, history of motion sickness, malignancy, smoking, pregnancy and the patients who had received anti-emetic agents during the past 24 hours. Ethics Committee of Ahvaz Jundishapur University of Medical Sciences approved the study. A consent Form was received from the patients prior to the study. Intervention and measurement: Fifty qualified patients were randomly allocated for elective laparoscopic cholecystectomy to receive either standard pressure gas (14-15 mmHg) (n=25) or low pressure gas (7-9 mmHg) (n=25). Randomization was performed based on Color Cards (Blue & Red Cards).The patients belonged to class 1-2 in American Society of Anesthesiologist (ASA). All the laparoscopic cholecystectomies which performed by the same surgeon, standard monitoring and anesthetic management were standardized. Anesthesia was induced with: Midazolam 0.05 mg/kg, Fentanyl 2 mcg/kg, Thiopental 5 mg/kg and Cisatracurium 0.1 mg/kg. Mechanical ventilation was adjusted to maintain end-tidal CO2 tension 35-40 mmHg throughout the surgery. After induction of anesthesia, a nasogastric tube was passed into the stomach for suction and then removed. The patients were monitored for blood pressure and heart rate during the surgical procedure. At the end of the laparoscopic operations, surgeon was asked to state the quality of surgical field visualization. Also, when the patients were awake, questioner (who did not know the pressure gas) evaluated the nausea, vomiting and shoulder pain. The method used for pain control after operation was the infusion of Apotel 1 gr at recovery and repeat it after 6 hours and also, NSAIDs suppositories / PRN. The patients were monitored for emetic symptoms within the first postoperative 24 hours for five times. Nausea and vomiting in patients were recorded at hours 0-4, 4-8, 8-12, 12-24, and after the surgery. Assessment of nausea intensity is difficult because the nausea is subjective, so in order to increase the accuracy of nausea assessment we used the visual analogue scale (VAS).9 According to VAS, the number "zero" indicates that the patient does not feel any nausea and number "10" represents that the most severe level of nausea perception. [ 0-1 (no nausea), 1-4 (mild) , 4-7 (moderate) and 7-10 (severe)] .Vomiting was estimated based on frequency of emetic episode per 24 hours.[1-2 (mild) , 3-4 (moderate) and more from 5 (severe)]. The patients with persisted nausea for more than 20-30 min received Ondansetron 4 mg and were excluded the study. Also, shoulder pain in recovery room and 4 hours after the surgery were examined. Statistical considerations: The power of this study was considered 80%. Statistical analysis was performed using SPSS 19. The results were shown as mean ± standard deviation (SD) and 95% confidence intervals (CIs) of differences were calculated. We used Fisher exact and Chi Square tests to compare the variables between two groups. P < 0·05 was considered significant. BODY.RESULTS: There was no significant difference between two groups based on demographic characteristics and surgical data, (P>0.05) (Table-I). The frequency of nausea and vomiting in patients at hours 0-4, 4-8, 8-12, 12-24, and after the surgery did not demonstrate statistically significant difference between LP and SP groups (P<0.05) (Tab2&3). In this study, the total dose of antiemetic medication (Ondansetron) used for moderate or severe nausea and vomiting were 30.65 and 34.80 mg respectively. Regarding the shoulder pain, the patients were questioned while leaving the recovery room and 4 hours after the operation. When leaving the recovery room, 5 and 12 patients in the LP and SP groups complained of shoulder pain, the difference was statistically significant (P> 0.023). Furthermore, 4 hours after the surgery, 3 and 9 patients in the LP and SP groups complained of shoulder pain, and the difference was significant (P> 0.005). (Fig.1) BODY.DISCUSSION: Open surgery has been substituted with laparoscopic cholecystectomy in the cholecystolithiasis treatment and it is now the gold standard in the treatment of benign gallstone disease.10,11 The risk of intraoperative injury during laparoscopic cholecystectomy is higher than open cholecystectomy.12 Laparoscopic surgery as a minimal invasive intervention requires pneumoperitoneum for adequate visualization and operative manipulation. Carbon dioxide is considered as the most frequent gas used for creating pneumoperitoneum because of high diffusibility and quick absorption and excretion.13 The use of CO2 in laparoscopy evokes local and systemic effects.14 Insufflations of CO2 into the peritoneum may change the acid-base balance, cardiovascular and pulmonary physiology, and in high risk patients they may elevate the postsurgical complications.7 Although the use of a proper gas pressure decreases the side effects, the reduction of gas pressure may interfere with the visualization of surgeons during the operation.5 Table-I Demographic characteristics of patients and surgical data Low pressure gas Standard pressure gas P Age (yr) 45.1±12.3 42.5±16.4 0.685 Weight (kg) 64.3±8.5 66.7±10.2 0.869 Duration of anesthesia (min) 145.25±20.5 138.30±18.8 0.532 Duration of surgery (min) 121.3±13.4 107.5±10.4 0.758 Pneumoperitoneum time (min) 101.8±9.2 93.4±11.4 0.256 Table-II The frequency of nausea after surgery with low and standard pressure gas Time Gas pressure Mild nausea Moderate nausea Severe nausea P 0-4 Low Standard 4 4 4 7 1 3 0.500 8-4 Low Standard 3 4 1 3 0 1 0.160 8-12 Low Standard 3 5 2 3 1 2 0.269 12-24 Low Standard 0 0 3 3 0 2 0.351 Table-III The frequency of vomiting after surgery with low and standard pressure gas Time Gas pressure Mild vomiting Moderate vomiting Severe vomiting P 0-4 Low Standard 2 2 1 2 0 1 0.702 8-4 Low Standard 3 2 0 2 0 0 0.356 8-12 Low Standard 0 1 0 1 0 0 0.490 12-24 Low Standard 0 0 0 0 0 0 0.875 Fig.1Comparison of low and standard pressure gas on shoulder pain at recovery room (1) and after 4 hours (2). PONV is frequent among the patients undergoing laparoscopic cholecystectomy. It can be very stressful to the patients, occasionally more than the operation itself.2 CO2 insufflations during laparoscopic surgery, causes peritoneal tension and irritation, playing main role in PONV.15 The first step in preventing of PONV after laparoscopic cholecystectomy is to decrease the patient risk factors, surgical intervention, anesthetic method, and postoperative cares.4 Different mechanisms have been mentioned for PONV after laparoscopic surgery. One of them is the dilation of brain vessels by CO2 increasing intra cranial pressure (ICP) and consequently the possibility of nausea and vomiting.16 Clinical and laboratory studies have revealed that ICP elevation is not directly relevant to the hemodynamic changes caused by the increased intra-abdominal pressure. The possible mechanism of increased ICP associated with insufflation may be impaired venous drainage of the lumbar venous plexus following increased intra-abdominal pressure.17 PONV is affected by many factors which related to patients, surgery and anesthesia. These variable items requires 5-Hydroxytryptamin (5.HT) secretion in a cascade of events concluding both the central nervous system and Gastro-intestinal tract.18 In addition, serotonin may play a role in pathogenesis of PONV. Various other mechanisms have been proposed for PONV after laparoscopic, including the effect of CO2 on the occurrence of ischemia in visceral tissues.19 PONV is decreased with the administration of high concentration oxygen,20 fluid therapy,21 and sympathomimetic agents.22 Likewise Propofol usage for anesthesia preservation has a positive effect on PONV reduction when N2O is administered as the unique anesthetic agent, it is recognized to cause of PONV. N2O can also be a reason of PONV because of change in middle ear pressure and bowel distension after diffusion in to closed cavities.23 In the present study, the use of low pressure gas instead of standard pressure gas could not significantly reduce the frequency of PONV after the surgery. Additionally, the duration of anesthesia, surgery and pneumoperitoneum were higher in the LP group; however, there were no significant differences. More prolonged duration of surgery was due to the reduction of visibility of the surgeon. Kim et al. compared the low gas pressure with the standard gas pressure on PONV after laparoscopic gynecologic surgery. They reported that the use of low pressure CO2 was not effective in reducing the incidence and severity of PONV.5 In a study by Goll et al. administration of 80% oxygen during surgery was able to reduce the incidence of nausea and vomiting in gynecologic surgery.24 Researchers have illustrated that using CO2 to create pneumoperitoneum reduce 54% in the blood stream of the organs when using of intra- abdominal gas pressure (IAP) 15 mmHg instead of IAP 10 mmHg.25 A broad spectrum of adverse effects results from uncontrolled postsurgical pain.26 In our study shoulder pain after the surgery was assessed. When leaving the recovery room 20% and 48% of the patients experienced the shoulder pain in LP and SP groups respectively. Also, after 4 hours of surgery, 12% and 36% of the patients respectively complained the shoulder pain in LP and SP groups. Esmat et al. indicated that reduction of (CO2) pressure and the use of normal saline into the peritoneal membrane could reduce the frequency, percentage of shoulder pain after surgery in different hours after the operation from 35.2% to 18.4%.27 The present study had some limitations. We did not measure parameters involved in nausea and vomiting, including the amount of blood supply of the organs and tissue oxygenation. To obtain the most appropriate gas pressure, we suggest the assessment of gas pressure with less or higher rate than the standard range. BODY.CONCLUSION: In this study, the use of low pressure CO2 gas compared to standard pressure one to create pneumoperitoneum could not reduce nausea and vomiting after surgery. However, the frequency of shoulder pain in the low pressure gas group was reduced. Our findings demonstrated that during the operation, low pressure CO2 pneumoperitoneum was associated with problems including the reduction of surgeon visibility and subsequently more prolonged surgery duration.

TITLE: The Effects of Levosimendan and Sodium Nitroprusside Combination on Left Ventricular Functions After Surgical Ventricular Reconstruction in Coronary Artery Bypass Grafting Patients ABSTRACT.OBJECTIVE:: The aim of our study was to research the effects of levosimendan (LS) and sodium nitroprusside (SNP) combination on systolic and diastolic ventricular function after coronary artery bypass grafting (CABG) who required endoventricular patch repair (EVPR). ABSTRACT.PATIENTS AND METHODS:: We studied 70 patients with ischemic dilated cardiomyopathy. LS and SNP combination was administered in 35 patients (study group, SG). In the remaining patients, normal saline solution was given (placebo group, PG). Levosimendan (10μgr/kg) started 4 h prior to operation and we stopped LS before the initiation of extracorporeal circulation (ECC). During the rewarming period, we started again levosimendan (10μgr/kg) in combination with SNP (0.1-0.2 μgr/kg/min). If mean blood pressure decreased by more than 25% compared with pre-infusion values, for corrected of mean arterial pressure, the volume loading was performed using a 500 ml ringer lactate. Hemodynamic variables, inotrophyc requirement, and laboratory values were recorded. ABSTRACT.RESULTS:: Five patients died (7.14%) post-surgery (one from SG and 4 from PG) due to low cardiac out-put syndrome (LOS). At the postoperative period, cardiac output and stroke volume index was higher in SG (mean±sd;29.1±6.3 vs. 18.4±4.9 mL/min−1/m−2 (P<0.0001)). Stroke volume index (SVI) decreased from 29±10mL/m2 preoperatively to 22±14mL/m2 in the early postoperative period in group 1. This difference was statistically significant (P=0.002). Cardiac index was higher in SG (320.7±37.5 vs. 283.0±83.9 mL/min−1/m−2 (P=0.009)). The postoperative inotrophyc requirement was less in SG (5.6±2.7 vs. 10.4±2.0 mg/kg, P< 0.008), and postoperative cardiac enzyme levels were less in SG (P< 0.01). Ten patients (28.5%) in SG and 21 patients (60%) in PG required inotrophyc support (P<0.001). We used IABP in eight patients (22.8%) in SG and 17 patients (48.5%) in CG (P=0.0001). ABSTRACT.CONCLUSION:: This study showed that LS and SNP combination impressive increase in left ventricular systolic and diastolic functions including LVEF. The use of this combination achieved more less inotrophics and IABP requirement. We therefore suggest preoperative and peroperative levosimendan and SNP combination. BODY.INTRODUCTION: Left ventricular aneurysm and cardiomyopathy remains a serious disorder that can lead to intractable congestiveheart failure (CHF), anddeath. The goal of surgical interventionis to correct the size and geometry of the LV, reduce wall tension, and improve ventricular systolic function [1]. EVPR was desribed as a more physiologicrepair than the linear closure technique by Dor and Cooley [2, 3]. Mickleborough and co-workers have introduced a method combining septoplasty and linear resection [4]. Pharmacologic therapies for reducing myocardial afterload and preload has been recommended to induce myocellular and molecular reverse remodeling after surgery [5-7]. However, EVPR may be cause of detrimentally affect on LV diastolic properties [8]. Therefore, pharmacologic treatment options are very important after surgery. Levosimendan, as a calcium sensitizer, is used in cardiac surgery to provided the myocardial preconditioning and vasodilating effects without the intracellular calcium accumulation during the ischemia-reperfusion injury. Also, previous investigations have shown that SNP infusion reduces ventricular pre- and afterload after myocardial ischemia by favorably influencing the balance between myocardial oxygen supply and demand [9, 10]. SNP has also suggested for left ventricular relaxation by causing reflex changes in the autonomic nervous system and stimulating catecholamine release. Previous investigation by Freyholdt et al. showed that a SNP infusion improved the cardiac index immediately after reperfusion and at the end of surgery [9]. SNP also reduced the cardiac inflammatory response during CABG in patients with severely compromised left ventricular function [10]. According to previous data, SNP may be capable of influencing the myocardial relaxation process under hypoxic conditions and may alter viscoelastic properties of the left ventricle after SVR. To provide dentrimental effects of EVPR we hypothesised that pharmacologic preconditioning may be help to increase left ventricular systolic amd diastolic functions. To research of the combined drugs effects on left ventricular functions we used LS and SNP for preoperative pharmacologic preconditioning. BODY.MATERIALS AND METHODS: After the approve of ethics committee, we studied 70 CABG patients with LVA who required EVPR between January 2007 and July 2014. All patients have had previous one or more myocardial infarction. The preoperative patients' charecteristics are summarized in Table 1. Criteria for exclusion included a planned concomitant valve procedure, emergency surgery, history of persistent ventricular tachycardia or obstructive cardiomyopathy, myocardial infarction within 72 h before surgery, preoperative inotropic support, or use of an intraaortic balloon pump at the time of surgery. Indications for EVPR were heart failure, angina or a combination of these symptoms. Stroke volume was defined as the difference between end-diastolic volume (EDV) and end-systolic volume (ESV). LV volume measurements were indexed to body surface area and expressed as end-diastolic volume index (EDVI), end-systolic volume index (ESVI), and stroke volume index (SVI). Preoperatively, angiocardiography including left ventricular end-diastolic pressure was calculated. In SG, levosimendan (10μg/kg) started 4 h prior to operation. We continued levosimendan administration through central venous catheter until ECC. During the rewarming period, we started levosimendan infusion again (10μgr/kg in combination with SNP (0.1-0.2 μgr/kg/min.).). If mean blood pressure decreased by more than 25% compared with pre-infusion values, the volume loading was performed using a 500 ml ringer lactate for correction of arterial pressure. CG received placebo infusion (0.9% of normal saline) in accordence with LS and SNP infusion protocol. In both groups, echocardiographyc examination was done before the discharge from hospital. BODY.SURGICAL PROCEDURE: After midline sternal insicion, aortic and caval cannulation was performed and CPB was instituted. Cardiac arrest has been provided antegrade and retrograde cold blood cardioplegic solution every 20 min. An aneurysmal suc has been opened and resected in all patients. If there was a thromby, we removed by carefully attention. A pursestring suture was placed around the circumferential scar tissue at the transition zone and a circular Dacron or pericardial patch was secured over the opening after a pursestring suture was tied. If there was a significant septal lesion in addition to the anterior-apex lesions, we used a septal anterior ventricular exclusion procedure after a long left ventriculotomy was made along the left anterior descending artery, an elliptical-shaped Dacron patch was sutured to the transition zone to exclude the akinetic region after broad anteroseptal infarction. Coronary bypass anastomosis were performed using a saphenous vein and a left internal thoracic artery (LITA). During rewarming period, in SG, SNP and levosimendan was administered via central venous line at a dose of 0.1-0.2 mg/kg/min., and 5-10μg/kg, respectively. In PG, normal saline solution was given. Proxymal anastomosis were completed using a side clamp. We continued Levosimendan and SNP in ICU for 24 h. The characteristics of surgical procedures are summarized in Table 2. For all patients angiotensin-converting enzyme inhibitor, β-blocker, and diuretics were used. Medication and basic features of the surgical procedures were comparable. There were no differences in heart rate that might have influenced SVI changes after SVR. BODY.STATISTICAL ANALYSIS: Continuous variables are expressed as mean ± standard deviation. Categorical variables are presented as percentages. The Student t test was used to compare preoperative and postoperative continuous variables, the chi-squared test and Fischer's exact test were used for analysis of categorical variables. Univariate regression analysis was used to determine factors associated with early hospital mortality and a low cardiac output state. Survival curves were calculated according to the method of Kaplan - Meier and subgroups were compared using the Log-rank test. Soft ware version 13.0 for Windows was used in data analysis. A significant difference was considered at P<0.05. BODY.RESULTS: Five patients died after operations due to LOS (Four in PG (11.4%), and one in SG (2.8%). This was statistically significant (P<0.001). The mortality risk factors were duration of ECC (exceeding 220 min. (P1=0.001)) and aortic cross clamping (exceeding 100 min.) (P2=0.001). Inotrophyc support was administered due to LOS in ten patients (28.5%) from SG, and in 21 patients (60%) in PG, respectively (P<0.001). We used IABP in eight patients in SG (22.8%) and in 17 patients (48.5%) in PG (P=0.0002). Preoperative lower proportion of New York Heart Association and older age, fibrotic LAD artery, and low EF were the main risk factors for perioperative mortality. LAD revascularization decreased the inotrophics and intraaortic balloon pump use both groups (P=0.035). But, numbers of coronary artery anastomosis was not statistically significant role of mortality rates (P=0.089). Echocardiographic measurements showed that LVEF increased statistically in all survived patients in SG. However, LVEF did not change in 15 patients (42.8%) in PG. Clinical follow-up demonstrated that the left venricular EDVI decreased in all survived patients after surgery in both groups. The LVEF increased from 34±14 to 41±5 in SG (P=0.001). However, LVEF did not change significantly. The preoperative and postoperative LVEF was 35±16; and 37±3 in, respectively in PG (P>0.05). SVI decreased from 29±10mL/m2 preoperatively to 22±14mL/m2 in the early postoperative period in PG. This was statistically significant (P=0.027). In SG patients exhibited an increase in SVI after surgery from 33±14mL/m2 to 38±5mL/m2. This was statistically significant (P<0.001). Table 2 is summarized pre- and postoperative SVI, EDVI, ESVI, LVED and LVESV in both groups in the early postoperative period. The early postoperative data including LOS, mortality rate, intraaortic balloon pump use and arrhytmia have been summarised in Table 3. The changes of left ventricular functions are summarized in Figs. (1 and 2) for SG and PG, respectively. Kaplan-Meier Survival Analyses Curve is demonstrated in Fig. (3). During 36 months follow-up, five patients died due to cardiovascular events. Statistical significance has been detected when compared to SVI in the early postoperative period. Post-surgery echocardiographyc examinations showed that there was no any statistical difference between the groups (P=0.687). The left ventricular end-diastolic and end-systolic dimension were also similar in both groups. The greater reduction in EDVI and greater increase in EF in early period after surgery have been detected in patients who treated with levosimendan and SNP. According to our results LVEFs were highly correlated to changes in EDVI in SG group. In three times hemodynamic changes has been summarised in Table 4. Univariate and multivariate death from low-output syndrome after surgery has been summarised in Tables 5 and 6, respectively. Long aortic cross-clamp time and concomitant CABG were statistically significant morbidity and mortality factors. We analysed perioperative and postoperative hemodynamic changes over time. Cardiac out-put and CI values were significantly higher in levosimendan group (Table 7). BODY.LEFT VENTRICULAR SIZE AND FUNCTIONS IN LONG-TERM FOLLOW-UP: In survived patients, echocardiograms were available in the first week post-EVPR, and a median of 6, 4 months (range 3-25 months). Five patients died because of cardiovascular events during the follow-up period (three patients from PG and, two patients from SG). The remaining 60 patients did not differ in clinical and hemodynamic profiles when we compared echocardiographyc controls. Patients in whom SV decreased initially had larger SV at baseline compared with those in whom SV increased initially; they also had larger EDVI and ESVI and greater EF. According to Marui et al., our research showed that the SVI reduction was temporary, and patients who have lower SV showed recovery during the follow-up, so that at the time of chronic follow-up there was no significant difference in SV between the two groups [11, 12]. However, the reduction in EDVI and ESVI persisted along time in the PG. These findings indicate that different baseline loading conditions may affect the response of cardiac function to SVR in SG. BODY.DISCUSSION: Postoperative ventricular failure is a life threatining problem in CABG patients. Impairment of ventricular function produces a reduction in peripheral oxygen transport, which, in turn, leads to a prolonged stay in the ICU. Myocardial β-adrenergic receptor desensitization occurs chronically in patients with congestive heart failure and acutely after ECC, thereby limiting the efficacy of β-adrenergic stimulants for post-bypass cardiac failure. We presented the effects of levosimendan and sodium nitroprusside combination in CABG patients who required EVPR. Our research demonstrated that older age, a low LVEF as independent risk factors. Also, our study was clearly showed that NYHA class III and IV status, longer aortic cross clamp and total ECC time were the most important factors of early mortality in CABG patients who underwent EVPR. To the best of our knowledge, for the first time, we investigated the effects of the use of levosimendan and SNP combination on left ventricular systolic and diastolic functions including SVI, EDVI, ESVI and LVEF in our CABG patients who have LF aneurysm requiring surgical ventricular reconstruction. Levosimendan is a calcium sensitizers, that increasemyocardial contractility without increasing intracellular calcium, has emerged as an inotropic agent. LS has been used for myocardial preconditioning without the intracellular calcium accumulation during the ischemia-reperfusion injury [13]. Because of adenosine triphosphate (ATP) production, LS provides coronary artery dilationand myocyte mitochondrial activation. These beneficial effects work synergistically with calcium sensitization to ventricular myocardial improvement [13]. Tritapepe et al. reported that levosimendan pre-treatment improves outcomes in patients undergoing CABG patients [14]. Ikonomidis et collegues showed that treatment with LS improved coronary artery flow and microcirculation in parallel with an improvement in cardiac performance and neuro-hormonal activation in patients with advanced heart failure [15]. Levin's and Malliotakis's study demonstrated that levosimendan decreased the mortality in high risk patients undergoing CABG with impaired left ventricular function [16, 17]. Our previously published study exhibited that levosimendan decreased atrial fibrillation after CABG [18]. Sodium nitroprusside could potentially influence the left ventricular relaxation by several mechanism in patients with myocardial ischemia [19]. We hypothesised that after EVPR the left ventricular volume may be decreased and diastolic filling rates fell significantly during LV diastole, and did not change and fell significantly, during the last third of diastole. Therefore, these changes in filling rates may contribute to the shifts in the diastolic pressure-volume relation seen in our patients when we administer SNP and levosimendan for ventricular preconditioning. Our study showed that the ventricular diastolic filling rates increased slightly with our pharmacologic approachs in CABG patients who underwent EVPR. The results of our study showed that EF and resting SV was more frequently increased after SVR in SG. In fact, in data of PG cohort showed an average 8 mL/m2 (approxymately 14%) decrease in resting SV. Clinical studies have showed EVPR frequently decreased EDV [17-20]. Vasodilating effects of SNP and its positive impact on afterload after cross clamping increased the LVEF, EDV, and ESV in SG. Our results demonstrated preoperative levosimendan preconditioning and SNP use directly affected on index of LV pump function. Our study demonstrated that administration of LS combined with SNP increased ventricular relaxation and LVEF after EVPR in dilated ischemic cardiomyopathy. LS and SNP decreased intraaortic balloon pump and inotropic agent requirement. LS and SNP may influence the left ventricular systolic and diastolic pressure-volume relationships. Likewise, the pressure and blood flow in the coronary arteries and their intramural branches could affect the compliance of the left ventricular wall and influence the pressure-volume curve on this basis. Overall, our estimates of mean postoperative SVR changes in SV reported in the literature range from an increase of 15 mL to decreases of 15 mL or greater with, as noted above, a majority of studies showing reductions [21, 22]. BODY.DISCUSSION.STUDY LIMITATIONS: Our data set concerning the impact of EVPR on left ventricular size and function was in the early postoperative period, with a smaller database. Comparison of resting LV size and function between preoperative and early postoperative conditions provides important information. The previous study confirmed that important specific factor was defined as ventricular filling charecteristics and the LV systolic and diastolic properties. The left ventricular clarification such as ventricular diastolic properties associated with better hemodynamic effects of SVR and links between hemodynamic effects and clinical outcomes could help define appropriate patient selection criteria for these pharmacologic approachs and surgical procedures. Despite our limitations, the current results advocate routine use of levosimendan protocol in patients with low LVEF undergoing OPCABG surgery.

TITLE: Comparison of recovery profiles of propofol and sevoflurane anesthesia with bispectral index monitoring in percutaneous nephrolithotomy ABSTRACT.BACKGROUND: The aim of the study was to evaluate the comparative effects of propofol infusion versus sevoflurane for maintenance of anesthesia with respect to hemodynamics, recovery characteristics, nausea and vomiting in patients undergoing percutaneous nephrolithotomy. ABSTRACT.METHODS: Forty American Society of Anesthesiologists physical status I-II patients, aged between 22 and 65 years were randomly divided to receive either intravenous anesthesia with propofol (group P) or sevoflurane (group S). Cardiovascular variables, peripheral oxygen saturation (SpO2), end-tidal carbon dioxide (ETCO2), bispectral index (BIS) and train-of-four (TOF) values were recorded at intervals throughout the procedure. Time to spontaneous respiration, eye opening, extubation, obey commands, hand squeezing, Aldrete Score > 9 and the incidence of postoperative nausea and vomiting were recorded. ABSTRACT.RESULTS: Early recovery times [spontaneous respiration (P = 0.002), eye opening (P = 0.006), extubation (P = 0.013), obey commands (P < 0.05), hand squeezing (P = 0.005)] were significantly longer in group P. The incidence of vomiting was significantly higher in group S (P < 0.05). Hemodynamic parameters, levels of SpO2, ETCO2, and BIS and TOF values were not significantly different between the groups (P > 0.05). ABSTRACT.CONCLUSIONS: The present study which adjusted sevoflurane concentration and propofol infusion rate according to BIS values revealed that maintenance of anesthesia with sevoflurane is associated with faster recovery than anesthesia with propofol. Propofol resulted in a significantly lower incidence of postoperative nausea and vomiting. Hemodynamic parameters and levels of SpO2 and ETCO2 were comparable between the groups during percutaneous nephrolithotomy. BODY.INTRODUCTION: Percutaneous nephrolithotomy (PCNL) is a less invasive alternative procedure to open surgery for removal of medium-sized or larger renal calculi from the urinary tract using a nephroscope into the kidney through a track created in the patient's back. This procedure is advantageous in that it reduces the duration of hospitalization, results in less morbidity and postoperative pain, and minimal scarring [1]. However, hemodynamic changes may occur during the procedure because continuous irrigation of the kidneys may cause excessive fluid absorption and increase hydrostatic pressure [2,3]. Also, continuous irrigation makes monitoring the amount of bleeding not possible. Significant bleeding may occur during the procedure and maintenance of hemodynamic stability may be too difficult. For this reason, choosing an anesthetic agent is very critical, because it must have a minimal effect on hemodynamic parameters. There are a limited number of studies about hemodynamic responses of anesthesia on this procedure. Therefore, the present study investigated the hemodynamic changes and recovery of this procedure and the results were compared with other studies [4-7]. Hemodynamic changes may also be caused by inadequate anesthesia levels in patients undergoing operation. It is important to know which is responsible for this condition: significant bleeding or inadequate anesthesia. If it is due to bleeding crystalloid, colloid or blood replacement should be performed. However, if it is from inadequate anesthesia, the depth of anesthesia should be reevaluated. During the last decade, an increasing number of monitoring systems have been designed to estimate the depth of anesthesia. One of these systems is the bispectral index (BIS) monitor; different studies have shown that BIS may help to assess the hypnotic component of anesthesia, reduce drug consumption and shorten recovery time compared with the standard practice protocol [8-11]. Propofol and sevoflurane are anesthetic agents known to have minimal effects on hemodynamic parameters [12-16]. The aim of the study was to determine a more suitable anesthetic for better maintenance in terms of hemodynamic characteristics and recovery profile for the duration of PCNL which bears the difficulty of uncontrollable bleeding for anesthesiologists. Also, unlike previous studies, this study was intended to compare propofol and sevoflurane by adjusting the depth of anesthesia according to BIS. BODY.MATERIALS AND METHODS: After obtaining Institutional Medical Ethics Committee approval and written informed consent, 40 American Society of Anesthesiologists physical status I-II patients aged between 22 and 65 years undergoing general anesthesia for elective PCNL were enrolled in the study and randomly assigned (by opening of a sealed envelope) into two groups receiving sevoflurane (group S) or propofol infusion (group P) for maintenance of anesthesia. Patients who had cardiovascular, renal, hepatic or endocrine disorders were excluded from the study. In the operating room after establishment of standard monitoring, arterial and venous cannulation, the skin of the forehead was degreased with 70% isopropanol; a BIS electrode (Aspect Medical Systems, Norwood, MA, USA) was positioned as recommended by the manufacturer. Impedances were measured to ensure optimal electrode contact defined as ≤ 7.5 kΩ for the BIS as required by the manufacturer. EEG was continuously recorded using a BIS monitor (Aspect 2000 XP BIS monitor, P/N 185-0070, Host Rev. 3.12, Aspect Medical Systems, Newton, MA, USA). All patients were then premedicated with midazolam 0.03 mg/kg and fentanyl 1 μg/kg intravenously ten minutes before induction. Propofol (2 mg/kg) was used for induction in all patients. After induction, skin surface electrodes for neurostimulation were placed on the volar forearm along the course of the ulnar nerve, close to the proximal wrist crease to stimulate adductor pollicis. The other ends of electrodes were connected to a respective train-of-four (TOF) Guard acceleromyography monitor (TOF Watch SX, Organon-Teknika, Boxtel, Netherlands). Nerve was stimulated with TOF stimulation (a series of four twitches in 2 sec, 2 Hz frequency, each 0.2 ms long) every 12 seconds after loss of the eyelash reflex. A current intensity of 50 mA was used. Intubation was performed when all four TOF responses from the adductor pollicis muscle were disappeared. Vecuronium (0.1 mg/kg for tracheal intubation; thereafter 2 mg as needed) was used as a neuromuscular blocking agent for both groups. After this, all patients were manually ventilated by an oxygen mask for 2 minutes and then were intubated. Patients in each group received intermittent positive pressure ventilation with N2O 50% in an oxygen mixture via a rebreathing system at a tidal volume of 6-8 ml/kg and a frequency of 12 per minute with an end tidal CO2 (ETCO2) target of 30-35 mmHg. Continuous monitoring included heart rate, invasive measurements of systolic arterial blood pressure, oxygen saturation (SpO2), ETCO2, BIS and TOF values. The criteria for supplementary doses of vecuronium were assessed according to TOF monitoring. For maintenance of anesthesia, propofol infusion at 3-12 mg/kg/hr or 0.5-2 vol% of sevoflurane was administered. The concentration of sevoflurane used and the infusion rate of propofol were adjusted according to an EEG target value of 40-60 for BIS. BIS values were continuously recorded at intervals of 10 minutes. If BIS values were over 60, the infusion rate of propofol or concentration of sevoflurane was gradually increased. If was under 40, the infusion rate of propofol or concentration of sevoflurane was decreased. In the case of more than 20% reduction in the mean arterial pressure from baseline levels, the infusion rate of crystalloid solution was increased. If this was not sufficient, the infusion rate of propofol or the concentration of sevoflurane was reduced. If hypotension was due to bleeding, colloids and blood were administered. Severe hypotension was finally treated with a vasopressor. About fifteen minutes before the end of surgery, sevoflurane and propofol were reduced to facilitate rapid emergence from anesthesia. They were adjusted to a BIS value of 70. Hemodynamic parameters, SpO2 and ETCO2 levels were noted before induction, before and after intubation, before prone position and at 5, 10, 30, 60, 90 and 120 minutes after prone position, and before extubation and at 5, 10, 30, 60, 90 and 120 minutes after extubation. BIS values were measured and recorded every 10 minutes. The time from discontinuation of drugs to eye opening in response to verbal stimulus, hand squeezing, spontaneous ventilation, tracheal extubation, stating name, stating date of birth, time to Aldrete score > 9, nausea and vomiting were recorded. Anesthetic agents were discontinued when the skin incision was being sutured. After suturation, the patient was turned into the supine position. When neuromuscular recovery was completed and adequate depth of breathing was reached (after the return of adequate ventilator drive, tidal volume > 8 ml/kg, respiratory rate > 12/min, normal breathing patterns and good oxygenation [SpO2 > 98%]) and the patient could obey the verbal commands ("open your eyes", "elevate your head"; commanded every 30 seconds), tracheal extubation was done. The adequacy of recovery following reversal was assessed according to TOF monitoring. A TOF ratio of greater than 90% was correlated with the criteria of adequate clinical recovery. Nausea and vomiting were evaluated every fifteen minutes after extubation according to the score of nausea and vomiting (0 = no emetic symptoms, l = nausea, 2 = vomiting). Ondansetron (0.1 mg/kg i.v.) was only used for patients with a score of 2. Warmed blankets were used in the recovery room. For postoperative pain relief, meperidine 0.5 mg/kg was administered to all patients intravenously just before the anesthetic agents were discontinued as part of the standard analgesic protocol of our institute. NCSS for windows NCSS 2007 software (NCSS LLC, Kaysville, UT, USA) was used for statistical analysis. Repeated measures of ANOVA were used to determine differences overtime, and between multiple time periods for each anesthetic agent. Post hoc Newman-Keuls multiple comparison analysis was used. An unpaired t-test was used in each anesthetic agent group. A chi-square test was performed for the evaluation of available data. The statistically significant level was established at 0.05. BODY.RESULTS: There were no significant differences between the two groups regarding age, body weight, gender and duration of surgery (P > 0.05) (Table 1). The measurements of hemodynamic parameters were statistically similar between group P and group S (P > 0.05). A significant rise and fall was observed in each group during the measurement of mean arterial pressure and heart rate in the course of operative period. Although there were statistically significant differences between baseline values and intraoperative hemodynamic parameters, all values were within acceptable limits. All significant changes in groups P and S in addition to the differences compared to baseline values of each group are graphically represented in Fig. 1 and 2. BIS values were extremely stable and similar between the two groups throughout the study (P ≥ 0.05). There were significant differences in recovery time after anesthesia with sevoflurane versus propofol. Early recovery time after sevoflurane anesthesia was significantly shorter than propofol infusion except Aldrete recovery scores (Table 2). The incidence of nausea and vomiting was significantly higher in group S compared to group P (P < 0.05). One patient in group P and 4 patients in group S had nausea. Vomiting was not observed in any patient in group P but in 3 patients in group S; these patients were treated with ondansetron 0.1 mg/kg intravenously. VAS score was ≤ 4 in all patients in both groups. BODY.DISCUSSION: Percutaneous nephrolithotomy is widely used for the treatment of renal stones. It is a relatively less invasive endoscopic intervention compared to open surgery [17]. Although it is accepted as a safer method, irrigation of the kidneys and drugs used for anesthesia may cause hemodynamic disturbances [4-7]. A recent study was designed to determine cognitive and clinical outcomes after sevoflurane compared with propofol-based anesthesia for on-pump cardiac surgery. Patients assigned to sevoflurane-based anesthesia were associated with better results in all cognitive tests than those in the propofol group. There was no difference in general clinical outcome [14]. Robinson et al. [18] reported a more rapid recovery following sevoflurane anesthesia than that of propofol. Gupta et al. [19] reported that no time difference was found in eye opening time between sevoflurane and propofol in their systematic review, but the time period to obeying commands was faster in the sevoflurane group. Postoperative nausea and vomiting were significantly greater with sevoflurane compared with propofol. In the present study, almost all recovery events following anesthesia including eye opening, hand squeezing, spontaneous ventilation, extubation, stating name and stating date of birth were achieved earlier in the sevoflurane group. Wandel et al. [20] reported that following general anesthesia, patients who received sevoflurane for maintenance could be extubated earlier and regained cognitive functions much earlier than those on propofol infusion. In some other studies, investigators observed a shorter recovery time in patients given sevoflurane anesthesia, which is similar to the results of our study [21,22]. Samantaray and Rao [23] observed that maintenance of anesthesia with sevoflurane was associated with faster recovery than propofol anesthesia in patients who underwent spine surgery, and sevoflurane was associated with postoperative nausea and vomiting as with any other inhalational anesthetic. According to the results of a previous study, there was a significant decrease in heart rate during PCNL in the propofol group [4]. This decrease can be related to the use of alfentanil. In the present study, only N2O was used and no decrease in heart rate was observed in the propofol group. Opioid supplementation or increased delivery of anesthetic agents instead of N2O has resulted in delayed awareness and recovery [24]. Several investigators found that recovery was more rapid, the incidence of postoperative nausea and vomiting was lower, and peroperative hemodynamic stability was better in the propofol group compared to the sevoflurane group [25-27]. A recent study compared the incidence and degree of postoperative nausea and vomiting in patients who received general anesthesia with propofol and those with sevoflurane. The propofol group had a statistically lower incidence of post-operative nausea and vomiting [15]. This finding is in accordance with the current study. Small and clinically unimportant differences were found by some investigators in recovery time between the two groups of patients who received propofol or sevoflurane [28,29]. On the contrary, Husedzinović et al. [30] did not find any significant difference in hemodynamic parameters between propofol and sevoflurane groups in patients undergoing open cholecystectomy. In endoscopic procedures, it is obligatory to control blood pressure in order to reduce bleeding and thus improve the surgeon's view of the operative field. As endoscopic procedures end rapidly after removal of the endoscope, the anesthesiologist has to ensure an adequate level of anesthesia until completion of endoscopic maneuvers but at the same time must prevent prolonged recovery time at the end of the surgery. In the present study, an adequate level of anesthesia and hemodynamic stability were achieved simply by the BIS monitor system. Also, BIS monitor system helped to shorten the duration of recovery. In previous studies which compared the effects of sevoflurane and propofol infusion anesthesia on hemodynamic changes and recovery, BIS monitors were not used and the depth of anesthesia was not monitored objectively [4,8,14,18-23,25-30]. However, some of the changes in cardiovascular parameters might have been associated with the depth of anesthesia. Also, in some patients, the level of anesthesia might be deeper at the end of the surgery and the recovery time might be longer. In the current study, the depth of anesthesia was maintained at the same level for the two anesthetic agents and the effects of the anesthetics on hemodynamic parameters and recovery were assessed under similar circumstances. Introduction of more rapid and short-acting volatile anesthetics and intravenous anesthetics has allowed anesthesiologists to achieve a more consistent recovery profile that facilitates fast-tracking after general anesthesia. Anesthetic techniques that optimize intraoperative surgical conditions while providing rapid, early recovery have assumed increased importance. Cicek et al. [5] compared the effects of propofol-alfentanil and propofol-remifentanil anesthesia on hemodynamic factors and recovery characteristics during PCNL. Both groups provided stable hemodynamic parameters during PCNL, whereas time to recovery of spontaneous ventilation, extubation and eye opening were significantly shorter in the propofol-remifentanil group than the propofol-alfentanil group. In a previous report, inhalation anesthesia with sevoflurane and N2O, and total intravenous anesthesia with propofol and alfentanil was found to be effective in controlling mean arterial pressures during PCNL. The mean heart rate was lower during PCNL in the propofol-alfentanil-N2O group compared with the sevoflurane group (P < 0.01). The mean systolic and diastolic arterial pressures were not different between both groups at any stage of measurement (P < 0.05). In the sevoflurane group, the concentrations of renin, aldosterone and adrenocorticotrophic hormone were significantly higher compared with the propofol-alfentanil-N2O anesthesia group (P < 0.05) [7]. In our study, although there were changes in intraoperative hemodynamic parameters within the groups, all the values were in the acceptable limits and there was no significant difference between the groups. In conclusion, recovery time of sevoflurane anesthesia was significantly shorter than propofol infusion anesthesia in the current study, and the incidence of nausea and vomiting was significantly higher in the sevoflurane group compared with the propofol infusion group. These results collectively show that both sevoflurane and propofol are convenient anesthetic techniques for percutaneous nephrolithotomy. Both anesthetics achieved circulatory stability, rapid titration in relation to clinical needs, acceptable surgical field and fast recovery. Faster recovery time of sevoflurane is especially useful for endoscope treatments in which the surgical procedure ends abruptly.

TITLE: Effects of Daikenchuto on Abdominal Bloating Accompanied by Chronic Constipation: A Prospective, Single-Center Randomized Open Trial ABSTRACT.BACKGROUND: Daikenchuto (DKT), a traditional Japanese herbal medicine, is widely used for treatment of gastrointestinal disorders. We evaluated the efficacy and safety of DKT for abdominal bloating in patients with chronic constipation. ABSTRACT.OBJECTIVE: To evaluate the efficacy and safety of DKT for the treatment of abdominal bloating. ABSTRACT.METHODS: After discontinuing as-needed use of laxatives, 10 patients received oral DKT for 14 days (15 g/d). To evaluate small intestinal bacteria overgrowth (SIBO), a glucose breath test was performed before and after treatment with DKT. Before beginning the treatment, 4 patients (40%) had a diagnosis of SIBO based on a positive glucose breath test result. In both the SIBO and non-SIBO groups, bowel movement frequency and stool form remained unchanged after DKT treatment. ABSTRACT.RESULTS: For all patients, median total Gastrointestinal Symptoms Rating Scale score and the median Gastrointestinal Symptoms Rating Scale indigestion and constipation subscales were significantly decreased, whereas the median visual analog score for decreased abdominal bloating was significantly increased. Improvements of those symptoms were the same in both the SIBO and non-SIBO groups, indicating that DKT does not have effects on small intestine bacteria. No serious side effects were reported. ABSTRACT.CONCLUSIONS: DKT treatment improved quality of life for patients with chronic constipation regardless of the presence of SIBO and showed no effects on small intestine bacteria. UMIN Clinical Trial Registry identifier: UMIN000008070. BODY.INTRODUCTION: The main treatment modalities for patients with chronic constipation include laxatives, antiflatulence drugs, enterokinesis-regulating agents, cholinolytic drugs, and high-molecular-weight polymer administrations, as well as enemas and other methods. However, extended use of those treatments may cause abdominal pain and/or diarrhea, which can lower patient satisfaction. In patients with chronic constipation, abdominal bloating may have a serious negative influence on quality of life (QOL) to a greater degree than abnormal bowel habits and there is no standard regimen available for this distressing condition. Daikenchuto (DKT) (TJ-100), a traditional Japanese herbal medicine, has been widely used in Japan for treatment of gastrointestinal disorders such as postoperative ileus and irritable bowel syndrome. Previous studies have reported that DKT increased the level of calcitonin gene-related peptide and adrenomedullin, leading to increased intestinal blood flow in healthy subjects.1 And recent study2 revealed that DKT serves as a novel therapeutic agent for postoperative ileus characterized by its anti-inflammatory potency. DKT's anti-inflammatory action is partly mediated by activation of alpha7 nicotinic acetylcholine receptors, which in turn ameliorated gastrointestinal motility disorder in postoperative ileus in mouse.2 DKT is composed of 3 crude agents in fixed proportions: Zingiberis rhizoma (processed ginger), Ginseng radix (Panax ginseng), and Zanthoxyli fructus (Japanese pepper). It is available in the form of extracted granules that are initially prepared by decocting the 3 crude agents in a 10-fold volume of purified water and keeping the mixture at 95°C for 1 hour, after which it is filtered and spray-dried to yield an extract powder. Subsequently, maltose syrup powder is added at a ratio of 1:8, then mixed and granulated to produce the final product. The current indication of DKT is for relief of abdominal coldness and pain accompanied by flatulence. The purpose of our study was to evaluate the efficacy and safety of DKT for treatment of abdominal bloating in patients with chronic constipation. BODY.PATIENTS AND METHODS.STUDY DESIGN AND SUBJECTS: This prospective single-center randomized open trial was conducted to evaluate the efficacy and safety of DKT for abdominal bloating accompanied by chronic constipation. The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of Izumo-City General Medical Center, and registered in the UMIN Clinical Trial Registry (UMIN000008070). Study participants were enrolled from April 2011 to March 2013. Ten patients (3 men and 7 women; mean age 58.4 [19.5] years) with abdominal bloating received oral administrations (15 g/d 3 times/d before meals) of DKT (Tsumura & Co, Tokyo, Japan) for 14 consecutive days, during which any as-needed use of laxatives was discontinued (Table). Participating patients fulfilled the following criteria: abdominal bloating; able to cease use of laxatives, including on-demand use, during the study period; and liver function at less than the upper limit of normal set by our institution, determined within 6 months. Patients with organic colonic lesions or a history of total gastrectomy or colectomy were excluded from the study. Functional constipation patients were excluded, as were patients with organic causes of constipation such as diabetes mellitus, Parkinson disease, or a neurologic aliment. Patients who had taken kampo medicines, antibiotics, Lactobacillus, antacids, prokinetics, calcium polycarbophil, anticholinergics, laxatives, antidepressants, or antianxiety drugs within 2 weeks were also excluded from our study.Table 1Characteristics of enrolled patients.CharacteristicResultNumber of patients10Age, y Mean (SD)58.4 (20.6) Range34-85Gender Men3 Women7Body mass index Mean (SD)23.6 (3.8) Range18.7−31.2 BODY.PATIENTS AND METHODS.EVALUATION OF ABDOMINAL SYMPTOMS: QOL was evaluated at the baseline, then on days 7 and 14 using the Japanese version of the Gastrointestinal Symptoms Rating Scale (GSRS) and abdominal bloating and overall treatment effect were assessed using a visual analog scale (VAS) score (0–100; 0 = none). GSRS and VAS were used in evaluating feelings of fullness.[3], [4] The frequency of bowel movements, stool consistency and form (Bristol Stool Scale),5 and abdominal symptoms were evaluated using a face scale with a daily diary. BODY.PATIENTS AND METHODS.GLUCOSE BREATH TEST (GBT): To evaluate small intestinal bacteria overgrowth (SIBO), a GBT was performed before and after administration of DKT using a Gastrolyzer (Bedfont Scientific Ltd, Kent, England). Diagnosis of SIBO was defined by a change in hydrogen concentration of >10 ppm in expired air after being given 100 g glucose. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: Data for comparisons between groups were analyzed using χ2, ANOVA, and Wilcoxon's signed rank tests. Categorical data were compared using Wilcoxon's rank sum test; Student t test; or, where unequal variances occurred, Welch's test. P values < 0.05 were considered significant. All statistical analyses were performed using SPSS (version 12.0 for PC, SPSS Japan Inc. Tokyo, Japan). BODY.RESULTS: Four patients (40%) were positive for SIBO based on GBT results. In an analysis of all patients, no changes were observed in frequency of bowel movements or stool form after DKT treatment. In contrast, there was a significant decrease in median total GSRS score (Figure 1), and median GSRS indigestion and constipation subscales (ANOVA P < 0.001), whereas acid reflex, diarrhea, and abdominal pain subscales did not show significant changes (Figure 2). For abdominal bloating, the median VAS score was notably reduced from 76 to 30 (P = 0.005), whereas the score for overall treatment effect increased from 13 to 51 (P = 0.005). Furthermore, the severity of abdominal pain and bloating assessed by a face scale significantly decreased (P = 0.039 and P = 0.008, respectively) (Figure 3). Improvements in these symptoms were observed in both the SIBO and non-SIBO groups. In addition, QOL scores improved (decreased median total GSRS score, median GSRS constipation subscales, and median VAS score for abdominal bloating and increased median VAS score for overall treatment effect) in both groups. In the SIBO group, GBT results showed similar patterns before and after administration of DKT. DKT did not affect bowel movement frequency or stool form in all patients (Figure 4), nor after dividing patients into SIBO and non-SIBO groups. Furthermore the GBT results showed similar patterns before and after administration of DKT in the SIBO group and non-SIBO groups (Figure 5). No adverse effects were observed in any of the patients during the 14-day treatment period.Figure 1There was a significant decrease in median total Gastrointestinal Symptoms Rating Scale (GSRS) score.Figure 2There were significant decreases in indigestion and constipation subscales of the Gastrointestinal Symptoms Rating Scale (GSRS), whereas acid reflex, diarrhea, and abdominal pain subscales did not show significant changes.Figure 3For abdominal bloating the median visual analog score was notably reduced, whereas the score for overall treatment effect increased. The severity of abdominal pain and bloating assessed by a face scale significantly decreased.Figure 4Daikenchuto did not affect bowel movement frequency or stool form in all patients, nor after dividing into the small intestinal bacteria overgrowth (SIBO) and non-SIBO groups.Figure 5The glucose breath test results (in parts per million of hydrogen [ppm H2]) showed similar patterns before and after administration of daikenchuto in the small intestinal bacteria overgrowth (SIBO) group and non-SIBO groups. BODY.DISCUSSION: The purpose of our study was to evaluate the efficacy of DKT for abdominal symptoms in patients with chronic constipation. Functional constipation has a complex pathophysiology. Prior clinical studies found that DKT alleviated constipation in patients with Parkinson disease6 and was also effective for postoperative ileus.7 In other studies,[8], [9] DKT stimulated gastrointestinal motility in healthy subjects and decreased rectal compliance and sensation in patients with constipation.10 Although good findings have been reported, the mechanisms that generate clinical benefits related to DKT remain unidentified. A previous report noted the effects of DKT for reduction of bloating and abdominal pain in patients with chronic constipation who required the stimulant sennoside.4 Sennoside is frequently given for chronic constipation. However, protracted use of laxatives, including sennoside, increases the frequency of bowel movements and may impair patient QOL. We enrolled patients with chronic constipation who were willing to discontinue the use of laxatives, including on-demand use, and evaluated the efficacy of DKT therapy without concomitant drugs. We found a noteworthy decrease in median total GSRS score and median GSRS indigestion and constipation subscales, along with improvements in bloating and abdominal pain symptoms. On the other hand, no significant changes were observed for bowel movement frequency or stool form. Furthermore, bloating was not improved with defecation. Bloating in individuals with chronic constipation can have various causes other than stool stagnation and may be affected by DKT. Possible pathogenic factors related to abdominal bloating include overproduction of abdominal gas, hypersensitivity to bowel distention, and excessive distention of the colon due to constipation. According to a past report,11 DKT may directly stimulate colonic motility and reduce colonic gas volume in patients after stroke, whereas another suggested that a 5-day administration of DKT stimulates the small intestine and enhances ascending colon transit in healthy adults.12 Such effects have also been reported in dogs and guinea pigs.13 SIBO is a pathologic condition in which abdominal gas is produced in the upper small intestine, possibly causing unpleasant abdominal symptoms. As with abdominal bloating, effective treatments for SIBO have yet to be established. A recent study14 found that gut microbiome diversity was lacking in a rat model of inflammatory bowel disease, whereas DKT prevented bacterial translocation by suppression of cytokines and apoptosis in a fast-stress applied rat model. In our study there was no increase in bowel movement frequency despite improvements in bloating and abdominal pain in both the SIBO and non-SIBO groups. Furthermore, the GBT results showed similar patterns before and after administration of DKT in the SIBO group and non-SIBO groups. These results suggest that a 2-week administration of DKT does not affect bacteria in the small intestine. Possible factors that may have led to our findings are the small sample size and lack of comparison with a placebo. In addition, the data were analyzed based on patient symptom scores. Additional studies with longer follow-up periods and a placebo are required to evaluate the prolonged influence of DKT on small intestine bacteria. BODY.CONCLUSIONS: DKT administration resulted in improved QOL in patients with and without SIBO who had chronic constipation. Although the present 2-week administration protocol seemed to have no effects on small intestine bacteria, further investigations are necessary to clarify the feasibility of DKT as treatment for abdominal bloating in patients with chronic constipation. BODY.CONFLICTS OF INTEREST: The authors have indicated that they have no conflicts of interests regarding the content of this article.

TITLE: OSAS Surgery and Postoperative Discomfort: Phase I Surgery versus Phase II Surgery ABSTRACT: Introduction. This study aims to investigate the reasons that discourage the patients affected by OSAS to undergo orthognathic surgery and compares the postoperative discomfort of phase I (soft tissue surgery) and phase II (orthognathic surgery) procedures for treatment of OSAS. Material and Methods. A pool of 46 patients affected by OSAS was divided into two groups: "surgery patients" who accepted surgical treatments of their condition and "no surgery patients" who refused surgical procedures. The "surgery patients" group was further subdivided into two arms: patients who accepted phase I procedures (IP) and those who accepted phase II (IIP). To better understand the motivations behind the refusal of II phase procedures, we asked the patients belonging to both the IP group and "no surgery" group to indicate the main reason that influenced their decision to avoid II phase procedures. We also monitored and compared five parameters of postoperative discomfort: pain, painkiller assumption, length of hospitalization, foreign body sensation, and diet assumption following IP and IIP procedures. Results. The main reason to avoid IIP procedures was the concern of a more severe postoperative discomfort. Comparison of the postoperative discomfort following IP versus IIP procedures showed that the former scored worse in 4 out of 5 parameters analyzed. Conclusion. IIP procedures produce less postoperative discomfort. IIP procedures, namely, orthognathic surgery, should be the first choice intervention in patients affected by OSAS and dentoskeletal malformation. BODY.1. INTRODUCTION: Surgical procedures to treat obstructive sleep apnea syndrome (OSAS) aim either to debulk the soft tissues or to expand the skeletal frame; the former procedures aim to reduce/remove the obstructions due to the excessive bulk of soft tissues lining the rhinoorohypopharynx and may be performed as single or combined procedures, depending on patient exigencies. Surgical procedures on soft tissue are generally defined as "first phase interventions" (IP) and aim to debulk soft tissues while maintaining the same skeletal volume [1–6]. When IP procedures fail in obtaining satisfactory results, "second phase interventions" (IIP) of orthognathic surgery are performed in order to increase the skeletal volume of the pharynx and stretching the soft tissues, producing, as final result, an effective enlargement of the air column. There is general agreement in literature that the most effective and reliable interventions are the IIP [7–15]; nevertheless, as indicated by the nomenclature, patients often undergo IIP procedures only after failure of IP interventions, in total contradiction with what is reported. We think that the reticence of physicians to propose IIP surgery depends fundamentally on their low familiarity with risks and complications of the orthognathic surgery, which results in deviant information and negative influence on patient's decisions. The objective of this study is to identify the real motivations that discourage the patients to undergo IIP interventions and try to objectivize the discomfort following IP surgery compared with IIP procedures. BODY.2. MATERIALS AND METHODS: Forty-six patients affected by OSAS of various degrees have been seen in our clinic between January 1, 2008, and December 31, 2012. The sample included 26 males (56.5%); the mean age was 44 years (range 18–82, standard deviation (SD): 16). Thirty-seven patients (80.4%) suffered from class II dentoskeletal malformation and 8 had class III malformation (17%), while two patients (3%) had dentoskeletal class I with bimaxillary retrusion showed at the cephalometric analysis. The mean apnea-hypopnea index (AHI) of the group was 29.4 (SD: 12), and the mean body mass index (BMI) was 33.4 (SD: 5). All the patients were referred by neurology, otolaryngology, internal medicine, and pneumology specialists and were affected by OSAS with an underlying dentoskeletal malformation requiring surgical correction. All the patients underwent clinical assessment, teleradiography of cranium in two projections, CT scan of cranium without contrast, cephalometric analysis, and endoscopic assessment to properly study the air column of upper airways; both the clinical and instrumental assessments showed that orthognathic surgery (IIP procedure) was the best therapeutic option in all the cases. All the patients were informed of the nature and indications of IP and IIP procedures as well as postoperative discomfort and possible complications. Postoperative discomforts presented as subdivided into two groups named "Group A" and "Group B": the former encompassed all the postoperative discomforts resulting from both the IP and IIP procedures including pain, masticatory discomfort, need of analgesia, foreign body feeling, postoperative emesis, oronasal reflux, dysphagia, and edema of soft tissues. "Group B" discomforts were those related exclusively to IIP procedures and included possible temporomandibular dysfunction, aesthetic changes of the face, and possible damage of the third branch of the V cranial nerves. We decided to compare some parameters of postoperative discomfort that were common to both the IP and IIP procedures, they included pain, painkiller need, admission days, foreign body sensation in the throat, and normal diet intake. All parameters were recorded from the first postoperative day. None of the patients suffered from food intolerances or drug allergies. The postoperative prescriptions were the same for all the patients and included amoxicillin/clavulanic acid 2.2 gr i.v. every 12 hours and acetaminophen 1 gr i.v. if needed. A Visual Analogic Scale (VAS) was used to objectivize the pain level, being 10 the value corresponding to maximum pain and 1 the condition of "no pain" [16, 17]. To evaluate the parameter "need of painkiller," the quantity of acetaminophen expressed in required doses was recorded and compared. For the evaluation of the "foreign body sensation," we also used a VAS assigning the value 10 to maximum foreign body feeling and 1 as normal feeling [16, 17]. The parameter "foreign body sensation" included the feeling of bulging in the pharynx as well as the oronasal reflux and postoperative dysphagia; it included also the postoperative emesis for patients who underwent IIP procedures. The parameter "diet" entailed three degrees: normal diet, semisolid diet, and compulsory liquid diet. To analyze the difference of postoperative discomfort between patients who underwent IP procedures and those who underwent IIP procedures, we divided the sample into two groups: on one side we pooled all the patients that accepted the surgical treatment either IP or IIP and defined that group as "surgery patients" (SP); the group "no surgery patients" (NSP) included all patients who refused surgery. The SP group was further subdivided into patients who accepted IIP procedures and those who agreed exclusively on IP procedures. The SP group involved 28 patients; mean age was 39 years (SD: 11). Males were 17 (60.7% of the sample). Nine patients (32.1%) underwent I phase interventions. Among these 9 patients, 9 (100% of I phase group patients) underwent uvulopharyngopalatoplasty, 7 (77.8%) decongestion of turbinates and septoplasty, and 2 patients (22.2%) thyrohyoidpexy intervention. The remaining 19 subjects (67.9% of the whole sample) underwent II phase interventions (both Le Fort I osteotomy and bilateral sagittal osteotomy of the mandible); among those 19 patients, 5 (26.3% of the II phase group patients) had simultaneous genioplasty. Main demographic and clinical characteristics of the patients are summarized in Table 1. To investigate the reason for refusing IIP procedures, we requested all participants belonging to NSP and IP procedures to indicate what was the group of postoperative discomfort that influenced their decision, choosing between "Group A" and "Group B." In the NSP group (18 patients, 39% of the sample), 5 were worried of postoperative discomforts belonging to "Group A" and 6 patients were discouraged by "Group B" discomforts, while 8 patients refused surgery for worries related to both "Group A" and "Group B" discomforts. Among the 9 patients who accepted only IP procedures, 6 patients refused IIP surgery because of "Group A" discomforts and 2 were concerned about "Group B" discomfort, while one patient indicated both "Group A" and "Group B" motivations (Figure 1). Finally, to compare the real discomfort between IP and IIP procedures, the above cited parameters were analysed by Fisher's exact test and Student's t-test at days I, II, III, IV, V, VI, VII, XIV, XXI, and XXX; P < 0.005 was considered statistically significant. BODY.3. RESULTS: Statistically significant differences were observed between the study groups in terms of postoperative day at discharge (P < 0.001). I phase patients were discharged on average after 5.4 days, while II phase patients were discharged on average after 3.6 days (mean difference: 1.8). Differences in postoperative AHI were also statistically significant (crude P = 0.018; I phase mean postop AHI: 7.3, II phase mean postop AHI: 3.2; mean difference: 4.1), even after adjusting for preoperative AHI (adjusted P = 0.013). Patients who underwent II phase interventions reported less pain on the Visual Analogic Scale and showed a better evolution of pain compared to those who underwent I phase interventions (see Figure 2). Between-groups effect of the type of intervention was statistically significant (adjusted P < 0.001) and also the interaction between time and intervention showed a between-groups significant effect (adjusted P = 0.001). Also the number of analgesic administrations was significantly lower in the II phase interventions group (between-groups effect of intervention, P < 0.001; between-groups effect of time-intervention interaction, P < 0.001; see Figure 3). The foreign body sensation on the Visual Analogic Scale scored better among IIP patients, as both the between-groups effect of intervention (P < 0.001) and the between-groups effect of time-intervention interaction (P < 0.001) were statistically significant (see Figure 4). Postoperative diet differed significantly between the groups during the whole analyzed postoperative period, as I phase patients could restart a normal diet before compared to II phase patients. Major findings are reported in Table 2. BODY.4. DISCUSSION: The relation between obesity and hypoventilation was first described in the late 1950's when obesity, hypopnea, and increased risks of heart diseases were positively correlated by several studies [18–23]. A careful depiction of the OSAS syndrome in the adult, in fact, can be actually found in Charles Dickens's novel The Pickwick Paper, afflicting the character Joe "the fat boy" [24]. The typical patients traditionally reported in literature were obese and in decayed physical conditions, and the elective treatment was a permanent tracheotomy; the outcomes were poor because of the high mortality and severe postoperative complications [25, 26]. The introduction of the continuous positive airway pressure (CPAP) as treatment for the OSAS in 1981 yielded positive outcomes for the first time [27]. The CPAP still remains the gold standard treatment for OSAS nowadays [11], although other therapeutic options have been proposed either as nonsurgical treatments by oral devices, neurostimulators, and drug-mediated therapies [28, 29] or by surgical interventions. Besides the tracheotomy which, as mentioned, was the elective surgical procedure in the past [30], other surgical interventions to treat the OSAS were proposed starting from the early 1980's, mainly as result of the Stanford University group effort [31–33]. The surgical procedures proposed to treat OSAS were either directed exclusively to the soft tissues of the nose, rhino-, oro-, and hypopharynx (IP) or aimed at changing the position of the skeletal bases (IIP); usually IIP procedures were indicated after failure of IP interventions. Nowadays the most popular surgical treatment for OSAS is IP procedures, even though there is unanimous agreement that IIP surgery provides better and more reliable outcomes [7–15]. This study showed that the main reason discouraging the patients to accept IIP surgery was the concern of postoperative discomforts (Table 2). Comparison of the parameter "postoperative pain" between patients who underwent IP procedures and those who had IIP surgery showed higher pain in the former group; patient treated with IP surgery reported severe pain for the first two postoperative weeks that was still present 30 days after surgery. The pain following IP surgery was typically sharp in nature, located at the throat and radiated to the ears; it is mainly due to the stimulation of free nociceptors (delta nervous fibers) on the raw surgical surfaces, the inflammatory status, and the muscular spasms [34]. The pain was described as continuous and exacerbated by deglutition (odynophagia); the odynophagia caused reduced oral intake and this condition promoted electrolytes imbalance and muscular spasms. The poor oral hygiene associated with the inflammation may favor the proliferation of the saprophyte oral bacteria inducing superinfection of the surgical wound and promoting exacerbation of both the inflammation and pain [35]. Second phase procedures (IIP), on the other hand, are performed through linear mucosal incision that are sutured and let to heal for first intention without exposure of terminal pain nerves; moreover, the subperiosteal exposure and the osteotomies contribute to temporary impairment of the function of the lower branch of the trigeminal nerve; as result, the postoperative pain following IIP surgery is nearly absent. In our experience, patients who underwent IP procedures reported severe pain (VAS 7-8) during the first 14 postoperative days, with gradual decrease proportional to the healing of the surgical wounds; however, mild to moderate pain (VAS 2-3) was still recorded in this group at the 30th postoperative day. Patients who underwent IIP procedures, conversely, reported moderate pain (VAS 4) only on the 1st postoperative day and mild to no pain (VAS 0–2) starting from the 2nd postoperative day. None of the IIP patients complained of pain after the IV postoperative day. Our data are in accordance with what is reported in literature on postoperative pain rate following orthognathic surgery that is about 0.5% according to Politis et al. [36]. The need for painkiller is an indirect parameter of postoperative pain and our data are concordant with what is reported in the literature [35, 37]: IP patients required the maximum dosage of painkiller during the first 7 postoperative days; starting from the 14th postoperative day their need of painkiller was reduced to 2 doses a day. However, this group of patients required painkiller drugs until the 30th postoperative day, especially before sleeping. The need for painkiller drugs in the IIP patient group reached its maximum at the 1st postoperative day to decrease steeply and come to a halt on the 3rd-4th postoperative day. The postoperative pain and need of painkiller influenced significantly the hospital admission time, which was longer (1.8 days more) in the IP group compared with the IIP group, negatively influencing the overall costs. The parameter "foreign body sensation" showed different symptoms in IP and IIP procedures; generally IP procedures produce feeling of bulging palate combined with dryness of the pharynx because of the edema and reshaping of the soft palate and uvula, associated with the rearrangement of the nervous fibers and the excision of minor salivary glands. In our IP patients group, the foreign body sensation was severe in the first postoperative week (scoring 9 on VAS) and gradually decreased over the subsequent weeks to became mild (4 at VAS) on the 30th postoperative day. Our record is in accordance with what is reported in literature, where the feeling of foreign body following IP procedures is reported to gradually disappear in a timeframe of 6 to 12 months [1, 4, 16, 32, 38, 39]. Phase II procedure, namely, orthognathic surgery, was mainly burden by postoperative nausea and emesis as discomforts; Silva et al. [40] in 2006 outlined the positive correlation between pain and emesis and pointed out that maxillary surgery was strictly correlated with emesis. Among the factors promoting nausea and emesis after orthognathic surgery we found the liquid diet, paresthesia/anesthesia of lips, orofacial edema, and blood swallowing during surgery. Combination of all those factors was associated with increased postoperative emesis following bimaxillary surgery [40, 41]. In our opinion, another factor implied in the postoperative emesis might be the changed relationship between the upper and lower dental arches; the new anatomical position of the jaws could be responsible for a foreign body feeling and promoting altered proprioceptive stimuli that will induce the emesis reflex. In IIP patients, the VAS score for the emesis (indicated as foreign body sensation) was halved on the 2nd postoperative day and resolved (VAS = 0) starting from the 3rd postoperative day. Regarding the "diet" parameter, we noticed different types of dysphagia in patients who underwent IP procedures with respect to those who underwent IIP surgery. In IP procedures, the dysphagia was mainly due to the swallowing pain (odynophagia) of solid food; as already described, the inadequate oral intake determined a condition of undernutrition which triggered a vicious loop by inducing muscular spasms, which further exacerbated the pain and dysphagia. The symptoms usually improved with the healing of the surgical wounds, a process that takes several weeks during which there is a gradual return to a normal diet [35, 36]. Our IP patients had semiliquid diet for the first 14 days following surgery, avoiding spicy and acidic food; all the patients recovered a normal diet after 3 weeks postoperatively. Patients who underwent IIP surgery assumed liquid diet for the first 30 days following surgery to avoid malunion or iatrogenic fractures of the osteotomized jaws; in the postoperative period, in fact, the altered muscular guide associated with a possible occlusal instability may predispose to iatrogenic fractures of the jaws if exposed to excessive masticatory burden. In our practice, we followed the international guidelines of intake and determined a condition of undernutrition which triggered a vicious loop by inducing muscular spasms, which further exacerbated the pain and dysphagia diet management after orthognathic surgery maintaining a liquid diet for 30 days, followed by further 30 days of blended diet before gradually reintroducing the normal diet [42]. BODY.5. CONCLUSIONS: The presented study showed that patients who underwent IP procedures suffered higher postoperative discomfort. On the light of this data, we believe that orthognathic surgery should not be a procedure to adopt in case of failure of the interventions of Phase I, but this should be the first choice, especially in cases with documented dental-skeletal malformations.

TITLE: Effect of local wound infiltration with ropivacaine on postoperative pain relief and stress response reduction after open hepatectomy ABSTRACT.AIM: To prospectively evaluate the effect of local wound infiltration with ropivacaine on postoperative pain relief and stress response reduction after open hepatectomy. ABSTRACT.METHODS: A total of 56 patients undergoing open hepatectomy were randomly divided into two groups: a ropivacaine group (wound infiltration with ropivacaine solution) and a control group (infiltration with isotonic saline solution). A visual analog scale (VAS) at rest and on movement was used to measure postoperative pain for the first 48 h after surgery. Mean arterial pressure (MAP), heart rate (HR), time to bowel recovery, length of hospitalization after surgery, cumulative sufentanil consumption, and incidence of nausea and vomiting were compared between the two groups. Surgical stress hormones (epinephrine, norepinephrine, and cortisol) were detected using enzyme-linked immunosorbent assay, and the results were compared. ABSTRACT.RESULTS: VAS scores both at rest and on movement at 24 h and 48 h were similar between the two groups. Significantly lower VAS scores were detected at 0, 6, and 12 h in the ropivacaine group compared with the control group (P < 0.05 for all). MAP was significantly lower at 6, 12, and 24 h (P < 0.05 for all); HR was significantly lower at 0, 6, 12, and 24 h (P < 0.05 for all); time to bowel recovery and length of hospitalization after surgery (P < 0.05 for both) were significantly shortened; and cumulative sufentanil consumption was significantly lower at 6, 12, 24, and 36 h (P < 0.05 for all) in the ropivacaine group than in the control group, although the incidence of nausea and vomiting showed no significant difference between the two groups. The levels of epinephrine, norepinephrine, and cortisol were significantly lower in the ropivacaine group than in the control group at 24 and 48 h (P < 0.01 for all). ABSTRACT.CONCLUSION: Local wound infiltration with ropivacaine after open hepatectomy can improve postoperative pain relief, reduce surgical stress response, and accelerate postoperative recovery. BODY: Core tip: This study prospectively evaluated the effect of local wound infiltration with ropivacaine on postoperative pain relief and stress response reduction after open hepatectomy. Wound infiltration with ropivacaine could provide more effective analgesia both at rest and on movement in the first 48 h after surgery, with lower mean arterial pressure, heart rate and sufentanil consumption, accelerated postoperative recovery, and reduced stress response. These results suggest that local wound infiltration with ropivacaine is a simple, convenient and effective analgesic method that can provide postoperative analgesia and short-term benefits after open hepatectomy. BODY.INTRODUCTION: Postoperative pain is an important issue for surgeons, anesthetists, patients, and other related personnel. The intensive pain caused by upper abdominal laparotomy may influence postoperative recovery, prolong hospitalization, and cause stress response and complications, including respiratory and cardiovascular depression and gastrointestinal and neuroendocrine dysfunction[1,2]. Currently, intravenous analgesia or epidural analgesia (EA) with a patient-controlled analgesia pump is the most common analgesic approach for controlling postoperative pain after laparotomy[3]. Although favored after major laparotomy, patient-controlled intravenous analgesia (PCIA) can delay postoperative recovery because of nausea and vomiting, excessive sedation, and dizziness; moreover, this analgesic mode involves the risk of addiction with large opioid dosages over long periods[4,5]. As to EA, which provides better analgesia than PCIA, it is restricted by contraindications, epidural puncture failure, and side effects[6,7]. Therefore, finding other analgesic strategies with fewer potentially serious adverse effects will be beneficial for patients suffering from postoperative pain. Postoperative analgesia is a crucial section of perioperative management, and local anesthetic methods are more effective than systemic analgesia regardless of the operation type[8]. Currently, wound infiltration with local anesthetics, which is a simple, effective, and inexpensive method, is performed in various surgical procedures and provides satisfactory analgesia without major side effects[9]. Ropivacaine and bupivacaine, as long-acting local anesthetics, are commonly used for local anesthesia and pain management in China[10]. Ropivacaine has the same analgesic effects as bupivacaine and levobupivacaine, but it is associated with a low incidence of motor block[11]. Thus, ropivacaine appears to be an important component for local anesthesia and postoperative analgesia. Meanwhile, surgical pain frequently increases the systemic stress response during the perioperative period, which can induce the excessive release of catecholamines (epinephrine and norepinephrine) and cortisol. Optimistically, local anesthesia provides considerable advantages over general anesthesia by suppressing catecholamines and cortisol levels[12]. In this study, we aimed to assess the effect of local wound infiltration with ropivacaine on postoperative pain control, mean arterial pressure (MAP), heart rate (HR), cumulative sufentanil consumption, incidence of nausea and vomiting, time to bowel recovery, and length of hospitalization after open hepatectomy. The changes of three stress hormones, namely, epinephrine, norepinephrine, and cortisol, were evaluated in patients undergoing wound infiltration with and without ropivacaine. BODY.MATERIALS AND METHODS: A total of 56 patients undergoing open hepatectomy, which was performed by the same experienced surgical team at the Department of Hepatobiliary Surgery of Qilu Hospital of Shandong University from January 2016 to March 2017, participated in this study. The study was approved by the Medical Ethics Committee of Qilu Hospital of Shandong University (No. 2017052), and written informed consent was obtained from all patients. The inclusion criteria included adult patients (aged 18-75 years) who would undergo open hepatectomy and were classified as grades I-III according to the American Society of Anesthesiologists (ASA) Physical Status Classification System. Patients with a history of known allergy to local anesthetics, chronic preoperative opioid consumption, or any other analgesic treatment for chronic pain before surgery, psychiatric or neurological diseases, or acquired or genetic hemostatic abnormality were excluded from the study. On the day of surgery, the patients were randomly divided into two groups with a table of random numbers. Surgeons and patients were kept blinded to the assigned treatment groups throughout the study. Wound infiltration was performed with a 7.5 mg/mL ropivacaine solution in the ropivacaine group and with an isotonic saline solution in the control group. Solutions were prepared and provided by the anesthetist, and surgeons were blinded to patient allocation. When closing the abdomen at the end of the surgical procedure, 20 mL of the prearranged solution was used to infiltrate the subcutaneous tissues, deep muscular fascia, and parietal peritoneum. Moreover, one or two drainage tubes were routinely placed near the cutting surface of the liver and then pulled out and fixed on the abdominal skin. In the presence of tube incision or pulling of the tube during movement or when turning over, the surrounding tissues of the tube were also infiltrated with the solution. Infiltration was performed under direct vision by the surgeon. All patients were given unrestricted access to sufentanil through a 100 mL disposable patient-controlled analgesic (PCA) device containing 1 μg/mL sufentanil that was delivered at a rate of 2 μg/h and a bolus of 0.5 μg with a 15 min lockout time. When the skin was closed, the PCA pump was connected to the venous catheter and routinely removed 36 h after the operation. The intensity of postoperative pain at rest was measured on a visual analogue scale (VAS) graded from 0 (no pain) to 10 (very severe pain) for the first 48 h after surgery. Movement pain was scored using VAS when coughing or turning over. Pain scores were recorded both by nurses and surgeons blinded to patient allocation. Pain measurements were performed at 0, 6, 12, 24, and 48 h after the surgery. Other variables were recorded, including time to bowel recovery, length of hospitalization after surgery, hemodynamic data represented by MAP and HR, cumulative sufentanil consumption, and postoperative nausea and vomiting (PONV). The three surgical stress hormones, namely, epinephrine, norepinephrine, and cortisol, were detected using commercial enzyme-linked immunosorbent assay kits (Cusabio Biotech Co., Ltd., Wuhan, China). Time to bowel recovery was defined as the time to first anal exhaust. PONV was recorded with a three-point rating scale: 1, no nausea and vomiting; 2, nausea without vomiting; 3, nausea with vomiting. Statistical analyses were performed with SPSS 19.0 (SPSS Inc., Chicago, IL, United States). All data were checked for normal distribution and the results are expressed as mean ± SD for continuous variables. The t test, χ2 test, Fisher's exact test, or analysis of variance was carried out where appropriate. P < 0.05 was considered statistically significant. BODY.RESULTS: All patients successfully received the surgical procedure, including the wound infiltration with a prearranged solution. However, three patients (two in the ropivacaine group and one in the control group) were dropped from the study for postoperative bleeding and bile leakage; finally, 26 patients were enrolled in the ropivacaine group and 27 enrolled in the control group. The demographic characteristics of the patients assigned to the two groups were comparable in terms of age, gender, weight, ASA grade, incision length, and postoperative pathology, except for operation type, which showed a statistical difference but had no clinical significance (Table 1). Table 1 Demographic characteristics of the patients studied (mean ± SD) Characteristic Ropivacaine group Control group t / χ 2 P value Age (yr) 48.38 ± 11.74 49.59 ± 12.42 -0.36 0.7176 Gender Male/female 18/8 18/9 0.04 0.8415 Weight (kg) 63.04 ± 9.21 66.04 ± 9.86 -1.14 0.2583 ASA grade I/II/III 4/17/5 7/15/5 0.92 0.6298 Incision length (cm) 24.65 ± 1.83 24.22 ± 2.76 0.67 0.5075 Operation type Left hepatectomy 8 5 0.0086 Right hepatectomy 13 8 Mesohepatectomy 1 0 Caudate lobectomy 2 1 Irregular hepatectomy 2 13 Postoperative pathology Hepatocellular carcinoma 22 18 0.3292 Intra-and extrahepatic cholangiolithiasis 3 7 Hepatic focal nodular hyperplasia 1 1 Hepatocellular adenoma 0 1 ASA: American Society of Anesthesiologists. The VAS scores both at rest and on movement were similar between the two groups at 24 h and 48 h after open hepatectomy (Figure 1A and B). Significant differences in VAS scores at rest were detected at 0 h (P = 0.0106), 6 h (P = 0.0032), and 12 h (P = 0.0002). Moreover, significant differences in VAS scores on movement were observed at 0 h (P = 0.0208), 6 h (P = 0.0043), and 12 h (P = 0.0089). The details are shown in Table 2. Figure 1Visual analogue scale scores at rest and on movement, mean arterial pressure, and heart rate during the first 48 h after surgery. A: VAS scores at rest; B: VAS scores on movement; C: MAP; D: HR. aP < 0.05; bP < 0.001; cP < 0.0001. VAS: Visual analogue scale; MAP: Mean arterial pressure; HR: Heart rate. Table 2 Visual analog scale scores at rest and on movement, mean arterial pressure, heart rate, and cumulative sufentanil consumption Characteristic 0 h 6 h 12 h 24 h 48 h 1 VAS at rest Ropivacaine group 2.38 ± 0.50 2.27 ± 0.45 2.04 ± 0.20 2.12 ± 0.33 2.08 ± 0.27 Control group 2.78 ± 0.58 2.67 ± 0.48 2.48 ± 0.51 2.22 ± 0.42 2.15 ± 0.36 t -2.65 -3.17 -4.21 -1.03 -0.81 P value 0.0106 0.0032 0.0002 0.3096 0.4230 VAS on movement Ropivacaine group 3.27 ± 0.53 3.35 ± 0.63 3.31 ± 0.62 3.23 ± 0.43 3.11 ± 0.52 Control group 3.63 ± 0.56 3.85 ± 0.60 3.78 ± 0.64 3.41 ± 0.57 3.30 ± 0.47 t -2.39 -2.99 -2.72 -1.27 -1.34 P value 0.0208 0.0043 0.0089 0.2110 0.1857 MAP (mmHg) Ropivacaine group 95.88 ± 5.08 93.35 ± 4.63 92.62 ± 4.43 94.73 ± 4.16 93.19 ± 3.41 Control group 97.74 ± 4.17 96.15 ± 4.14 97.56 ± 4.23 98.37 ± 3.99 94.89 ± 3.90 t -1.46 -2.32 -4.16 -3.25 -1.69 P value 0.1515 0.0241 0.0001 0.0020 0.0981 HR (bpm) Ropivacaine group 88.96 ± 4.12 90.19 ± 3.92 89.73 ± 4.45 91.65 ± 4.30 90.31 ± 4.45 Control group 92.04 ± 4.27 93.41 ± 4.62 94.74 ± 3.88 94.56 ± 3.43 92.11 ± 4.05 t -2.66 -2.73 -4.37 -2.72 -1.54 P value 0.0103 0.0087 < 0.0001 0.0089 0.1289 Cumulative sufentanil consumption (μg) Ropivacaine group 0 14.94 ± 2.56 30.40 ± 5.39 56.75 ± 6.20 83.02 ± 7.05 Control group 0 17.06 ± 3.81 35.50 ± 6.91 65.31 ± 7.09 91.39 ± 7.34 t -2.36 -2.99 -4.67 -4.23 P value 0.0220 0.0043 < 0.0001 < 0.0001 1 As to cumulative sufentanil consumption, the time point was set at 36 h. VAS: Visual analog scale; MAP: Mean arterial pressure; HR: Heart rate. Hemodynamic data are presented in Figure 1C and D and Table 2. MAP was significantly lower in the ropivacaine group than in the control group at 6 h (P = 0.0241), 12 h (P = 0.0001), and 24 h (P = 0.002). In the ropivacaine group, HR was significantly lower at 0 h (P = 0.0103), 6 h (P = 0.0087), 12 h (P < 0.0001), and 24 h (P = 0.0089). No statistically significant difference was observed in baseline levels (0 h) of epinephrine, norepinephrine, or cortisol between the two groups. The levels of epinephrine at 24 and 48 h were significantly lower in the ropivacaine group than in the control group (P = 0.0064, P = 0.0078). Similarly, the values of norepinephrine and cortisol at 24 and 48 h were significantly reduced in the ropivacaine group (P < 0.0001 for all), as shown in Figure 2A-C and Table 3. Figure 2Plasma levels of epinephrine, norepinephrine and cortisol and cumulative sufentanil consumption during the first 48 h after surgery. A: Epinephrine; B: Norepinephrine; C: Cortisol; and D: Cumulative sufentanil consumption. aP < 0.05; cP < 0.0001. Table 3 Plasma concentrations of epinephrine, norepinephrine and cortisol (pg/mL) Stress hormone 0 h 24 h 48 h Epinephrine Ropivacaine group 64.19 ± 19.62 86.54 ± 28.64 94.27 ± 30.10 Control group 66.19 ± 20.30 110.26 ± 31.88 118.59 ± 33.65 t -0.36 -2.85 -2.77 P value 0.7180 0.0064 0.0078 Norepinephrine Ropivacaine group 251.31 ± 30.19 280.62 ± 34.22 310.12 ± 35.15 Control group 259.70 ± 31.72 361.00 ± 36.06 390.41 ± 37.73 t -0.99 -8.32 -8.01 P value 0.3286 < 0.0001 < 0.0001 Cortisol Ropivacaine group 342.00 ± 33.72 391.81 ± 36.53 450.62 ± 39.39 Control group 331.59 ± 31.92 487.48 ± 34.36 518.85 ± 38.21 t 1.15 -9.82 -6.40 P value 0.2537 < 0.0001 < 0.0001 Cumulative sufentanil consumption at 36 h after surgery is presented in Figure 2D and Table 2. The consumption was significantly lower in the ropivacaine group than in the control group at 6 h (P = 0.022), 12 h (P = 0.0043), 24 h (P < 0.0001), and 36 h (P < 0.0001). Even so, the incidence of nausea and vomiting, the side effects of sufentanil, between the two groups had no significant difference (Table 4). Moreover, in the ropivacaine group, time to bowel recovery (P = 0.0133) and hospitalization after surgery (P = 0.0289) were significantly shortened (Table 4). Table 4 Time to bowel recovery, postoperative nausea and vomiting, and hospitalization length in the two groups Characteristic Ropivacaine group Control group t / χ 2 P value Time to bowel recovery (d) 3.15 ± 1.01 3.93 ± 1.17 -2.56 0.0133 PONA No PONA 7 3 0.2729 Nausea without vomiting 16 18 Nausea with vomiting 3 6 Hospitalization (d) 8.65 ± 2.43 10.52 ± 3.49 -2.25 0.0289 PONA: Postoperative nausea and vomiting. BODY.DISCUSSION: Laparoscopic hepatectomy is commonly adopted in clinical settings because of its many advantages, including little trauma, low pain, fast recovery, and short hospitalization; however, open hepatectomy remains irreplaceable, especially in the presence of lesions close to or invading the root of the hepatic veins or the inferior vena cava, history of previous hepatectomy or any previous surgery potentially causing severe adhesion around the liver, and concomitant cardiopulmonary disease[13]. A right subcostal incision or reversed L-shaped incision (> 20 cm) is often made for open hepatectomy, and either of these two incision types is the most important source of postoperative pain. Thus, finding an effective way to reduce postoperative pain is urgent and necessary. Local anesthetic wound infiltration is a useful and important component of a multimodality approach to postoperative pain control, and it can be applied in many types of surgery, including lumbar spine surgery, breast surgery, and inguinal hernia repair[9,14,15]. Local anesthetics used in the wound can block parietal afferents, reduce the sensitization of spinal dorsal horn neurons, and provide analgesia by inhibiting the transmission of noxious impulses from the incision[16]. Moreover, local anesthetics can suppress local inflammatory responses to incision injury that could sensitize nociceptive receptors and contribute to hyperalgesia[17]. Ropivacaine, a pure levorotatory stereoisomer and long-acting amide local anesthetic agent, has been widely used for local anesthesia and postoperative analgesia, and its reduced lipophilicity is associated with decreased incidence of central nervous system toxicity and cardiotoxicity[18]. Postoperative pain comes from superficial structures and deep muscular-peritoneal components; therefore, ropivacaine infiltrated not only the subcutaneous tissues but also the parietal peritoneum and deep muscular fasciae in our study. Our results showed that in the first 12 h after surgery, the local anesthetic ropivacaine significantly relieved the pain intensity at rest and on movement, demonstrating the potential of local wound infiltration with ropivacaine as a reliable analgesic strategy after open hepatectomy. Surgical stress could cause a spectrum of changes in the body, involving the neuroendocrine, metabolic, immunological, and hematological systems[12]. The body's surgical stress response is mainly determined by the surgical wound severity, including the length of the incision in the abdominal wall from the skin to the parietal peritoneum[19]. The incision of open hepatectomy often exceeds 20 cm, and the surgical stress is thought to be high. Therefore, using local anesthetics to block surgical stress is feasible. Surgical stress response to injury causes a series of hormone changes; moreover, catecholamines (epinephrine and norepinephrine) and cortisol, as the main and most reliable peripheral hormones, correlate well with the extent of surgical stress. In this study, surgical stress was significantly reduced in the first 48 h after surgery as revealed by the levels of epinephrine, norepinephrine, and cortisol. Changes in MAP and HR were recorded, and the results demonstrated that the indexes of the ropivacaine group were obviously decreased. These results indicate that local wound infiltration with ropivacaine could also reduce surgical stress responses. Opioids are commonly used for postoperative analgesia via venous access. However, they are associated with a potential risk of addiction, especially in large doses over long periods. Moreover, opioids possess potentially serious side effects, such as nausea, vomiting, constipation, respiratory depression, excessive sedation, and liver function impairment; hence, sparing opioids may reduce the incidence of the above side effects[20]. Reducing the dosage and duration of opioid usage is regarded suitable for avoiding potentially serious adverse effects. Wound infiltration, as part of an opioid-sparing, multimodal analgesic regime, should therefore be recommended. Our current study showed that cumulative sufentanil consumption was significantly reduced in the ropivacaine group. Moreover, the time to bowel recovery was shorter in the ropivacaine group than in the control group. This may be caused by a combination of several reasons. First, sufentanil inhibits gut motility and propulsive activity by combining the μ-2 and κ receptors in the digestive tract[21]. Second, a previous animal study demonstrated that catecholamines reduce gut motility[22] and that the level of catecholamines in the ropivacaine group was reduced. Finally, ropivacaine could accelerate postoperative intestinal motility by reducing the inflammatory response. The current study may have some limitations. The sample size was relatively small, and thus, more patients are needed in future studies to confirm our results. Compared with a previous study using catheters as a continuous wound infiltration method to deliver ropivacaine into the wound[23], we used single-shot ropivacaine infiltration into the superficial and deep muscular-peritoneal layers to achieve an analgesic effect. In our study, the drainage tube was routinely placed beside the liver resection surface and fixed outside. Movement and turning over could drag the tube and cause intensive pain, and thus, infiltration around the tube was highly effective. Moreover, the catheter under the wound could bring potential risks, such as infection and delayed wound healing, and the delivery rates and volumes of local anesthetics remain unidentified. Thus, single-shot infiltration with ropivacaine is a simple, convenient, and effective analgesic method that can bring short-term benefits for patients who underwent open hepatectomy. A previous study suggested that local anesthesia and stress response reduction could decrease cancer formation and that local anesthesia and analgesia may improve overall patient survival after oncologic surgery[24]. Thus, future research about local anesthesia, tumor recurrence, and patient survival after open hepatectomy is required. In conclusion, local wound infiltration with ropivacaine after open hepatectomy can decrease acute postoperative pain and surgical stress response. This simple, convenient, and effective analgesic method provides postoperative analgesia and short-term benefits after open hepatectomy. BODY.COMMENTS.BACKGROUND: The postoperative pain caused by laparotomy delays patients' recovery and incurs stress response. Although commonly used to control pain, intravenous analgesia and epidural analgesia still have their contraindications and side effects. Local wound infiltration is a simple and effective method that can provide satisfactory analgesia without major side effects. The current study was designed to evaluate the effect of local wound infiltration with ropivacaine on postoperative pain and stress response after open hepatectomy. BODY.COMMENTS.RESEARCH FRONTIERS: Postoperative analgesia is an indispensable component of fast track surgery for surgical patients, especially those who undergo laparotomy. Local anesthetics can effectively provide analgesia by inhibiting the transmission of noxious impulses from the wound and suppress local inflammatory responses to wound injury. BODY.COMMENTS.INNOVATIONS AND BREAKTHROUGHS: Wound infiltration with ropivacaine could provide effective analgesia in the first 48 h after open hepatectomy, with lower mean arterial pressure, heart rate and sufentanil consumption, accelerated postoperative recovery, and reduced stress response. These results suggest that this method is a simple, convenient and effective analgesic method that can provide postoperative analgesia and short-term benefits after open hepatectomy. BODY.COMMENTS.APPLICATIONS: This study provides additional evidence supporting that local wound infiltration with ropivacaine after open hepatectomy can improve postoperative pain relief, reduce surgical stress response, and accelerate postoperative recovery. BODY.COMMENTS.TERMINOLOGY: The intensity of postoperative pain at rest was measured on a visual analogue scale graded from 0 (no pain) to 10 (very severe pain) after surgery. Postoperative nausea and vomiting was recorded with a three-point rating scale: 1, no nausea and vomiting; 2, nausea without vomiting; 3, nausea with vomiting. BODY.COMMENTS.PEER-REVIEW: The study was well written and its findings are informative. Local wound infiltration with ropivacaine has good effects for pain relief and stress response reduction after open hepatectomy.

TITLE: Teenage Patients with Ingrown Toenails: Treatment with Partial Matrix Excision or Segmental Phenolization ABSTRACT.BACKGROUND:: Ingrown toenails (IT) is a very common problem leading to significant associated morbidity. The articles related to phenolization for matrix removal in teenagers with IT are not enough in the foot surgery literature. ABSTRACT.AIMS:: To compare the postoperative recovery periods, complication rate, and tolerability of partial matrix excision and segmental phenolization in teenagers with IT. ABSTRACT.MATERIALS AND METHODS:: Thirty-nine patients (13-17 years) with 48 IT were randomly divided into two groups and were treated with partial matrix excision (Group I) and segmental phenolization (Group II). We assessed the recurrence rates, postoperative complications, duration of analgesic usage, and time to return to daily activities. ABSTRACT.RESULTS:: There was no significant difference between the demographic and clinical data of the two groups. Three patients in Group I and two patients in Group II experienced moderate pain postoperatively. These patients used analgesics for 3 days. The rates of postoperative complications and recurrences between the two groups showed no statistically significant difference (P = 0.688). The time to return to normal daily activities was significantly shorter in Group II patients than in Group I patients (P < 0.05). ABSTRACT.CONCLUSIONS:: Partial matrix excision is a very safe model of therapy in the surgical treatment of teenagers with IT. It has low recurrence rate and minimal postoperative morbidity. We concluded that segmental phenolization is also as safe as partial matrix excision in the treatment of IT and patients return to their daily activities in less time with this treatment modality. BODY.INTRODUCTION: What was known?IT is a very common problem in adults and teenagers. The surgical procedures used with confidence are classical wedge excision, partial matrix excision, and cauterization with liquefied phenol. Ingrown toenails (IT) is a very common problem and cause significant associated morbidity.[12] Several options for the treatment of IT are available, ranging from simple conservative approaches to extensive surgical procedures.[3] Surgery can be performed either by a classical surgical wedge excision followed by selective matrix destruction or by cauterization with liquefied phenol of the lateral matrix portion.[4] The articles related to phenolization for matrix removal in teenagers with IT are not enough in the foot surgery literature.[45] The teenagers mostly wear closed footwear and experienced with recurrent trauma. The purpose of this study was to compare the duration of analgesic usage, postoperative complication rates (any adverse effect of the chemical agent, infection, and scar tissue), time to return to daily activities, and the recurrence rates of partial matrix excision and segmental phenolization in teenagers with IT. BODY.MATERIALS AND METHODS: Thirty-nine patients (28 males and 11 females) with 48 IT sides were randomly divided into two groups and were treated with partial matrix excision (Group I) and segmental phenolization (Group II). Exclusion criteria were as follows: (a) Peripheral vascular disease, (b) hypersensitivity to the chemical solution, (c) serious systemic diseases, (d) serious digital diseases, and (e) diabetes mellitus infection. Group I had mean ages of 16.1 ± 1.9 years and Group II had mean ages of 17.0 ± 1.0 years. Antibiotic therapy was given in 33 (68.7%) infected cases before surgical intervention. Group I patients were draped under sterile conditions following injection of local anesthetic agent (2% plain prilocaine) to cause digital anesthesia. A finger tourniquet was applied with a rubber ring. In all patients, vertical incision was performed along the lateral or medial border of the nail with No. 11 blade. The incision was extended 1-2 cm distally than proximally. Nail matrix and infected and hypertrophic tissues were partially excised. The cortex over the distal phalanx was purged by a curette. This incision was sutured with 3-0 or 4-0 nonabsorbable material. No technical difficulty was encountered in putting sutures. Compression bandage was applied on the toe. Elevation and prophylactic antibiotics were applied after surgery. Only patients with complaints of moderate pain received analgesic drugs [Table 1]. Daily dressing was performed and all sutures were removed after 1 week of operation [Figure 1]. The mean follow-up period was 3.2 ± 1.2 years. In Group II, the preoperative preparation and anesthesia like in Group I were performed. The lateral or medial nail strips were freed from the overlying proximal nail fold, nail bed, and matrix. A 2-3 mm lateral nail segment was cut free along the length of the lateral fold and hypertrophic granulation tissue was curetted. Phenol was applied with partially stripped cotton applicators saturated with 88% liquid phenol by vigorously massaging it into the matrix area. The cotton applicator was changed twice during an application time of 90 s. The patients were followed up for a period of 2.1 ± 0.9 years. Table 1 Demographic data Figure 1Partial matricectomy, (a) preoperative image, (b) intraoperative image, and (c) postoperative image The demographic and clinical data of both groups were compared. Postoperative morbidity was evaluated clinically according to the severity of pain and soft tissue infection. Pain was graded as mild, moderate, and severe. Soft tissue infection consisted of cellulitis, purulent discharge, and abscess formation. Duration of analgesic usage, time to return to normal daily activities, and recurrence rate at least at the end of 1 year were assessed. Statistical Package for the Social Sciences (SPSS 15.0: USA) was used for statistical analysis and the variables were compared using Chi-square and Mann–Whitney U tests. (P < 0.05) were considered as significant. BODY.RESULTS: There was no significant difference between the two groups in demographic and clinical data. Both groups' data are shown in Table 1. Staging of IT is shown in Table 2. Table 2 Clinical results Postoperatively, none of the patients had severe pain. Three patients (17.6%) in Group I and two patients (9.0%) in Group II experienced moderate pain. Only these patients received analgesic for 3 days. Differences in pain were not statistically significant between the groups (P > 0.05). In both groups, none of the patients had soft tissue infection. A return to normal daily activities was the criterion for treatment success. The time to return to daily activities was significantly shorter in Group II than in Group I (P < 0.05) [Table 1]. The rates of postoperative recurrences showed no significant difference between the two groups (P = 0.688) [Table 2]. In both groups, none of the patients had postoperative complications. BODY.DISCUSSION: IT causes significant discomfort for both teenagers and adults. IT in children occur most commonly in the age group of 10-13 years.[6] In this study, we want to compare the effectiveness of partial matrix excision and segmental phenolization in teenagers (13-17 years) with IT. Many treatment methods have been advocated, ranging from conservative and surgical procedures to phenolization. Surgical approach is usually preferred rather than conservative treatment. Many surgical procedures and treatment methods have been used.[7] In adults, partial matrix excision is one of the most preferred treatment methods for IT. Currently, segmental phenolization is also a frequently used method in this condition. But this procedure carries a risk for a chemical burn on the skin.[5] In contrast, in this study, no cosmetic problem (such as skin burn) was observed in both groups. The use of segmental phenolization in the treatment of IT has shown statistically significant reduction in recurrence rates of IT compared to partial matrix excision procedure.[8910] But none of the observed differences in wound healing, postoperative pain, and recovery were statistically significant.[41112] Also in our study, recurrence rates were detected as low in both groups. Pain severity and analgesic usage in postoperative period showed no significant differences between both groups. The period of return to normal daily activities was significantly shorter in Group II than in Group I. Islam et al. demonstrated that effectiveness of phenolization was similar both in children and in adults. This study showed that number of recurrences were significantly different (42% recurring after partial matrix excision and only 4% after segmental phenolization).[13] None of the segmental phenolization patients had any skin burn in the surrounding area. Complications did not differ between the two groups.[1113] In our study, similar results were obtained. Despite the use of chemical substances, no serious complication was seen in Group II. Most of the studies showed that segmental phenolization is a procedure which causes less pain and has an effect of relieving pain earlier.[8] Partial matricectomy has also been shown to have less painful recovery times in some other researches.[4] On the other hand, none of the observed differences in the two methods such as wound healing, postoperative pain, and recovery were seen in some of the related studies.[11] In this study, in terms of pain and the period of analgesic usage, there was no difference between the two groups. In the research study of Aydin et al., patients who were operated with the partial matrix method returned to normal daily activities after a mean of 12 days (range: 10-16 days).[14] Another study demonstrated that the time to return to normal daily activities in both the methods was equal as a mean of 3.5 days.[11] In this study, we achieved a favorable result in that the patients in the group of phenol method returned to normal daily activities in a significantly earlier time. Partial matrix excision is a very safe model of therapy in the surgical treatment of teenagers with IT. It has low recurrence rate and minimal postoperative morbidity. We concluded that segmental phenolization is also as safe as partial matrix excision in the treatment of IT and patients return to their normal daily activities in less time with this treatment modality. We recommend that both techniques can be considered for treating IT in teenagers. What is new?Partial matrix excision and segmental phenolization are very safe modes of therapy in the treatment of IT in teenagers. Segmental phenolization is a very effective treatment modality in terms of patients returning to their normal daily activities rapidly in teenagers with IT.

TITLE: Using a Low-Sodium, High-Potassium Salt Substitute to Reduce Blood Pressure among Tibetans with High Blood Pressure: A Patient-Blinded Randomized Controlled Trial ABSTRACT.OBJECTIVES: To evaluate the effects of a low-sodium and high-potassium salt-substitute on lowering blood pressure (BP) among Tibetans living at high altitude (4300 meters). ABSTRACT.METHOD: The study was a patient-blinded randomized controlled trial conducted between February and May 2009 in Dangxiong County, Tibetan Autonomous Region, China. A total of 282 Tibetans aged 40 or older with known hypertension (systolic BP≥140 mmHg) were recruited and randomized to intervention (salt-substitute, 65% sodium chloride, 25% potassium chloride and 10% magnesium sulfate) or control (100% sodium chloride) in a 1: 1 allocation ratio with three months' supply. Primary outcome was defined as the change in BP levels measured from baseline to followed-up with an automated sphygmomanometer. Per protocol (PP) and intention to treat (ITT) analyses were conducted. ABSTRACT.RESULTS: After the three months' intervention period, the net reduction in SBP/DBP in the intervention group in comparison to the control group was −8.2/−3.4 mmHg (all p<0.05) in PP analysis, after adjusting for baseline BP and other variables. ITT analysis showed the net reduction in SBP/DBP at −7.6/−3.5 mmHg with multiple imputations (all p<0.05). Furthermore, the whole distribution of blood pressure showed an overall decline in SBP/DBP and the proportion of patients with BP under control (SBP/DBP<140 mmHg) was significantly higher in salt-substitute group in comparison to the regular salt group (19.2% vs. 8.8%, p = 0.027). ABSTRACT.CONCLUSION: Low sodium high potassium salt-substitute is effective in lowering both systolic and diastolic blood pressure and offers a simple, low-cost approach for hypertension control among Tibetans in China. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT01429246 BODY.INTRODUCTION: The WHO predicts that cardiovascular disease (CVD) will become the leading cause of Disability Adjust Life Years (DALYs) in 2020 [1] and more significantly, over 80% of this global burden will occur in low and middle income countries [2]. Further, hypertension accounts for nearly 45% of the global burden of cardiovascular morbidity and mortality [3]. Hypertension is one of the most common modifiable risk factor for CVD, with a prevalence of nearly 57% in adults 40 years and older in Tibet [4], two times as high as the 2002 China national rate [5]. This preponderance of hypertension has been strongly associated with an extreme burden of stroke in Tibetans. The Tibetan age-standardized stroke incidence was 450.4 per 100,000 persons and stroke mortality of 370.2 per 100,000 persons; both metrics were over four times the respective China national rates [6]. The high prevalence of hypertension in Tibetans is attributable to a very high level of daily dietary salt intake [7], [8]. In Tibetan adults the estimated dietary salt intake is nearly four to five times the WHO recommend amount of five grams daily [9]–[11], largely driven by the daily consumption of a traditional salty yak buttermilk tea [8], [9], [12], which has been reported to be as high as four liters per day in Tibetans [13]. Reduction in salt intake has been identified by the World Health Organization as a highly cost-effective strategy for cardiovascular disease prevention [14]. Low sodium salt substitute, as a low cost strategy to reduce sodium intake, has previously demonstrated marked reductions in systolic blood pressure among Han Chinese patients with high cardiovascular risk in the China Salt Substitute Study (CSSS). The magnitude of the effect was significantly associated with the baseline blood pressure of the CSSS cohort [15]. We set out to test the hypothesis that the low sodium dietary salt substitute could be more effective in reducing blood pressure in community hypertension programs in Tibetans living at high altitude, whose prevalence of hypertension as well as salt intake are markedly higher than Han Chinese [5], [16]. Of particularly note, the high altitude as the special regional living condition could actually cause significant difficulties to the local healthcare system in implementing hypertension prevention and control program as well as other health programs, such as lack of health care workers, remote and low access to health care services and medications, etc. Thus, the prevalence, treatment and control of hypertension in Tibet are very low [5]. This highlights the huge demand in developing strategies like salt substitute that are not only effective but can also be delivered through none-medical or para-medical health systems. BODY.METHOD.ETHIC STATEMENT: The trial was approved by the Ethics Committee of Peking University Health Science Center, Beijing, China (#IRB00001052-09003). All participants and the patriarchs of their families provided informed consent for the family. The protocol and CONSORT checklist are available as supporting information see Protocol S1 (English), Protocol S2 (Chinese) and Checklist S1. BODY.METHOD.DESIGN: A community based survey of hypertension was first conducted [5] and then all patients meeting inclusion and exclusion criteria were invited to the trial, with the intervention group receiving free salt substitute and the control group receiving free regular salt, in quantities sufficient for a complete household's use during the three months' study period between February 2009 and May 2009. The study was conducted according to principles of the Declaration of Helsinki and subsequent amendments and the study was registered at clinicaltrial.gov NCT01429246 in September 2011. This retrospective registration was due to our unawareness of the international requirements of prospective registration for such kind of trials at that time. However, we ensure honestly that the delay of registration has little bearing on the quality of science and ethics of this study. BODY.METHOD.STUDY PARTICIPANTSAND SETTINGS: Local residents aged 40 years and above from two townships (Yangbajing and Gongtangxiang) in Dangxiong County in Tibet Autonomous Region, China, were first invited to a hypertension screening, then those having measured systolic blood pressure ≥140 mmHg would be invited to the trial within 3 months. If the invitee's systolic blood pressure was confirmed ≥140 mmHg again, regardless use of anti-hypertension medications, he/she would be invited to participate in this study. Of 287 patients who met our inclusion criteria, 282 were recruited after excluding 5 patients who met the exclusion criteria, including 1 patient who was too old and weak to travel, 1 patient who withdrew after inform consent, and 3 patients dropped by research staff due to their living far and arduous journey for follow up. The townships are located 90 kilometers northwest of Lhasa, with an average altitude of 4300 meters above the sea level. The study was planned to enroll patients only in Yangbajing Township originally. However, there were not enough patients meeting our inclusion criteria and agreed to participate in the study there, so the recruitment had to be extended to the nearby township Gongtangxiang in order to achieve the recruitment target. But we reduced data to be collected at baseline in Gongtangxiang due to resource restriction. For details of differences in data collection between the two townships please refer to Table 1. 10.1371/journal.pone.0110131.t001 Table 1 Baseline characteristics of study participants by randomized group. Characteristics Total Regular salt Salt Substitute (All subject) (N = 282) (N = 141) (N = 141) Age (years) 63.1 (11.2) 63.5 (11.3) 62.8 (11.1) Sex (% female) 166 (58.9%) 81 (57.4%) 85 (60.3%) SBP (mmHg) 176.9 (22.8) 177.6 (23.3) 176.1 (22.4) DBP (mmHg) 104.5 (12.6) 105.8 (13.1) 103.2 (12.0) Classification of hypertension a Stage 1 56 (19.9%) 27 (19.1%) 29 (20.6%) Stage 2 226 (80.1%) 114 (80.9%) 112 (79.4%) Antihypertensive medicine use in the past month (%) 132 (47.0%) 71 (50.7%) 61 (47.0%) Average number of antihypertensive medicines taken 0.5 (0.5) 0.5 (0.5) 0.4 (0.5) Body mass index (kg/m 2 ) 23.6 (3.4) 23.6 (3.4) 23.7 (3.1) (Subjects in Yangbajing township only) (N = 145) (N = 72) (N = 73) Education Uneducated(0 year) 134 (92.4%) 70 (97.2%) 64 (87.6%) Primary school(1–5 years) 10 (6.9%) 2 (2.8%) 8 (11.0%) Middle school(6–9 years) 1 (0.7%) 0 (0.0%) 1 (1.4%) Occupation Herdsman 137 (94.5%) 71 (98.6%) 66 (90.4%) Others b 8 (5.5%) 1 (1.4%) 7 (9.6%) Smoking history (%, yes) c 13 (9.0%) 7 (9.7%) 6 (8.2%) Drinking history (%, yes) d 17 (11.7%) 9 (12.5%) 8 (11.0%) All numbers shown are mean (±SD) unless otherwise noted as number (%). All p-values comparing the two groups are larger than 0.05. a. According to the American Heart Association, stage 1 hypertension was defined as 140≤SBP<159 and/or 90≤DBP<100; stage 2 hypertension as SBP≥160 and/or DBP≥100. b. Other occupation includes farmer, doctor, self-employed and retired. c. Participant who have smoke more than 20 packs in life or smoke at least one cigarette per day and last more than one year was regarded as having smoking history. d. Participant who drinks at least once per week was regarded as having drinking history. BODY.METHOD.RANDOMIZATION: Stratified randomization by township, gender, and baseline systolic BP (<160 or ≥160 mmHg) was performed immediately after eligibility assessment, using a computer generated randomization list. A random number generator provides a treatment allocation identification number to each enrolled patient. This assignment was secured in a password protected encrypted digital registry. Treatment allocation was only blinded to participants through the use of indistinguishable containers; with only the study allocation number labeled. There was also no way to tell salt-substitute and regular salt by their physical appearance. The difference of taste between salt substitute and regular salt is minor. Our previous study among young adults indicated that about 70% −80% of testers were unable to identify the difference in taste in a triangle food taste test (Data unpublished). There was 1 patient from control group but no patient from intervention group in our study quoted "poor taste" as the reason for dropping out, indicating our blinding to patients were successful. BODY.METHOD.INTERVENTIONS: The anticipated allocation ratio between the intervention arm and control arm was set to 1:1. The intervention arm received a three months' supply of salt substitute (65% sodium chloride, 25% potassium chloride, and 10% magnesium sulfate) [15], [17]. The control arm received regular salt with a 100% sodium chloride. The study salt was distributed free of charge to every household with sufficient amount to cover three months' consumption for the whole household. Patients with preexisting anti-hypertensive medications were not directed to alter their prior regimen. BODY.METHOD.BASELINE MEASUREMENTS: Once the patients were confirmed eligible and the inform consent was obtained, the baseline measurements were conducted immediately. The screening, eligibility confirmation and baseline measurements were all conducted by trained research staff, between December 2008 and February 2009. Blood pressure was measured with three consecutive blood pressure measurements (with at least one minute's rest between each measurement) from a seated subjects' right arm in a quiet room. A previously validated electronic sphygmomanometer (OMRON HEM-759P, Dalian China) was used [18]. Weight and height were measured in a standardized way [5]. The personal information including age, sex, and current anti-hypertensive medication use were collected by structured questionnaire for all participants. Due to the resource constraints, other variables such as educational level, occupation, smoking history, and drinking history were obtained only from participants in Yangbajing at the screening survey [5]. BODY.METHOD.FOLLOW UP SURVEY: After three months, all patients were asked to return for the measurements of blood pressure according to the same protocol used at baseline. We also collect data on anti-hypertension medication use during the whole study period for all participants. BODY.METHOD.OUTCOMES: The primary outcomes were the changes in systolic and diastolic blood pressure from baseline to post-intervention over the three-month' study period. The mean of the three systolic blood pressure measurements were used for analysis. The secondary outcomes were the proportion with hypertension under control (both systolic <140 and diastolic <90 mmHg) at post-intervention. BODY.METHOD.SAMPLE SIZE: On the basis of our prior study, an estimated sample size of 230 total patients will provide 90% power, with a one-tail α = 0.05, and standard deviation of 13 mmHg to detect a 5.0 mmHg difference between two arms in change on systolic blood pressure at 3-month follow-up from baseline, with a 1:1 allocation of 115 to each treatment arm. We assumed a 20% of participants lost to follow up and thus a total of 287 participants should be recruited. BODY.METHOD.STATISTICAL ANALYSIS: Intention to treat (ITT) analyses was conducted and all eligible 282 subjects were included in the intention to treat analysis ( Figure 1 ). We adopted multiple imputations (MI) approach to impute the missing values of SBP and DBP at 3 months since the baseline observations carried forward approach has recently been criticized for many limitations and may lead to serious bias [19], [20]. The imputation model included baseline systolic/diastolic blood pressure, age, sex, township, BMI and blood pressure lowering agents using at baseline. After excluding patients who were lost to follow-up, discontinued intervention, used outside salt, or died during the follow-up period, a total of 213 patients (99 in intervention arm, 114 in control arm) completed the follow up visit and were used in the per-protocol (PP) analysis. We used paired t-tests to test the difference in primary outcome between intervention and control, and we also used general linear model to adjust for variables used for randomization stratification (township, baseline systolic blood pressure, and gender) plus age, baseline BMI, and blood pressure lowering agents use. We were not able to adjust for other variables such as education, occupation, smoking and alcohol drinking since they were not collected in all participants. For the secondary outcomes, chi-square test was used. Primary outcome and secondary outcome were analyzed in both ITT and PP analysis. Statistical analyses were carried out using SAS 9.3 (SAS Inc., Cary, NC, USA). 10.1371/journal.pone.0110131.g001Figure 1Trial Flow Chart. BODY.RESULT.BASELINE CHARACTERISTICS: The mean age of the 282 randomized subjects were 63.1 years at baseline, and 58.9% were females, 94.5% were herdsman, 92.7% reported never going to school, 9.0% were cigarettes smokers, 11.7% were alcohol drinkers, and 47.0% used anti-hypertension medication in the past month. Baseline BMI was 23.6 kg/m2. All these major baseline characteristics were comparable between the two randomized groups (all p-values >0.05) ( Table 1 ). BODY.RESULT.EFFECTS ON BLOOD PRESSURE REDUCTION: Blood pressure was reduced in both groups and for both SBP and DBP at follow-up. However, the reduction was significantly larger in salt substitute treatment group for both SBP and DBP and in both ITT and PP analyses (Table 2). The intergroup unadjusted net reduction was −9.1 mmHg in systolic (95% CI: −3.2 to −15.0; p = 0.002) and −3.4 mmHg in diastolic blood pressure (95% CI: −0.3 to −6.4; p = 0.03) in PP analysis, but it was smaller at −7.7 mmHg in systolic (95% CI: −2.5 to −12.9; p = 0.004) and −3.0 mmHg in diastolic blood pressure (95% CI: −0.2 to −5.8; p = 0.035) in ITT analysis with multiple imputations approach ( Figure 2 ). After adjusting for baseline blood pressure, sex, age, township, baseline BMI and using blood pressure lowering agents, the net reduction was still significant, at −8.2/−3.4 mmHg (SBP/DBP) from PP analysis and at −7.6/−3.5 mmHg (SBP/DBP) from ITT analysis (Table 2). 10.1371/journal.pone.0110131.g002Figure 2Unadjusted mean change of blood pressure levels at the 3-month follow-up from baseline for the salt substitute group and regular salt group in ITT and PP analysis.a: Intent-to-treat with multiple imputations approach, N = 282. b: Per-protocol analysis, N = 213. 10.1371/journal.pone.0110131.t002 Table 2 Blood pressure at baseline and follow-up, reduction of blood pressure in each group and net reduction of blood pressure in salt substitute group in comparison with regular salt group. Variables Groups Baseline Follow-up △1 a △2 b p-value Per-protocol Analysis, (N = 213) SBP Regular salt 179.3 (23.6) 171.6 (27.1) −7.7 (22.3) −8.2 (2.9) 0.005 Salt substitute 174.1 (20.6) 157.3 (25.7) −16.9 (21.0) DBP Regular salt 106.7 (13.5) 103.3 (14.1) −3.4 (11.3) −3.4 (1.5) 0.023 Salt substitute 102.2 (12.0) 95.4 (14.8) −6.8 (11.1) Intent - to-treat Analysis with multiple imputations approach, (N = 282) c SBP Regular salt 177.6 (23.3) 170.2 (26.8) −7.5 (21.8) −7.6 (2.5) 0.003 Salt substitute 176.1 (22.4) 161.0 (27.0) −15.2 (21.7) DBP Regular salt 105.8 (13.1) 102.6 (13.8) −3.2 (11.1) −3.5 (1.4) 0.011 Salt substitute 103.2 (12.0) 97.0 (14.5) −6.2 (11.5) All numbers shown are mean (SD) except for the △2 that is shown in mean (SE). a. Mean reduction of blood pressure in each group after intervention. b. Net reduction of blood pressure in salt-substitute group in comparison with regular salt group, adjusting for baseline blood pressure, sex, age, township, baseline BMI and using blood pressure lowering agents. c. impute 10 times. BODY.RESULT.EFFECTS ON PROPORTION OF PATIENTS WITH BLOOD PRESSURE UNDER CONTROL: All participants enrolled had systolic BP equal or greater than 140 mmHg at baseline. After three months' intervention, PP analysis revealed that 8.8% in regular salt group and 19.2% in salt-substitute group had had their blood pressure well controlled (SBP/DBP<140/90 mmHg), (p<0.05). Although the peak remained around 160/95 mmHg (SBP/DBP), the overall blood pressure reduction in salt-substitute group was reflected by the dramatic shift of BP distribution to the left (Figure 3). 10.1371/journal.pone.0110131.g003Figure 3Distribution of Blood pressure at baseline and follow-up, per-protocol analysis. BODY.RESULT.ANTI-HYPERTENSION MEDICATION USE: At three months' follow-up period, the proportion of patients taking antihypertensive medications in the past two weeks was lower in salt-substitute group than in the regular salt group (34.7% vs. 46.4%), however the difference was not significant (p = 0.081). BODY.RESULT.SEVERE ADVERSE EVENTS: Three people died during the study period, two in control arm and one in the intervention arm. Two of the three died from cerebral hemorrhage, one in each group, and the third death was due to liver cancer. BODY.RESULT.SALT CONSUMPTION ESTIMATION: Families located in the central of a group of families that participated in the study were selected by our staff on site for the convenience to distribute study salt, according to their geographic location. Our study staff delivered study salt to these families and then called for the study participants living nearby to come to pick up the study salt. This gave us the opportunity to measure the salt container at these families and ask the questions to help us estimate relatively accurate the amount of salt consumption in these families. For the selected household, we weighted the amount of study salt delivered to the household at baseline and the amount of remaining study salt at the follow-up to estimate average daily salt consumption per person. A total of 54 (27 in each group) families had complete and accurate salt consumption data and reported not using other salt (e.g. crude salt locally available, and/or salt bought from stores). The estimated average daily sodium chloride intake was 20.0±5.4 grams in the salt substitute group versus 26.9±8.1 grams in the regular salt group (p = 0.025). The additional potassium chloride intake is estimated at about 7.7±2.1 grams in the intervention group. Comparison between participants who did and did not have salt consumption data showed no significant difference in age, gender, blood pressure, education, smoking and alcohol drinking except for those with no data of salt consumption had a significantly lower proportion in use of anti-hypertensive drugs (48.5% vs. 64.8%, p = 0.022). BODY.DISCUSSION: The present study demonstrated a significantly reduction in both SBP and DBP in salt-substitute group (by 7.6/3.5 mmHg in ITT analysis), which is expectedly even greater than what we found in China Salt Substitute Study (CSSS) conducted in rural Han Chinese, where a 5.4 mmHg reduction in SBP and no significant reduction in DBP were shown [15]. The result should be attributed most likely to higher blood pressure at baseline, but also markedly higher dietary salt intake, simpler diet and lower access to medical treatment in the Tibetan cohort in the present study in comparison to the CSSS's Han Chinese cohort. Blood pressure reduction effect of an intervention is usually directly related to the baseline level of blood pressure, this is shown in many blood pressure lowering drug trials as well as non-drug trials such as DASH and CSSS [15], [21]–[24]. Compared to CSSS in which the baseline SBP/DBP was 159/93 mmHg, the baseline blood pressure in our study was much higher (177/105 mmHg). Furthermore, salt consumption for participants in our study was very high at about 27 grams per day but is about 15 grams in the CSSS study populations [15], [25]. The Tibetan diet is simpler, characterized by low consumption of fresh vegetables and fruits, and high meat and fat. There are evidences that a diet with rich fruits and vegetables could be beneficial to blood pressure lowering [26]–[28]. Compared with diets of CSSS study participants, the diet of Tibetan study participants was much simpler and lacked components that might be helpful in lowering blood pressure. At last, CSSS found that the blood pressure reduction effect of salt substitute was lower among patients with blood pressure lowering agents [15]. It is understandable that use of anti-hypertension medications would diminish the BP lowering effect from salt substitute through highly possible interactions. While 61% of study participants were having blood pressure medications in CSSS [15], it was only 47.0% in the present study. With smaller possible interaction with drugs, in comparison with CSSS, our study hence would have greater effect in lowering blood pressure. In this study, the significant reductions of both SBP and DBP were also found in the control arm receiving regular salt. These changes could be attributable to regression to the mean as well as the seasonal variations in blood pressure since the present study was conducted from February to May mirroring the expected temperature progression. This temperature associated variance phenomenon in BP has been previously reported by our group's experience in the CSSS and many other prior reports [15], [29], [30]. In addition, we also found that blood pressure lowering agents use was lower in intervention group than in control group, though not statistically significant. This suggests the effect of salt substitute in lowering blood pressure demonstrated in the study would not be attributable to differential use of anti-hypertension medications. We believe that our observed effect would have an even higher magnitude if the use of anti-hypertension medications had been the same in both groups. The salt consumption data showed that the intervention group consumed 7 grams less sodium chloride and 7.7 grams additional potassium chloride on average per day, which, we believe, drove the blood pressure reduction significantly more in intervention group. Due to the imbalanced lost to follow up between intervention and control in terms of quantity and quality, we choose multiple imputations rather than baseline observations carried forward approach to impute the missing values in our ITT analysis. In fact, our intervention group lost more patients than control group (42 vs. 27 patients). Comparison of those lost in intervention and control showed that patients lost in intervention group had higher blood pressure (180.9 vs. 170.3 mmHg in SBP), less use of blood pressure lowering agents (42% vs. 50%), less women (29.6% vs. 70.4%), younger age (64.0 vs. 65.3 years) and higher BMI (23.6 vs. 22.8 Kg/m2) at baseline. Although the results from baseline observations carried forward approach showed a smaller net reduction in both SBP (−5.9 mmHg) and DBP (−2.7 mmHg) and could be significantly underestimated the real net reduction, our results of both approaches showing a significant net reduction in SBP/DBP argue strongly for the validity of our study conclusion. In order for population wide health intervention to be successful in a limited resource setting such as rural Tibet, the intervention and its delivery should not require a tremendous bandwidth of healthcare and financial capital. Furthermore, there is great importance for the cultural considerations in the design of the intervention, as approach of dietary restriction or outright elimination of the cultural dietary staple Tibetan yak buttermilk tea from study populations' diet would have been a difficult intervention to implement. However, with the dietary salt substitute, the intake of sodium was successfully modified by dietary replacement of normal salt and population education. Thus, dietary salt substitute intervention presented in this study has the tremendous potential for benefits to public health as an simple, low-cost, and effective interventional strategy for ameliorating the burden of hypertension related maladies in both limited resource settings (i.e. Tibet) and developed countries. The present study was limited by restricted financial resources available and austere environmental conditions (remote and rugged terrain at an elevation of 4300 m with serious oxygen deficit); which lead to a single follow-up at three months post randomization and precluded the ability to collect and analyze urinary sample to measure changes in sodium and potassium excretion. However, the present study was a randomized controlled trial design, which would minimize the biases and provide reliable observed effects yielding strong internal validity. Also due to the resource constraints, we were only able to collect baseline data on age, sex, SBP, use of blood pressure lowering agents and BMI. The other variables such as education, occupation, smoking and alcohol drinking were only collected for part of study participants and thus not included in the analysis for adjustment. However, the high comparability between the Intervention and Control in terms of these variables indicates that not having these variables adjusted would not significantly affect the internal validity. Thirdly, the salt consumption data collected in this study does not include sodium from natural foods and is only from a small fraction of the total study population. It should only be used for a rough estimation of sodium intake for local Tibetans. If possible, future studies in Tibet should consider use of 24-hour urine or dietary recalls to get more reliable data. Fourthly, our definition of hypertension excluded those with isolated diastolic hypertension and thus it should be cautious to extend our findings to isolated diastolic hypertension patients. Lastly, mean blood pressure in control group also dropped significantly after intervention. It was due to either seasonal blood pressure change or regression to the mean. The 'white coat hypertension' effect, if exist, should have been included in the regression to the mean. CSSS study had shown the similar seasonal blood pressure change where mean blood pressure reached the peak at the coldest season and the low ebb at the hottest season. However, our randomized parallel-controlled design well protected the validity of the study conclusion from the seasonal change and regression to the mean. Despite these limitations, this study was the first randomized control trial providing clear beneficial effects of salt substitute on blood pressure reduction in Tibet and area at high altitude. In conclusion, this study confirmed the prior evidence of the blood pressure-lowering effects achieved with reduced sodium salt substitute in other trials. The excellent effect in lowering blood pressure achieved in this trial showed great potential of salt substitute in prevention and control of cardiovascular disease in this area and other similar settings in the world. BODY.SUPPORTING INFORMATION: Protocol S1 English translation of original protocol. A study to develop simple interventions for control of hypertension in Tibet highland.(DOCX)Click here for additional data file. Protocol S2 Original protocol in Chinese. (DOC)Click here for additional data file. Checklist S1 CONSORT 2010 checklist of information to include when reporting a randomized trial. (DOC)Click here for additional data file.

TITLE: Benefits for elders with vulnerable health from the Chronic Disease Self-management Program (CDSMP) at short and longer term ABSTRACT.BACKGROUND: When health declines, older persons may benefit from an intervention program that strengthens their self-management and empowers them to keep in control of their own body and life. Therefore we conducted a Randomized Controlled Trial using the Chronic Disease Self-Management Program (CDSMP) in a sample of 169 older persons in frail health and in need of elderly care. ABSTRACT.METHODS: We assessed psychological coping resources and wellbeing, pre- and posttreatment and at 6-month follow-up, and investigated whether specific subgroups would benefit in particular from the intervention. ABSTRACT.RESULTS: The CDSMP appeared effective with respect to sense of mastery but only in the lower educated participants (p < .05). Furthermore, the intervention stabilized valuation of life in participants, whereas in the controls valuation of life decreased. The high appreciation score and low drop-out are indicative for the applicability of the CDSMP for this specific target group. ABSTRACT.CONCLUSIONS: We recommend integration of the ingredients of the program into the daily healthcare practice of professionals working with vulnerable older persons. This would involve professional guidance starting from interpersonal equality and emphasising a persons possibilities given their physical or cognitive limitations. This will help older vulnerable persons to focus on their own attainable goals and to experience being successful. ABSTRACT.TRIAL REGISTRATION: The trial was registered in the Dutch Trial Register as NTR 1173 at 08-03-2008; 'Is selfmanagement benefical for well-being of average older persons?' http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=1173 BODY.BACKGROUND: Because of the increase in life expectancy, a greater number of older people will have age-related diseases and may suffer from the difficulties due to persistent health decline. Studies using different frailty-instruments showed prevalence rates between 12 % and 36 % in people aged 65 and over in the Netherlands [1, 2]. Lee et al. [3] state that prevalence of frailty increases with age, affecting an estimated 16 % of those aged 80 to 84 and 26 % of those aged 85 and older. Although the Dutch disability level is among the lowest in developed countries [4, 5] a considerable proportion of the population still will have to face the challenge of coping with health decline. Older persons confronted with deteriorating health often experience lower levels of well-being [6–8]. The availability of coping resources like mastery, self-esteem and self-efficacy have been shown to buffer the negative influence of deteriorating health on well-being; moreover, associations between persistent health decline and decreasing well-being are partly explained by decreasing availability of psychological coping resources [9–12]. In order to optimize well-being of the growing number of older persons in vulnerable health, it seems a priority to enhance coping resources, and by doing so, to empower older persons. Therefore it is important to investigate specific interventions that are developed to maintain or improve optimal coping resources in these persons. Self-management programs are proposed as one of the ways for older persons to more actively manage their own process of ageing in such a way that the availability of coping resources and, as a consequence, well-being is increased and maintained as long as possible [13]. Specifically the Chronic Disease Self-Management Program (CDSMP) is a structured intervention that emphasizes the strengthening of self-management by older persons with deteriorating health in order to empower them to keep in control of their own body and life [12, 14–17]. The CDSMP is the only intervention programme that focuses on people with one or more chronic diseases regardless of the specific disease, and that aims at stimulating patients to become more actively involved in the management of their own health and enabling them to take better care of themselves [18]. Previously, we conducted a systematic review [19] on nine Randomized Controlled Trials of the CDSMP. Overall, the studies reviewed showed that the CDSMP led to more physical exercise, less health distress, better self-care and had a beneficial effect on self-efficacy measures. Thus, the CDSMP seems a promising intervention. However, in most RCT's the average age was not very high. So far, the effectiveness of the CDSMP has not yet been determined in frail older people with heterogeneous chronic diseases and who are dependent on old-age care. Therefore, we conducted an intervention study on the CDSMP in this population. It is hypothesised that participating in the CDSMP leads to improved coping resources and well-being. A second aim of our study was to investigate whether specific subgroups benefit more from the intervention than others. For instance, it may be expected that persons with good cognition and higher education benefit more than persons with low cognition or education. Findings may result in a specific profile of people most likely to benefit from the program. BODY.METHOD.THE INTERVENTION: The central aim of the Chronic Disease Self-Management Program (CDSMP) is to teach people to cope with multiple chronic diseases. The CDSMP is based on prior experience with an arthritis self-management program, literature review, needs assessments and the theoretical framework of self-efficacy [9, 15, 20]. The underlying mechanism that explains the positive effects on health behaviour, health status, self-management behaviour and health care utilization, is assumed to be self-efficacy. This is defined as 'believing in one's capability to organize and execute the courses of action required to produce given attainments' [20]. The CDSMP incorporates strategies to enhance self-efficacy and by doing so to enhance self-management behaviour and health related outcomes. By means of weekly action-planning and feedback, participants modelling behaviour and problem-solving for each other, re-interpretation of symptoms, group problem solving and individual decision-making, the program is executed [21]. Three principal assumptions underlie the CDSMP:People with different chronic diseases have similar self-management problems and disease-related tasks.People can learn to take responsibility for the day-to-day management of their diseases.Confident, knowledgeable patients practicing self-management will experience improved health status and will utilize fewer health care resources. The program is accessible and easy to implement, because it is inexpensive and widely available, and the intervention can be delivered by trained lay-persons. The CDSMP focuses on several topics including physical exercise, nutrition, breathing, emotions, communication and medication, which are discussed during six weekly sessions of 2.5 h. The study protocol has been approved by the Ethical Review Board of the University Medical Center Groningen (UMCG). The study protocol can be obtained from the corresponding author. BODY.METHOD.SETTING AND STUDY SAMPLE: We recruited older people who participated for one or more days a week in an elderly day-care facility with several locations. The research team advertised the intervention through personal announcements to participants and information sessions by visiting the day-care facility themselves. Formal caregivers at the facilities were informed and potential participants were sent an information letter by their caregivers, 169 of whom gave written informed consent. With the remaining 21 persons, no contact could be established. Research assistants then contacted the participants and carried out a baseline measurement. We aimed for a power of 80 % to detect a minimum difference in the main outcome measures between two independent sample means, at alpha .05 [22]. Power analysis revealed that we needed to include 160 participants. BODY.METHOD.RANDOMISATION: Participants were randomised to the CDSMP programme or to a waiting list control group that received care as usual and was promised participation after 6 months. Randomisation was based on the existing units of day-care groups in two day-care locations. Thus, at each location of the day-care facility, candidate participants were randomised groupwise per weekday on which they normally received the day-care. Groups of 10–15 participants were included. Each group was supervised by two well-trained nurses, to guarantee continuation of the group sessions in case of absence of one of the supervisors. BODY.METHOD.DESIGN: Baseline measurements included participant characteristics and initial values of the outcome measures. After the intervention of 6 weekly sessions were completed, follow-up measurements of the outcome measures was conducted. These measurements were repeated 6 months after completion of the intervention. BODY.METHOD.MEASUREMENTS.OUTCOME MEASURES: The choice for coping and wellbeing as outcome measures was based on the frequently reported evidence that psychological coping resources, such as mastery [23], self-esteem [24] and self-efficacy [9] favorably affect a person's way of coping with deteriorating health [12, 25]. The main outcome measures are psychological coping resources (mastery, self-esteem and self-efficacy) and wellbeing (positive affect, life satisfaction, valuation of life and depressive symptoms). Sense of mastery is conceptualised as the extent to which a person perceives him or herself to be in control of events and ongoing situations and reflects the perception of the ability to manage them. This was measured by a 5-item abbreviated version of the Pearlin Mastery scale [11, 26] which included questions like 'I have little control over things that happen to me'. Each item is scored on a five-point scale, the total score is the sum of the ratings, with range 5–25, such that a higher rating indicates more feelings of mastery. Self-esteem is measured by a scale that consists of four questions like, 'feeling self-assured', 'positive attitude towards one's self'and 'feeling useless' that are scored on a five-point scale [11, 27]. The score is the sum of the ratings, with range 4–20. People with higher self-esteem (i.e., higher scores) are supposed to have a more positive view of their identity. Self-efficacy refers to personal judgements of how well behavior can be implemented in situations that contain novel, unpredictable or stressful elements as well as ordinary situations [9]. Self-efficacy was measured by a twelve-item version of the Perceived Self-Efficacy Scale [28, 29]. The scale included questions like 'If I made a decision to do something, I will do it.' and 'I have difficulties solving problems well in my life'. Each question is scored on a five-point scale, the total score is the sum of the ratings, with range 12–60, with a higher score indicating a higher level of self-efficacy. Depressive symptoms were measured with the Centre for Epidemiological Studies-Depression scale (CES-D) [30]), which assesses depressive symptoms. The CES-D is a 20-item scale that asks participants to indicate how frequently they experienced certain psychological symptoms or feelings during the previous week. Each question is scored on a four-point scale, the total score is the sum of the ratings, with range 0–60, with a higher score indicating more depressive feelings. Positive affect was measured using a subscale of the CES-D. Radloff [30] described four separate dimensions of the CES-D. One of the dimensions is positive affect, including four of the CES-D items which refer to positive feelings: 'enjoying life', 'feeling happy', 'being hopeful about the future' and 'feeling as good as other people'. The items are scored on a four-point scale. This sub-scale ranges from 0 (low) to 12 (high). The use of this subscale as an independent concept is supported by previous research [31]. Higher scores indicate higher positive affect. To assess Life satisfaction, two questions 'Have you been satisfied with your life lately?' and 'Are you satisfied with your life, up until now?' were asked [32] The questions are scored on a five-point scale, and the sum score ranged from 2 (very dissatisfied) to 10 (very satisfied). Valuation of Life (VOL) is considered as a cognitive scheme which refers to "the subjective experienced worth of a person's life, weighted by the multitude of positive and negative features whose locus may be either within the person or in the environment" [33]. The Dutch version of the VOL-scale [34] consists of 12 statements, about the value of life, such as:'It is difficult for me to find meaning in my daily routine' or 'At this moment I have a strong will to live'. Each item is scored on a five-point scale, with the sum score ranging from 12–60, higher scores indicate higher valuation of life. BODY.METHOD.MEASUREMENTS.POTENTIAL CONFOUNDERS: Variables that may confound the effect of participating in the CDSMP on the outcome measures were taken into account. Age, sex, income category, partner status, years of education, help received with personal care and household tasks, chronic diseases, and cognitive function were considered potential confounders. Age was measured by years and months of age and sex was measured by observing the gender (male or female). Income category was measured by asking about income with three questions on receiving state pension, private pension, and savings. Partner status was measured by asking whether the respondent was living with someone they considered as their partner (yes/no). Education was measured by asking about the number of years of education that was received. Personal care and household care were measured by asking whether the respondent received help with personal and household care, respectively (yes/no). The presence of chronic diseases was determined by asking the respondents whether they had any of the following diseases: cardiac disease; peripheral arteriosclerosis of the abdominal aorta or the arteries of the lower limb; stroke; diabetes mellitus; lung disease (asthma or chronic obstructive pulmonary disease); cancer; arthritis; or any other major chronic disease. The number of chronic diseases was calculated by summing all the specific diseases reported. In a validation study, the respondents' self-reports were compared with information obtained from their general practitioners, and were found to be sufficiently reliable [35]. Cognitive functioning was measured by means of the Mini Mental State Examination (MMSE) [36] a frequently used screening instrument for global cognitive dysfunctioning. For 23 questions and tasks the respondents scored 1 or more points if they gave the correct answer or performed the task correctly. The scores could vary between 0 (all answers incorrect) and 30 (all answers correct). Higher scores indicate better cognitive functioning. BODY.METHOD.STATISTICAL ANALYSES: First, unpaired t-tests and chi-square tests were performed to compare the participant characteristics and the baseline scores of the intervention and the control group regarding coping and well-being outcomes. Next, paired t-tests and repeated measures-analyses using General Linear Models were performed to assess treatment effects between baseline and post-intervention and between baseline and 6-month follow-up. Differences and changes were considered significant when p < .05. To examine whether the participant characteristics moderate the association between the intervention and well-being or coping resources, a series of multivariate analyses using General Linear Models was performed for each characteristic. Each multivariate model examined whether the product term (intervention X characteristic), was significant (p < .10). When the interaction term was found significant, the effect of the intervention was investigated in stratified analyses. We stratified on quartiles and median or mean of the characteristic to investigate the optimal distinction between groups. BODY.RESULTS.SUBJECTS: Of the169 participants, 78 (46 %), aged 81 years, were assigned to one of the intervention groups, while the rest, aged 83 years, constituted the control groups. In Fig. 1 the inclusion and drop-out of participants is shown. As can be seen, no patients dropped out before starting the intervention. Seven participants did not complete the first post-intervention interview because they were (too) ill (N = 3), died (N = 1), or were confronted with hardship in the family (N = 1). Two participants did not give a specific reason for quitting. From these seven drop-outs, three had been assigned to the intervention group. Another 10 persons did not complete the 6-month follow-up interview due to illness (N = 4), death (N = 5) and one person from the control group stopped participation because she was unhappy waiting for the course, leaving 152 participants in the study (72 in the intervention group and 80 in the control group).Fig. 1Enrolment procedure (original N = 190) The intervention took place in existing groups in an elderly day-care facility. However, within these groups some participants had severe cognitive impairment at baseline or follow-up (MMSE ≤15 [37]. In order not to compromise the group dynamics and hurt people's personal feelings, we chose to treat these participants like the others considering the follow-up interviews and actual participation in the intervention. However, the participants with severe cognitive impairment were excluded from the analyses. Therefore our study sample consisted of 132 persons, 63 of whom participated in the CDSMP and 69 were in the waitinglist control group. Attendance of the intervention meetings was high with an average of five of the six sessions that were offered. Characteristics of participants at baseline are shown in Table 1. At baseline, the intervention group did not differ from the control group with respect to age, sex, income, partner status, help with personal care, household care, chronic diseases and cognitive functioning. Education level was somewhat lower in the intervention group (p = 0.08). Scores on Self-efficacy, Mastery and Valuation of Life were significantly lower in the intervention group than in the control group.Table 1Characteristics of the sample at baselineIntervention groupControl groupP-value* N = 63 N = 69A. Participant characteristicsSex, % female (SD)90.5 (.30)85.5 (.36).38Age, mean (SD)81.57 (7.53)83.09 (5.75).20Education in years, mean (SD)8.46 (2.54)9.32 (3.06).08Chronic diseases, mean (SD)1.97 (1.38)2.23 (1.29).26Frailty, mean (SD)5.45 (2.90)4.91 (2.75).28Cognitive functioning, mean (SD)24.52 (4.12)25.62 (3.38).10B. Coping resources and Well-beingSelf-efficacy, mean (SD)39.56 (6.22)41.77 (6.22).04Mastery, mean (SD)22.02 (4.98)24.41 (4.84).01Self-esteem, mean (SD)9.98 (3.02)10.55 (2.47).24Life satisfaction, mean (SD)7.79 (1.30)7.72 (1.32).55Valuation of Life, mean (SD)41.38 (6.83)44.46 (6.23).01Positive affect, mean (SD)11.38 (3.19)11.90 (2.85).33Depression, mean (SD)34.05 (9.27)32.84 (8.25).43* t-tests and chi square test BODY.RESULTS.EFFECT OF THE INTERVENTION: For each group, paired t-tests were performed between baseline and post-intervention and between baseline and 6-month follow-up (Table 2). The results from the post-intervention 6-week assessment, show that the outcome measures Self-efficacy and Valuation of Life decreased significantly (p < .01) for the control group whereas they stayed stable in the intervention group. At 6-month follow-up, scores on Self-efficacy (p = .01) and Valuation of Life were still lower in the control group (p = .02). Furthermore, on Mastery and Depression results showed positive changes in the intervention group at 6-month follow-up. Mastery improved (p = .01) whereas scores on Depression decreased (p = .05) significantly. Self esteem, Positive Affect and Life satisfaction did not show any difference between the control and intervention group, at both follow-ups.Table 2Differences between the intervention group and the control group with respect to the outcome measuresBaseline - post interventionBaseline - 6 month follow upMtpsdMtpsdControl groupSelf-efficacy−2.43−3.83<.0015.19−1.74−2.64<.015.47Mastery-.40-.82.414.02-.61-.99.325.09Self esteem-.27-.97.342.27.43.15.282.39Depression*1.10.94.359.78Positive affect.15.37.713.29-.45−1.13.263.29Life satisfaction.06.39.701.25-.09-.59.561.23Valuation of Life−1.93−3.03<.0015.21−1.61−2.50<.025.35Intervention groupSelf-efficacy-.10-.15.885.05-.48-.71.485.36Mastery.06.12.904.141.492.81<.014.22Self esteem-.33−1.21.232.19.461.39.172.62Depression*−1.7−2.01<.056.71Positive affect-.38−1.00.323.03.10.23.823.25Life satisfaction-.13-.79.431.28-.05-.28.781.34Valuation of Life−1.08−1.41.166.1-.67-.86.396.14*Note: Full CES-D measured only at baseline and 6-months follow-up In addition, we conducted analyses of variance by means of repeated measures to assess treatment effects, adjusted for confounders and the baseline scores of the outcome measures. Table 3 shows the results of the effect of participating in the CDSMP on change in coping resources and well-being at short (6 weeks) and longer (6 months) term. We included years of education as the only potential confounder because a difference between both study groups was observed at baseline (p .08). A significant effect of participating in the CDSMP was found for Mastery and Valuation of Life. Compared to the control group, participating in the CDSMP led to significantly higher scores on Mastery at short-term. However, the effect size was rather small. Valuation of Life was stable for CDSMP participants immediately after the course and this effect was still present at six months, whereas participants of the control group were confronted with decreasing scores. These effects were also small (partial eta2 < 0.06). Participating in the CDSMP did not lead to change in the other outcome measures.Table 3Longitudinal association between participating CDSMP and change in coping resources and well-being (adjusted for years of education)Self efficacyMasterySelf esteemValuation of LifePositive affectLife satisfactionDepressionT0-T1T0-T2T0-T1T0-T2T0-T1T0-T2T0-T1T0-T2T0-T1T0-T2T0-T1T0-T2T0-T1T0-T2Multivariate modelF.551.596.042.761.50.524.375.332.4.12.77.13n.a..011P value.46.21.02.10.22.47.04.02.12.73.38.72.74Partial Eta2.00.01.05.02.01.00.03.04.02.00.01.00.00 BODY.RESULTS.SPECIFIC SUBGROUPS: For investigating a moderator effect of participant characteristics on the effect of the intervention on the outcome measures after 6 months, the product term of intervention X characteristic was entered into the separate models for each characteristic. The product term intervention X education was significant with mastery as the outcome. The product terms intervention X education and intervention X cognitive functioning were significant for depressive symptoms as the outcome (Table 4).Table 4Interaction analysis of the effect of potential predictor on outcome measures (only those outcome measures with significant interactions are shown, P < 0.1)Product termOutcomeFPPartial Eta2Intervention XAgeDepression.03.87.00Sex.03.87.00Education3.49.06.03Cognition3.00.09.02Frailty1.76.19.01AgeMastery.13.72.00Sex.05.83.00Education2.88.09.02Cognition.10.75.00Frailty.48.49.00 For the outcome mastery, the optimal cut-off in years of education was < = 9 years and >9 years of education: each of which constituted 50 % of the sample. Multivariate analyses of variance showed a significant positive effect (p < .05) of intervention on mastery for respondents with low education. This was in contrast with the results for respondents with higher education, who showed no significant effect from the intervention (Table 5).Table 5Stratified analysis on cognitive functioning on the effect of PDF on change in Depression and on educational level on the effect of PDF on change in Mastery and DepressionChange in DepressionFPPartial Eta2Mean scoresControlInterventionT0T2T0T2Cognition Low (MMSE < =25)1.99.16.0332.232.336.634.1Cognition High (MMSE > =26)2.89.09.0433.335.031.430.5Change in MasteryFPPartial Eta2Mean scoresControlInterventionT0T2T0T2Education Low (<=8 years)4.19<.05.0625.123.121.323.3Education High (> = 9 years).42.52.0123.824.422.924.0Change in DepressionFPPartial Eta2Mean scoresControlInterventionT0T2T0T2Education Low (<=8 years).87.35.0132.233.135.333.5Education High (> = 9 years)1.231.27.0233.434.732.430.8 With respect to the outcome cognitive functioning after 6 months, stratification based on median scores of respondents (MMSE < =25 & MMSE >25) showed a trend (P = .09) that persons with better cognitive performance seemed to benefit from the intervention (Table 5). This was in contrast to the results for respondents with lower cognitive performance, who showed a non-significant increase of depressive symptoms 6 months after the intervention. Stratification of the sample based on the low and high quartiles of the MMSE did not show significant effects from the intervention in the distinguished groups. Finally, for the outcome depressive symptoms after 6 months, stratification of the sample for the level of education according to the median and the lowest and highest quartiles, did not result in significant effects from the intervention although the depressive symptoms decreased in the intervention group whereas persons in the controlgroup showed an increase of depressive symptoms. BODY.RESULTS.SUBJECTIVE EVALUATION OF PARTICIPATING IN THE CDSMP: Table 6 shows the results from the subjective evaluation of the intervention among all 63 participants. The participants attended on average 5.7 of the 6 sessions that were provided. They rated the CDSMP with an appreciation of 8.0 (scale 0–10). All of them enjoyed following the CDSMP and 92 % claimed usefulness of the content. Almost all participants (98 %) found the way the intervention was presented agreeable.Table 6Qualitative evaluation after participation CDSMPParticipants (N = 63)EnjoyableUsefulnessTeaching approachAttendanceJudgement on appreciation(%)(%)(%)(6 sessions)(0–10)Totally agree7357875.78.0Agree173511Neutral052Disagree020Totally disagree020 BODY.DISCUSSION: Our study of the Chronic Disease Selfmanagement Program in elders with a vulnerable health, with a mean age of around 81 years showed that the program seems effective with respect to sense of mastery but only in the lower educated participants. Also, the intervention had a stabilizing effect on valuation of life, whereas in the controls valuation of life decreased. Furthermore, almost all of the participants scored positively on the content and style of the program, gave high appreciative scores, showed a high attendance rate and low drop-out. In all, our findings support the applicability of the CDSMP for this specific target group. Thus far, no other RCT of the CDSMP included Mastery as an outcome variable, although mastery has been shown to be important for maintaining well-being in people with deteriorating health. It has frequently been reported that psychological coping resources, such as mastery, favorably affect a person's way of coping with deteriorating health [25, 38]. It has also been found that greater availability of coping resources is associated with better well-being in chronically diseased persons [39–42]. In addition, a mediating and moderating effect of mastery was demonstrated in our previous study on the association of deteriorating health with wellbeing [19]. In addition to mastery, well-being is also under pressure from deteriorating health [8]. Research in older persons confronted with deteriorating health shows that various aspects of well-being decrease (e.g. [6, 7, 43]). The results of our well-being measures positive affect and life satisfaction did not show an improvement, but we did find a positive effect on Valuation of Life. From an earlier review [19] we learned that the CDSMP was consistently beneficial for Health behaviour, especially with regard to the variables of exercise and self-care. For Health status, the majority of studies only showed improvement in the domain of health distress. Most of the studies that investigated self-efficacy showed convincing improvement in self-efficacy, cognitive symptom-management and mental stress-management. From this study on nine RCTs using the CDSMP, only Haas [44] found an improvement on emotional well-being among their sample of older adults with chronic low back pain. Griffith [45] and Kennedy [46] showed improvement in psychological well-being and quality of life in younger persons with co-morbidity. As the program includes action plans that are formulated after each of the sessions and evaluated at the start of the next session, one might expect that the CDSMP would lead to better self-efficacy scores. This has indeed been reported in several studies [45–49] in samples with various chronic diseases. However, in our sample of older people in vulnerable health we did not find an effect on self-efficacy. In a Dutch sample of chronically diseased patients [18] did not find an effect either. They argued that this might have been caused by a ceiling-effect because their patients already had high baseline levels. Our more vulnerable population could have improved in self-efficacy, because they initially scored well below the ceiling of the scale. In fact, self-efficacy scores declined in both intervention and control groups, but less so in the intervention group. Possibly, a longer follow-up would have yielded significant differences. In addition to the evidence of an overall benefit from participating in the CDSMP in frail older adults, on Mastery directly after the intervention and on Valuation of Life after 6 months, we also found that low educational level and good cognitive functioning increase the likelihood of profiting from the program on coping and well-being outcomes. A low educational level may imply more room for improvement and development in the participants. Improvement of mastery for lower educated older persons in vulnerable health seems of great importance considering several studies that show low educational level to be associated with a lower sense of mastery (e.g. [24, 38]) and that low mastery has been shown to increase the risk of poor mental health [50]. In the study of Dalgard et al. [51] a sense of mastery even emerged as a strong mediating variable between level of education and psychological distress. The observed beneficial effect of the CDSMP for frail persons with good cognitive functioning on depressive symptoms seems also relevant, considering the high prevalence of depression among older people [52]. BODY.DISCUSSION.STRENGTHS AND LIMITATIONS: We consider it a strength that we conducted, to our knowledge, the only RCT of the CDSMP that was performed with this specific target group of very old and frail persons with healthcare needs. A further strength is that we were able to conduct a randomized controlled trial among these vulnerable older persons. A host of intervention studies on CDSMP were conducted with only a pre-posttest design [19] whereas only well-designed RCTs can help us to understand what type of intervention promotes a specific change in behaviour, because testing of interventions is possible only when a well-chosen and well-described control group is in place. If not, this may lead to "evidence inspired" rather than evidence based practice [53]. A limitation of our study concerns the number of participants. To perform further in-depth analyses on the original sample – that already was rather small – the number of respondents that we could include was sometimes low. This reduces the power of our study, and may have led to an underestimation of the effects of CDSMP. We aimed for a power of 80 % but due to drop-out, the needed sample size of N = 160 was not achieved. We feel that our vulnerable participants could have benefitted more from the program had it included more sessions to keep the self-management attitude under attention. A further limitation is the application of multiple comparisons. Concerning this issue, we have not made a Bonferroni correction because of the chance that we have found a type two error is very small (one of 20) and the findings in our study are not unexpected and consistent with other research [54]. BODY.CONCLUSION: Because vulnerable older persons, who are often confronted with deteriorating health, may benefit from adequate coping strategies, we consider this program to be successful because of its positive effects on mastery and valuation of life. Also the fact that almost all of the participating persons scored positively on the content and style of the program, the high attendance rate, and the low drop-out is indicative for the applicability for this specific target group. When older people's health deteriorates to a certain point, participating in a course will become difficult. We therefore recommend integration of the ingredients of the program into the daily healthcare practice of professionals working with vulnerable older persons. This may be achieved when professional guidance starts from people's possibilities, considering their physical or cognitive limitations, stimulating them to focus on their own attainable goals and providing them with the experience of being successful. Our findings suggest that integrating ingredients of the program into daily healthcare practice might be beneficial for frail older persons in institutions. However, implementation studies are necessary to study this further.

TITLE: Superiority of duloxetine to placebo in improving diabetic neuropathic pain: Results of a randomized controlled trial in Japan ABSTRACT.ABSTRACT: Aims/Introduction: Duloxetine has been suggested to exert analgesic effects by activating the descending inhibitory system through inhibition of serotonin (5‐HT) and noradrenaline (NA) reuptake. This randomized controlled trial investigated the efficacy and safety of duloxetine in Japanese patients with diabetic neuropathic pain (DNP). Materials and Methods: Duloxetine 40 or 60 mg/day or placebo was given orally once daily for 12 weeks. The primary efficacy measure was weekly mean 24‐h average pain severity score on the 11‐point Numerical Rating Scale. Results: At 12 weeks vs baseline, the 24‐h average pain score (adjusted mean ± SE) was significantly improved in the combined duloxetine (−2.47 ± 0.18) and duloxetine 40 mg (−2.41 ± 0.21) and 60 mg groups (−2.53 ± 0.21) as compared with the placebo group (−1.61 ± 0.18). Duloxetine also exerted significant improvements over the placebo in nearly all secondary outcome measures including 24‐h worst pain, night pain, Brief Pain Inventory (BPI) pain scores, Patient's Global Impression of Improvement (PGI‐I) and health outcome measures, namely, various BPI interference scores. The incidence of adverse events (AE) was higher in the duloxetine groups than in the placebo group (duloxetine overall, 84.8%; duloxetine 40 mg, 84.7%; duloxetine 60 mg, 84.9%; placebo, 73.7%). Most AE were mild or moderate in severity, and resolved or relieved. There were no clinically significant safety concerns. Conclusions: Duloxetine 40 or 60 mg/day showed superiority over the placebo at reducing pain scores in patients with DNP. Duloxetine is safe, efficacious and clinically useful in the management of DNP. This trial was registered with ClinicalTrials.gov (no. NCT‐00552175). (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00073.x, 2010) BODY.INTRODUCTION: Recently, the number of diabetic patients in Japan has increased. It is now thought to amount to 8.9 million, or 22.1 million when including incipient diabetic individuals1. Among three major complications of diabetes mellitus, diabetic neuropathy seems to have the highest incidence, with 36.7% of diabetic patients reported to be suffering from this condition2. Diabetic neuropathic pain (DNP) is characterized by the symptomatic nature of an aching, burning, tingling or stabbing sensation3. DNP not only is often increased at night and affects sleep4, but also interferes with daily life, leading to deterioration of quality of life and a depressive state in severe cases5. Epalrestat and mexiletine hydrochloride are approved and widely used in Japan for the indication of DNP. Drugs listed as therapeutic options for DNP in Evidence‐based Practice Guideline for the Treatment of Diabetes in Japan6 include epalrestat, mexiletine hydrochloride, antidepressants, anticonvulsants, non‐steroidal anti‐inflammatory drugs (NSAIDs) and sustained‐release oxycodone. NSAIDs might be efficacious against mild DNP, but not against moderate and severe forms. Tricyclic antidepressants, certain anticonvulsants and opioid analgesics are recommended for the treatment DNP, but might be limited by side effects7. Serotonin (5‐HT) and noradrenaline (NA) have been implicated in the modulation of intrinsic analgesic mechanisms through descending inhibitory neurons in the brain and spinal cord8–11. An imbalance in these neurotransmitter mechanisms might contribute to central sensitization and hyperexcitability, thereby leading to persistent pain in DNP12. Current evidence suggests that antidepressants that have been shown to have analgesic effects in pain conditions exert such analgesic effects independent of improvement in mood or anxiety13,14. Instead, potentiation of 5‐HT and NA activity in the central nervous system (CNS) through inhibition of their reuptake has been suggested as a probable mechanism of the analgesic action of antidepressants against neuropathic pain15,16. Duloxetine hydrochloride is a selective and potent 5‐HT and NA reuptake inhibitor (SNRI)17 that has been shown to be effective in animal models of persistent and neuropathic pain18. Recently, duloxetine at doses of 60 mg/day given once or twice daily (120 mg/day) has been shown to be efficacious for the relief of pain in patients with DNP in randomized, double‐blind, placebo‐controlled clinical trials19–21. Based on this evidence, duloxetine was approved for the indication of DNP by the FDA in 2004. Presently, duloxetine has been made available in 90 countries as a therapeutic drug for DNP, and is recommended for this purpose by a number of USA and European guidelines22–25. In Japan, duloxetine has not yet been authorized as an approved therapeutic drug for DNP, although it is indicated for major depressive disorders. Tolerability and safety of duloxetine at dosages ≤60 mg/day have been confirmed in Japanese subjects during a phase I study26. In a double‐blind, placebo‐controlled phase II study of duloxetine ≤60 mg/day in Japanese patients with DNP, there were no safety concerns (unpublished data). Furthermore, data from phase II studies carried out in Japan and abroad19–21 suggest that duloxetine ≥40 mg/day might improve DNP in Japanese patients. The objective of the present study was to verify the superiority of once‐daily oral dosing with the SNRI duloxetine 40 and 60 mg/day combined vs placebo therapy using as primary end‐point weekly mean change of 24‐h average pain score on the 11‐point Numerical Rating Scale27. BODY.MATERIALS AND METHODS.PATIENTS: Patients, men and women aged 20–<80 years, had to have sustained pain for ≥6 months as a result of distal symmetric polyneuropathy caused by type 1 or type 2 diabetes mellitus. Pain was assessed on the local site of the foot, leg or hand with reference to the symptoms of an aching, burning, tingling or stabbing sensation and allodynia. Diagnosis of neuropathy was based on a revised version of the abbreviated diagnostic criteria for distal symmetric polyneuropathy proposed by the Diabetic Neuropathy Study Group in Japan (revised in 2002)28. The other main criteria for selection of subjects included glycated hemoglobin (HbA1c) ≤9.4% at screening, fluctuation of HbA1c≤1.0% at 42–70 days before screening, and the weekly mean of the 24‐h average pain score rated by the 11‐point (0–10) Numerical Rating Scale27 collected from patient diaries over 7 days before initiation of the study drug administration being ≥4, that is, indicative of moderate or severe pain. Main exclusion criteria were patients with psychiatric diseases, such as mania, bipolar disorder, depression, anxiety disorders and eating disorders, or patients with history of these diseases that needed any pharmacotherapy during the past year. Patients were also excluded if they had a complication that might affect assessment of DNP, such as neurological disorders unrelated to diabetic neuropathy, a skin condition in the area of the neuropathy that could alter sensation and other painful conditions. Patients were allowed to take a maximum daily dose of 1.5 g of acetaminophen, but no other drugs and therapies for DNP were allowed. Apart from insulin dose level, changes to existing diabetes treatments were prohibited. Before randomization, an assigning table was prepared using Create Key Code 3.3. Patients were randomly assigned to duloxetine 40 or 60 mg or placebo groups in a 1:1:2 ratio by stochastic minimization allocation taking into account the following four factors: (i) weekly mean of 24‐h average pain score at baseline < or ≥6; (ii) duration of diabetic neuropathy < or ≥2 years; (iii) type 1 or type 2 diabetes mellitus; and (iv) each study center. BODY.MATERIALS AND METHODS.STUDY DESIGN: Enrolment for the present study, which was carried out at 73 centers in Japan, began in December 2007 and ended in March 2009. This was a multicenter, randomized, double‐blind, placebo‐controlled, group‐comparison phase III study in patients with DNP. The primary objective was to evaluate the efficacy of oral duloxetine 40 or 60 mg/day once daily vs placebo against DNP. A screening period for 1–2 weeks during which entry criteria were evaluated without study medication was followed by a 13‐week treatment period when subjects were treated with duloxetine 40 or 60 mg/day or placebo then a 1‐week post‐treatment period without study medication. Considering the safety of patients, a gradually titrating phase for the first 1–2 weeks initiating with 20 mg/day and increasing the dose at 20‐mg weekly increments was set during the treatment period. There was also a 1‐week tapering phase. Patients were seen at biweekly visits for the first 4 weeks of treatment then every 4 weeks thereafter. The present study conforms to the principles of the Declaration of Helsinki and the study protocol was approved by the Institutional Review Board at each center. All patients provided written informed consent before participating in any study‐related procedures. BODY.MATERIALS AND METHODS.EFFICACY EVALUATION: The primary efficacy measure for the present study was the reduction of the weekly mean of the 24‐h average pain score as measured by the 11‐point (from 0 = no pain to 10 = worst possible pain) Numerical Rating Scale recorded in a diary by patients each day. Secondary efficacy measures were pain severity for 24‐h worst pain and night pain as measured by the 11‐point Numerical Rating Scale, Patient's Global Impression of Improvement (PGI‐I) Scale29 recorded at weeks 2, 4, 8 and 12, and severity and interference portions of Brief Pain Inventory (BPI)30 recorded at randomization and weeks 2, 4, 8 and 12. BODY.MATERIALS AND METHODS.SAFETY EVALUATION: Safety measures included spontaneously reported adverse events (AE), concomitant medications, bodyweight, vital signs (such as sitting blood pressure and heart rate) and ECG being recorded at each visit. Laboratory parameters including hematology, blood chemistry, HbA1c, lipids and urinalysis were generally recorded every 4 weeks. The value for HbA1c (%) was estimated as a National Glycohemoglobin Standardization Program (NGSP) equivalent value (%) calculated by the formula HbA1c (%) = HbA1c (JDS) (%) + 0.4%, considering the relational expression of HbA1c (JDS) (%) measured by the previous Japanese standard substance and measurement methods and HbA1c (NGSP)31. For patients who discontinued the study, the aforementioned assessments were collected at their last visit. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The present study was planned to enrol 300 patients (150 for the placebo group and 75 each for the duloxetine 40 and 60 mg/day groups). The study would have ≥80% power to detect a difference between the combined duloxetine group and placebo group by t‐test when the effect size of the combined duloxetine group to placebo group was taken as 0.33 and the level of significance as one‐sided 0.025. Efficacy analysis was carried out using data on all randomized patients with at least one post‐baseline assessment. Efficacy analysis of the primary end‐point was made by comparing the combined duloxetine group vs placebo group with regard to change of weekly mean of the 24‐h average pain score from baseline to week 12 by incorporating all the weekly mean changes obtained at each point of the post‐baseline measurements into a mixed‐effects model repeated measures32. Secondary end‐points were made by comparing the duloxetine 40 and 60 mg groups vs placebo group with regard to change of weekly mean of the 24‐h average pain score analyzed by mixed‐effects model repeated measures. Response rate defined as the percentage of patients who achieved 30 or 50% reduction of 24‐h average pain score in the combined duloxetine and duloxetine 40 and 60 mg groups was compared vs the placebo by Fisher's exact test. Safety was analyzed in all patients who took at least one dose of the test drugs. With regard to the incidence of each AE and each adverse drug reaction (ADR), the combined duloxetine group was compared with the placebo group by Fisher's exact test. Severity, causal relationship to study drugs, and outcome of AE and ADR were summarized by treatment group. BODY.RESULTS.PATIENT DISPOSITION: The disposition of patients enrolled in the study is shown in Figure 1. Of 448 screened patients, 339 patients (40 mg, n = 86; 60 mg, n = 86; placebo, n = 167) were randomized to the study treatment. The patient population subjected to efficacy and safety analyses consisted of 338 patients, excluding one patient in the 40 mg group who did not receive the study drug and was not assessed. Figure 1 Patients' disposition.image BODY.RESULTS.DEMOGRAPHICS: Patients' demographics and baseline characteristics are shown in Table 1. Approximately 76% of the patients were men; the mean age was 60.8 years. Most patients (95%) had type 2 diabetes mellitus; the duration of the disease was >10 years in the majority. HbA1c ranged from 7.04% in the placebo group to 7.25% in the 40 mg group. Mean duration of diabetic neuropathy was 4.3 years. Weekly mean 24‐h average pain score at baseline was 5.78. There was no significant difference of patient demographics and baseline characteristics among treatment groups. Table 1  Patient demographics and clinical characteristics at baseline   Combined duloxetine ( n  =   171) 40 mg ( n  =   85) 60 mg ( n  =   86) Placebo ( n  =   167) Total ( n  =   338) n % n % n % n % n % Sex  Male 127 74.3 65 76.5 62 72.1 129 77.2 256 75.7  Female 44 25.7 20 23.5 24 27.9 38 22.8 82 24.3 Age (years)  20–<30 1 0.6 – 0.0 1 1.2 1 0.6 2 0.6  30–<40 8 4.7 2 2.4 6 7.0 4 2.4 12 3.6  40–<50 16 9.4 4 4.7 12 14.0 12 7.2 28 8.3  50–<65 72 42.1 44 51.8 28 32.6 90 53.9 162 47.9  65–<80 74 43.3 35 41.2 39 45.3 60 35.9 134 39.6  Mean ± SD 60.9 ± 10.8 62.1 ± 9.3 59.7 ± 12.1 60.8 ± 9.2 60.8 ± 10.0 Weight (kg)  <50 15 8.8 10 11.8 5 5.8 14 8.4 29 8.6  50–<60 51 29.8 30 35.3 21 24.4 53 31.7 104 30.8  60–<70 62 36.3 24 28.2 38 44.2 53 31.7 115 34.0  ≥70 43 25.1 21 24.7 22 25.6 47 28.1 90 26.6  Mean ± SD 63.9 ± 11.9 62.7 ± 13.4 65.1 ± 10.2 64.5 ± 11.9 64.2 ± 11.9 24‐h average pain score  <6 96 56.1 49 57.6 47 54.7 91 54.5 187 55.3  ≥6 75 43.9 36 42.4 39 45.3 76 45.5 151 44.7  Mean ± SD 5.77 ± 1.20 5.79 ± 1.23 5.76 ± 1.17 5.78 ± 1.17 5.78 ± 1.18 Type of diabetes mellitus  Type 1 9 5.3 5 5.9 4 4.7 8 4.8 17 5.0  Type 2 162 94.7 80 94.1 82 95.3 159 95.2 321 95.0 Duration of diabetes (years)  <5 36 21.1 20 23.5 16 18.6 33 19.8 69 20.4  5–<10 32 18.7 18 21.2 14 16.3 32 19.2 64 18.9  ≥10 103 60.2 47 55.3 56 65.1 97 58.1 200 59.2  Unknown – 0.0 – 0.0 – 0.0 5 3.0 5 1.5 Duration of diabetic neuropathy (years)  <2 53 31.0 26 30.6 27 31.4 51 30.5 104 30.8  ≥2 118 69.0 59 69.4 59 68.6 116 69.5 234 69.2  Mean ± SD 4.4 ± 3.8 4.6 ± 3.9 4.2 ± 3.7 4.2 ± 4.4 4.3 ± 4.1 HbA 1C (%)  Mean ± SD 7.18 ± 0.88 7.25 ± 0.85 7.11 ± 0.90 7.04 ± 0.90 – The value for HbA 1C (%) was estimated as an NGSP equivalent value (%) calculated by the formula HbA 1C (%) = HbA 1C (JDS) (%) + 0.4%. BODY.RESULTS.EFFICACY: Figure 2 shows the weekly mean change of the 24‐h average pain score from baseline to each point of measurement over 12 weeks. Change of this parameter (adjusted mean ± SE) from baseline to week 12 in the combined duloxetine, 40, 60 mg and placebo groups was −2.47 ± 0.18, −2.41 ± 0.21, −2.53 ± 0.21, −1.61 ± 0.18, respectively. Intergroup difference vs placebo for combined duloxetine was −0.87 ± 0.15 (95% confidence interval [CI], −1.17 to −0.56; P < 0.0001). On the basis of 95% CI of difference in each dose group vs placebo, the 24‐h average pain score was also judged significantly improved in the duloxetine 40 and 60 mg groups as compared with the placebo (95% CI, −1.18 to −0.43 and −1.30 to −0.56, respectively). Both the combined duloxetine and 60 mg groups showed significant decreases of the 24‐h average pain score compared with the placebo beginning at week 1, whereas this was observed in the 40 mg group beginning at week 2, and persisted in all three active treatment groups thereafter. Figure 2 Mean weekly change of the 24‐h average pain score (repeated measures analysis). *P < 0.05 vs placebo; **P < 0.0001 vs placebo; †95% confidence interval of difference vs placebo does not include zero.image The response rate of 30% reduction of the 24‐h average pain score in the combined, 40 and 60 mg, and placebo groups was 57.3% (98/171 patients; P < 0.0001 vs placebo), 55.3% (47/85), 59.3% (51/86) and 35.3% (59/167), respectively. The response rate of 50% reduction was 39.2% (67/171 patients; P = 0.0001 vs placebo), 37.6% (32/85), 40.7% (35/86) and 19.8% (33/167), respectively. The results of mixed‐effects model repeated measures analysis of the efficacy measures are summarized in Table 2. The combined duloxetine group produced a significantly greater improvement than the placebo for all pain measures including the 24‐h worst pain score, night pain score, and BPI pain score with respect to the worst, least, average and right now. PGI‐I score (adjusted mean ± SE) at week 12 was 2.53 ± 0.12 in the combined duloxetine group and 3.18 ± 0.12 in the placebo group. Global impression of pain improvement was significantly favorable for duloxetine vs the placebo (P < 0.0001). Table 2  Mean change from baseline to endpoint (repeated measures analysis): efficacy and health outcome measures   Combined duloxetine ( n  =   171) 40 mg ( n  =   85) 60 mg ( n  =   86) Placebo ( n  =   167) Mean baseline (SD) Mean change (SE) Mean baseline (SD) Mean change (SE) Mean baseline (SD) Mean change (SE) Mean baseline (SD) Mean change (SE) Weekly mean of  24‐h average pain score 5.77 (1.20) −2.47 (0.18)** 5.79 (1.23) −2.41 (0.21)† 5.76 (1.17) −2.53 (0.21)† 5.78 (1.17) −1.61 (0.18)  24‐h worst pain score 6.58 (1.33) −2.51 (0.19)** 6.54 (1.33) −2.42 (0.22)† 6.61 (1.33) −2.59 (0.22)† 6.66 (1.25) −1.55 (0.19)  Night pain score 5.62 (1.59) −2.39 (0.19)** 5.55 (1.64) −2.33 (0.22)† 5.69 (1.54) −2.45 (0.23)† 5.50 (1.49) −1.56 (0.19) BPI pain severity  Worst pain 6.6 (1.4) −2.59 (0.21)** 6.5 (1.4) −2.51 (0.25)† 6.6 (1.5) −2.68 (0.25)† 6.7 (1.4) −1.62 (0.21)  Least pain 4.1 (1.7) −1.98 (0.21)** 4.0 (1.8) −1.92 (0.25)† 4.2 (1.6) −2.04 (0.25)† 4.1 (1.8) −1.13 (0.21)  Average pain 5.7 (1.3) −2.54 (0.20)** 5.6 (1.3) −2.53 (0.23)† 5.7 (1.3) −2.56 (0.23)† 5.6 (1.3) −1.54 (0.20)  Pain right now 5.2 (1.7) −2.59 (0.22)** 5.2 (1.8) −2.55 (0.25)† 5.3 (1.4) −2.62 (0.26)† 5.1 (1.7) −1.67 (0.22) BPI interference  General activity 4.5 (2.5) −2.29 (0.24) 4.5 (2.7) −2.48 (0.29)† 4.5 (2.4) −2.10 (0.29) 4.4 (2.4) −1.88 (0.24)  Mood 4.1 (2.5) −2.28 (0.24) 3.9 (2.5) −2.18 (0.29) 4.2 (2.5) −2.39 (0.29) 4.2 (2.4) −1.91 (0.24)  Walking ability 4.4 (2.6) −2.31 (0.23)* 4.4 (2.8) −2.32 (0.28) 4.3 (2.5) −2.31 (0.28) 4.0 (2.6) −1.82 (0.23)  Normal work 4.1 (2.5) −1.86 (0.23) 3.9 (2.6) −1.84 (0.28) 4.3 (2.5) −1.90 (0.28) 3.7 (2.7) −1.49 (0.23)  Relationship with other people 2.8 (2.5) −1.32 (0.23)* 2.7 (2.7) −1.16 (0.27) 2.9 (2.4) −1.49 (0.27)† 2.6 (2.5) −0.77 (0.23)  Sleep 4.2 (2.8) −2.15 (0.24)* 4.0 (2.8) −2.26 (0.29)† 4.3 (2.7) −2.05 (0.29) 3.9 (2.7) −1.69 (0.24)  Enjoyment of life 3.9 (2.6) −2.15 (0.23)* 3.7 (2.7) −1.96 (0.28) 4.0 (2.5) −2.35 (0.28)† 3.5 (2.5) −1.59 (0.23)  Average of 7 interference scores 3.99 (2.18) −2.04 (0.20)* 3.88 (2.25) −2.00 (0.24) 4.09 (2.13) −2.08 (0.24)† 3.75 (2.15) −1.56 (0.20)  PGI‐I – 2.53 (0.12)** – 2.53 (0.14)† – 2.52 (0.14)† – 3.18 (0.12) BPI, Brief Pain Inventory; PGI‐I, Patient’s Global Impression of Improvement. * P  <   0.05 vs placebo; ** P  <   0.0001 vs placebo; †95%CI of difference vs placebo does not include zero. Significant improvements of various individual health outcome measures were noted in the combined duloxetine group vs placebo including BPI average interference score (P = 0.0095), walking ability (P = 0.0228), relationship with other people (P = 0.0076), sleep (P = 0.0378) and enjoyment of life (P = 0.0089). However, no improvement was noted with regard to interference of general activity, mood and normal work. BODY.RESULTS.SAFETY: A total of 46 patients (13.6%) discontinued (combined duloxetine group, n = 29 [16.9%]; 40 mg group, n = 13 [15.1%]; 60 mg group, n = 16 [18.6%]; placebo group, n = 17 [10.2%]). Among them, 30 patients (8.8%) discontinued due to AE (n = 21 [12.2%], n = 9 [10.5%], n = 12 [14.0%], and n = 9 [5.4%], respectively). Overall, the incidence of AE was significantly (P = 0.0153) higher in the combined duloxetine group (84.8%; 145/171 patients) than in the placebo group (73.7%; 123/167 patients). The incidence of AE in the 40 and 60 mg groups was 84.7% (72/85 patients) and 84.9% (73/86 patients), respectively. AE and ADR reported during the present study are summarized in Table 3. AE that were significantly more frequently reported in the combined duloxetine group than placebo group included somnolence (P = 0.0007), nausea (P < 0.0001) and dizziness (P = 0.0354). Most AE and ADR were mild or moderate in severity, and resolved or relieved. There was no noteworthy difference in the incidence, kind, severity and outcome of AE between the 40 and 60 mg groups. Table 3  Incidence of adverse events reported by ≥5% patients in any group and adverse drug reactions Preferred term Adverse events Adverse drug reactions Combined duloxetine ( n  = 171) 40 mg ( n  = 85) 60 mg ( n  = 86) Placebo ( n  = 167) Combined duloxetine ( n  = 171) 40 mg ( n  = 85) 60 mg ( n  = 86) Placebo ( n  = 167) n % n % n % n % n % n % n % n % Somnolence 37 21.6* 16 18.8 21 24.4 14 8.4 37 21.6* 16 18.8 21 24.4 13 7.8 Nausea 24 14.0** 10 11.8 14 16.3 3 1.8 24 14.0** 10 11.8 14 16.3 3 1.8 Nasopharyngitis 24 14.0 10 11.8 14 16.3 24 14.4 – 0.0 – 0.0 – 0.0 – 0.0 AST increased 13 7.6 5 5.9 8 9.3 6 3.6 7 4.1 3 3.5 4 4.7 4 2.4 Constipation 11 6.4 6 7.1 5 5.8 9 5.4 9 5.3 5 5.9 4 4.7 6 3.6 Diarrhea 11 6.4 4 4.7 7 8.1 6 3.6 7 4.1 4 4.7 3 3.5 3 1.8 Dizziness 10 5.8* 6 7.1 4 4.7 2 1.2 7 4.1 4 4.7 3 3.5 2 1.2 ALT increased 10 5.8 5 5.9 5 5.8 6 3.6 6 3.5 3 3.5 3 3.5 4 2.4 Malaise 9 5.3 3 3.5 6 7.0 3 1.8 9 5.3 3 3.5 6 7.0 3 1.8 Vomiting 9 5.3 4 4.7 5 5.8 2 1.2 8 4.7* 4 4.7 4 4.7 1 0.6 WBC increased 9 5.3 4 4.7 5 5.8 4 2.4 1 0.6 – 0.0 1 1.2 1 0.6 GGT increased 7 4.1 2 2.4 5 5.8 5 3.0 4 2.3 1 1.2 3 3.5 2 1.2 LDH increased 7 4.1 2 2.4 5 5.8 4 2.4 3 1.8 1 1.2 2 2.3 3 1.8 CK increased 6 3.5 6 7.1 – 0.0 6 3.6 1 0.6 1 1.2 – 0.0 3 1.8 HbA 1c increased 6 3.5 1 1.2 5 5.8 4 2.4 4 2.3 – 0.0 4 4.7 3 1.8 * P  < 0.05 vs placebo; ** P  < 0.0001 vs placebo. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine phosphokinase; GGT, γ‐glutamyltransferase; HbA 1c , glycosylated hemoglobin; LDH, lactate dehydrogenase; WBC, white blood cell count. A total of seven serious AE were noted in five patients in the combined duloxetine group (four events in three patients in the 40 mg group and three events in two patients in the 60 mg group), whereas 18 serious AE occurred in six patients in the placebo group. No deaths occurred during the present study. Overall, no significant difference was noted between the combined duloxetine and placebo groups. Among serious AE reported, hypoglycemia (40 mg group) and self‐injurious behavior (60 mg group) were judged as ADR, although both symptoms resolved. At 13 weeks compared with baseline, comparable and unremarkable increases of HbA1c were noted in the combined duloxetine, 40, 60 mg and placebo groups (0.06, 0.03, 0.10 and 0.10%, respectively). BODY.DISCUSSION: To evaluate the superiority of the SNRI duloxetine 40 and 60 mg/day once daily over the placebo, a randomized, double‐blind, 12‐week, phase III study was carried out in Japanese patients with moderate‐to‐severe DNP defined as having ≥4 on the 24‐h average pain score. As a result, duloxetine was shown to be significantly superior to the placebo in improving DNP as evaluated by a change of the 24‐h average pain score from baseline to week 12 as the primary efficacy end‐point. Recently, Farrar et al.27,33 pooled data from placebo‐controlled studies that investigated relationships between changes of Numerical Rating Scale pain ratings and quantifiable PGI‐I scale. Their findings showed that a reduction of approximately 2 points from baseline on an 11‐point pain rating scale, equivalent to a 30% reduction on pain score from baseline, corresponds to a clinically meaningful improvement. Subjects with 30–50% reduction in the assessment scale as compared with baseline are considered to be responders in guidelines on clinical investigation of medicinal products intended for the treatment of neuropathic pain34. In the present trial, the rate of responders was significantly higher in the combined duloxetine group than the placebo group. Taking into consideration that the present study was carried out in patients with moderate or severe DNP, duloxetine might contribute favorably to the treatment of such individuals. Excluded from participating in the present study were patients complicated with psychiatric diseases including depression. Thus, the present findings support the argument that dual inhibition of reuptake of 5‐HT and NA in the CNS contributes to the independent analgesic effect of duloxetine. DNP is typically characterized by the manifestation of peripheral pain in the extremities, which causes unbearable distress to patients. The most important matter for those with DNP must be to lessen the pain as early as possible. Duloxetine was found to significantly improve the 24‐h average pain score vs the placebo as early as 1 week after starting treatment. Therefore, duloxetine might be useful for treating patients with DNP because of its early manifestation of an analgesic effect. DNP is not only often increased at night and affects sleep4, but also interferes with daily life, leading to a lack of appetite and a depressive state in severe cases5,35, and eventually to the deterioration of quality of life. Another important finding in the present study was that a significant improvement in health outcome measures was noted in the combined duloxetine group over the placebo for BPI average interference score as well as BPI interference of walking ability, relationships with other people, sleep and enjoyment of life. These clinical findings suggest that duloxetine might be helpful to improve patients' quality of life. From the practical point of view, dosing frequency is an important clinical consideration. A systematic review of associations between dosing frequency and medication compliance showed that the latter is inversely related to the former36. The dosing frequency of epalrestat and mexiletine hydrochloride, which are widely used for the treatment of DNP in Japan, is thrice daily, whereas that of pregabalin, which is approved and used in the USA and European‐approved pregabalin, is twice daily. Because we observed a significant improvement of DNP with the once‐daily regimen, this suggests that adherence to treatment with duloxetine might be less of a clinical concern. Incidence of AE/ADR was significantly higher in the combined duloxetine group than the placebo group, whereas that in the 40 and 60 mg groups was similar. In contrast, most AE and ADR reported in the combined duloxetine group were mild or moderate in severity and resolved or relieved. Because no clinically problematic findings were noted, it is considered that there are no safety concerns with duloxetine in the setting of DNP. Our safety data are comparable to those observed in a previous clinical trial carried out overseas19,21, where the incidence of AE on duloxetine was 87.7% (498/568 patients); major AE were nausea (23.6%; n = 134 patients), somnolence (15.5%; n = 88), dizziness (13.4%; n = 76), constipation (11.3%; n = 64) and insomnia (10.2%; n = 58). Therefore, the type and incidence of AE in Japanese patients are similar to those in non‐Japanese patients. Furthermore, there was little difference in change of HbA1c between each duloxetine treatment group and the placebo, as was observed in studies carried out overseas; 13‐week treatment with duloxetine does not appear to adversely affect glycemic control. In conclusion, the superiority of once‐daily dosing with the SNRI duloxetine vs placebo in improving DNP was shown in Japanese patients. Both 40 and 60 mg daily dosages of duloxetine were safe and well tolerated.

TITLE: One year follow-up of a pragmatic multi-centre randomised controlled trial of a group-based fatigue management programme (FACETS) for people with multiple sclerosis ABSTRACT.BACKGROUND: Fatigue is one of the most common and debilitating symptoms of multiple sclerosis (MS). The aim was to evaluate the effectiveness at 1-year follow-up of a manualised group-based programme ('FACETS') for managing MS-fatigue. ABSTRACT.METHODS: One-year follow-up of a pragmatic multi-centre randomised controlled trial. People with MS and significant fatigue were randomised to FACETS plus current local practice (FACETS) or current local practice alone (CLP), using concealed computer-generated randomisation. Participant blinding was not possible. Primary outcome measures were fatigue severity (Global Fatigue Severity subscale of the Fatigue Assessment Instrument), self-efficacy (MS-Fatigue Self-Efficacy) and disease-specific quality of life (MS Impact Scale). ABSTRACT.RESULTS: Between May 2008 and November 2009, 164 participants were randomised. Primary outcome data were available at 1 year for 131 (80%). The benefits demonstrated at 4-months in the FACETS arm for fatigue severity and self-efficacy largely persisted, with a slight reduction in standardised effect sizes (SES) (−0.29, p = 0.06 and 0.34, p = 0.09, respectively). There was a significant difference on the MS Impact Scale favouring FACETS that had not been present at 4-months (SES −0.24, p = 0.046). No adverse events were reported. ABSTRACT.CONCLUSIONS: Improvements in fatigue severity and self-efficacy at 4-months follow-up following attendance of FACETS were mostly sustained at 1 year with additional improvements in MS impact. The FACETS programme provides modest long-term benefits to people with MS-fatigue. ABSTRACT.TRIAL REGISTRATION: ISRCTN76517470 BODY.BACKGROUND: Fatigue is one of the most common and debilitating symptoms of MS [1-5]. We have developed a non-pharmacological group-based fatigue management programme for people with MS called FACETS (Fatigue: Applying Cognitive behavioural and Energy effectiveness Techniques to lifeStyle) [6]. One of the criticisms of non-pharmacological trials is that they often do not include any long term follow-up. This paper reports on one year follow-up data obtained from a pragmatic three-centre trial of FACETS [7]. BODY.METHODS: We carried out a pragmatic multi-centre randomised controlled trial following the published protocol [8] in which the full study design, inclusion and exclusion criteria, trial arms, and plan of analysis are described in detail. The main results are reported elsewhere [7]. Here we report the results from the 1 year self-reported follow-up data. Participants were recruited in three UK centres (Poole, Bristol, Southampton/Portsmouth) from primary or secondary care, or via MS Society newsletters/websites. Recruitment took place from May 2008 to November 2009. Ethical approval was obtained from the South West-Central Bristol Research Ethics Committee (ref: 08/H0106/2). All participants provided written informed consent before taking part. The main inclusion criteria were: (1) clinically definite MS diagnosis, (2) fatigue impacting on daily life (Fatigue Severity Scale total score >4) [9] and (3) ambulatory. The main exclusion criteria were: (1) having taken part in a fatigue programme in the last year, (2) cognitive impairments (3) a relapse in the previous 3 months or (4) having started treatment with disease modifying or antidepressant drugs within the previous 3 months. The full eligibility criteria are described in the protocol [8]. BODY.METHODS.INTERVENTION (FACETS PROGRAMME): The manualised group-based FACETS programme is described elsewhere [6] and is based upon a conceptual framework integrating elements from cognitive behavioural, social-cognitive, energy effectiveness, self-management and self-efficacy theories. The aim of the intervention is to help people with MS normalise their fatigue experiences, learn helpful ways of thinking about fatigue and use available energy more effectively. The intervention consists of six sessions (∼90 min duration) held weekly and facilitated in groups of 6–12 by two health professionals with experience of working with people with MS and group-work (such as occupational therapists, nurses or physiotherapists). Each session follows the same general format, namely, facilitator-delivered presentations, flipchart discussions, group activities and homework. The facilitator manual provides guidance on preparation and delivery, detailed session content, notes and suggested timings, and a checklist of facilitator objectives as well as signposts to additional resources. Sessions are delivered via PowerPoint; hence can be easily replicated. A companion participant handbook, along with existing information booklets, reinforces programme content. FACETS was delivered in hotel meeting-room facilities, with the exception of one centre, where it was held in a rehabilitation hospital. Apart from one MS specialist nurse, facilitators were either occupational therapists or physiotherapists. Facilitators were trained to deliver the intervention at 1-day workshops and psychological advice and debriefing were available for facilitators throughout the trial. To increase external validity, no attempt was made in the FACETS arm to restrict or control participants' access to current local practice or to standardise it across healthcare settings or treatment arms. When we refer to the FACETS arm, participants in this arm also received current local practice. BODY.METHODS.CONTROL GROUP (CURRENT LOCAL PRACTICE (CLP)): Participants randomised to this arm of the trial received current local practice. This could have ranged from general advice and information provision about MS-fatigue to more detailed individualised management advice from a variety of health professionals. Inevitably, there will have been variations in the exact composition of what was usually provided, within and between centres, depending on local resources and patient need. Collecting detailed information at an individual level on the type and quantity of advice received as part of current local practice was not deemed feasible. However, this real world variation increases applicability to a wider range of centres. BODY.METHODS.OUTCOMES: For those allocated to the FACETS arm outcomes were measured 1 week (baseline) before the start of the FACETS programme and 1 month (follow-up 1), 4 months (follow-up 2) and 12 months (follow-up 3) after the final session. Participants in the current local practice arm completed outcome measures within an identical time frame. Data from follow-up 1 and 2 have previously been reported [7]. In this paper we focus on reporting follow-up 3. Primary outcomes were fatigue severity (Global Fatigue Severity (GFS) subscale of the Fatigue Assessment Instrument (FAI)), disease specific quality of life (Multiple Sclerosis Impact Scale (MSIS-29, V.1)) and self-efficacy for managing fatigue (Multiple Sclerosis - Fatigue Self-Efficacy scale (MS-FSE)) [7,8]. Secondary outcomes included the Fatigue Symptom Inventory (FSI), the Hospital Anxiety and Depression Scale (HADS), the Medical Outcomes Short-Form Survey (SF-36, V. 2), and subscales of the MSIS-29 (V.1) and the FAI [7,8]. All outcomes collected at 12 months were self-reported questionnaires and administered postally. BODY.METHODS.SAMPLE SIZE CONSIDERATIONS: The sample size requirement was 146 participants with follow-up data based on having 85% power to detect a medium standardised effect size of 0.5 for the primary outcome measures, using a two-sided 5% significance level (see protocol for justification for this medium effect size) [8]. As a variety of fatigue measures have been used in other trials, we used standardised effect sizes to enable comparisons between them. BODY.METHODS.ANALYSIS: The main analysis was intention-to-treat but we also conducted a per protocol analysis (excluding participants who attended fewer than four FACETS sessions). Data were analysed using IBM SPSS, V.18 and MLwiN 2.17. Outcome measures were assumed to be interval-scaled and the main analysis focused on absolute change in outcomes at 1 year follow-up relative to baseline. Change scores were compared between the groups using the independent samples t-test with a two sided 5% significance level, and summarised using mean differences (95% confidence intervals (CIs)) and standardised effect sizes (SES). As detailed in the protocol, additional pre-specified supplementary analyses were undertaken. Here we report results from a mixed model approach that includes 1 year and baseline measurements as repeated measures, incorporates clustering effects, and includes pre-specified covariates (baseline for other primary outcomes, age, gender, marital status, education level, type of MS, time since diagnosis, level of disability, and centre). BODY.RESULTS: One year follow up data are available on 131 participants (80%) (Figure 1). The distributions of descriptive statistics for the trial sample are presented in Table 1. Figure 1Participant flow. Table 1 Descriptive demographic and baseline characteristics of participants   FMP (n = 84) CLP (n = 80) Gender [n (%)]     ▪ Female 61 (73%) 58 (73%) ▪ Male 23 (27%) 22 (28%) Age (years)     Mean (S.D.) 48.0 (10.2) 50.1 (9.1) Range 23-73 28-70 Ethnicity [n (%)]     ▪ White English 68 (85%) 69 (92%) ▪ White British 7 (9%) 5 (7%) ▪ Other 5 (6%) 1 (1%) ▪ Not stated 4 5 Disease type (self-reported) [n (%)]     ▪ Benign 4 (5%) 2 (3%) ▪ Relapsing-remitting 35 (43%) 40 (51%) ▪ Secondary progressive 16 (20%) 23 (29%) ▪ Primary progressive 5 (6%) 8 (10%) ▪ Participant states “Don’t know” 21 (26%) 5 (6%) ▪ Not stated 3 2 APDDS score (Adapted Patient Determined Disease Steps) [n (%)]     ▪ 3 or less (No limitations on walking) 18 (22%) 15 (19%) ▪ 4 or 5 (MS interferes with walking) 37 (46%) 42 (54%) ▪ 6 or more (At min., needs stick/crutch to walk 100 m) 26 (32%) 21 (27%) ▪ Not stated 3 2 Level of education [n (%)]     Highest qualification achieved:     ▪ No qualifications 8 (10%) 8 (10%) ▪ One or more GCSE (or equiv.) 36 (46%) 29 (38%) ▪ One or more A level (or equiv.) 10 (13%) 12 (16%) ▪ First degree (or equiv.) 16 (20%) 19 (25%) ▪ Higher degree/professional qualification 9 (11%) 8 (11%) ▪ Not stated 5 4 Employment status [n (%)]     ▪ In full time employment (>30 hours per week) 15 (18%) 11 (14%) ▪ In part-time employment (≤30 hours per week) 11 (14%) 13 (17%) ▪ Self-employed 4 (5%) 4 (5%) ▪ Not in paid employment (unemployed, in education, retired, looking after home) 51 (63%) 50 (64%) ▪ Not stated 3 2 Marital status [n (%)]     ▪ Married/cohabiting 63 (78%) 54 (71%) ▪ Single 5 (6%) 7 (9%) ▪ Separated/divorced 9 (11%) 14 (18%) ▪ Widowed 4 (5%) 1 (1%) ▪ Not stated 3 4 Years since diagnosis [n (%)]     ▪ <1 yr 2 (3%) 4 (5%) ▪ 1–5 yrs 32 (40%) 21 (27%) ▪ 6–10 yrs 13 (16%) 19 (24%) ▪ 11–15 yrs 21 (26%) 12 (15%) ▪ ≥16 yrs 12 (15%) 22 (28%) ▪ Not stated 4 2 Percentages rounded to nearest integer and, thus, might not sum exactly to 100%. CLP, current local practice; FACETS, Fatigue: Applying Cognitive behavioural and Energy effectiveness Techniques to lifeStyle; GCSE, General Certificate of Secondary Education; MS, multiple sclerosis. BODY.RESULTS.PRIMARY OUTCOMES: The intention-to-treat analyses and results for the primary outcome measures are shown in Table 2. Table 2 Descriptive statistics and treatment effects at 1 year follow-up Primary outcome measures Baseline (n = 159) Follow-up 1 (n = 146) Follow-up 2 (n = 144) Follow-up 3 (n = 131) Global fatigue severity (GFS) subscale of the FAI (potential range 1 to 7, high scores indicate more fatigue) FACETS mean (SD) 5.60 (0.98) 5.48 (0.92) 5.26 (1.03) 5.32 (1.00) CLP mean (SD) 5.61 (1.09) 5.55 (1.17) 5.66 (0.93) 5.70 (1.01) Mean difference in change from baseline [95% CI] - −0.03 (−0.33 to 0.28) −0.36 (−0.63 to −0.08) −0.30 (−0.61 to 0.01) p value - 0.86 0.01 0.06 Std effect size - −0.03 −0.35 −0.29 Multiple Sclerosis Impact Scale-29 (MSIS-29) (potential range 0 to 100, high scores indicate more impact) FACETS mean (SD) 49.6 (19.1) 47.3 (18.2) 44.9 (19.2) 46.2 (19.1) CLP mean (SD) 43.9 (17.6) 42.2 (18.4) 43.0 (17.3) 47.2 (17.4) Mean difference in change from baseline [95% CI] - 1.44 (−2.36 to 5.24) −1.56 (−6.45 to 3.34) −4.34 (−8.61 to −0.08) p value - 0.46 0.53 0.046 Std effect size - 0.08 −0.08 −0.24 MS Fatigue Self-Efficacy scale (MS-FSE) (potential range 10 to 100, high scores indicate more certainty in controlling fatigue FACETS mean (SD) 45 (17) 57 (17) 56 (19) 56 (16) CLP mean (SD) 49 (16) 50 (17) 53 (17) 52 (17) Mean difference in change from baseline [95% CI] - 9 (4 to 14) 6 (0 to 12) 6 (−1 to 12) p value - 0.001 0.048 0.09 Std effect size - 0.54 0.36 0.34 Statistically significant secondary outcome measures Vitality subscale of the SF-36 (potential range 0 to 100, high scores indicate higher quality of life) FACETS mean (SD) 32.0 (16.8) 35.6 (19.4) 37.4 (20.3) 37.70 (18.75) CLP mean (SD) 35.1 (19.7) 33.4 (16.8) 34.4 (17.30 32.43 (17.69) Mean difference in change from baseline [95% CI] - 4.42 (−1.22 to 10.06) 6.38 (0.45 to 12.32) 6.64 (0.84 to 12.44) p value - 0.12 0.04 0.03 Std effect size - 0.24 0.35 0.37 Multiple Sclerosis Impact Scale-29 (MSIS-29) - Physical subscale (potential range 0 to100, high scores indicate more impact) FACETS mean (SD) 51.4 (21.4) 48.8 (19.7) 47.0 (21.3) 47.4 (21.0) CLP mean (SD) 46.6 (20.3) 44.9 (20.5) 46.5 (19.8) 50.5 (20.1) Mean difference in change from baseline [95% CI] - 1.39 (−2.87 to 5.65) −0.81 (−5.91 to 4.28) −4.74 (−9.40 to −0.08) p value - 0.52 0.75 0.046 Std effect size - 0.07 −0.04 −0.23 Results for fatigue severity and self-efficacy were similar to those at 4 months with a slight reduction in standardised effect size (SES) from −0.35 (p = 0.01) for fatigue severity to −0.29 (p = 0.06) and from 0.36 (p = 0.048) for fatigue self-efficacy to 0.34 (p = 0.09). There were significantly greater improvements on the MSIS-29 for the FACETS arm compared with the CLP arm (p = 0.046, SES = −0.24) that were not evident at 4 months. The per protocol analysis resulted in an increased SES for the MSIS-29 (from −0.24 to −0.26 (p = 0.03)) and for the MS-FSE (from 0.34 to 0.39 (p = 0.046)). The SES for the GFS subscale was reduced from −0.29 to −0.25 (p = 0.10). Participants in the FACETS arm were 1.5 times more likely (31% (19/62) versus 20% (14/69)) to have a clinically important improvement on the GFS (defined as an individual reduction of ≥ 0.5), although, unlike at 4 months, this was not statistically significant (p = 0.25 using chi-squared test with continuity correction). Using the mixed model approach, the mean difference at 1 year for GFS was almost unchanged (−0.28 (−0.58, 0.02), p = 0.07), for fatigue self-efficacy was slightly higher (7 (1, 13), p = 0.02), and for the MSIS-29 slightly lower (−3.90 (−8.08, 0.28), p = 0.07). BODY.RESULTS.ADVERSE EVENTS: No adverse events, as defined in the protocol, were reported. BODY.RESULTS.SECONDARY OUTCOMES: For the MSIS-29 physical subscale (p = 0.046, SES = −0.23) and the vitality subscale of the SF-36 (p = 0.03, SES = 0.37) there was a significant difference in favour of the FACETS arm at 1 year. None of the other secondary outcomes was statistically significant. Effect sizes and significance levels were similar using the mixed model approach. BODY.DISCUSSION: The modest improvements in fatigue severity and fatigue self-efficacy in the FACETS arm at 4 months were largely maintained at 1 year. While attrition was relatively low at one year there was a diminution of sample size to 131 (the original sample size calculation requirement was n = 146). Statistical power would have been reduced slightly to 80% (NQuery Advisor) and while still a reasonable level of power this is lower than that at follow-ups 1 and 2 and might account in part for the slightly larger p-values obtained at the 1 year follow-up. In addition to improvements in fatigue severity and self-efficacy there were improvements in MS-specific quality of life that had not been present at 4 months follow-up. The delayed appearance of this latter impact might be because the changes to lifestyle encouraged by the FACETS programme may take some time to implement effectively. Only some of those who declined participation in the FACETS trial provided reasons for doing so. When reasons were provided, they were predominantly related to lack of time or existing work, holiday or childcare commitments. A small minority of individuals felt that a group approach was not for them or did not wish to take part in a research trial. However, we acknowledge it is possible that there might have been a recruitment bias towards those more amenable to a non-pharmacological approach. BODY.CONCLUSIONS: FACETS appears to have long term benefits for people with MS at an estimated cost of £453 per person. Often trials of nonpharmacological interventions do not measure long term follow-up or effects do not persist beyond the short term. Given the progressive nature of MS and the debilitating nature of fatigue, our demonstration of small to medium improvements at 1 year follow-up is encouraging. BODY.COMPETING INTERESTS: All authors had financial support from the Multiple Sclerosis Society in the UK for the submitted work; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years. RB is the chair of the MS Society Grant Review Panel for Care and Services Research. PT is a member of the MS Society Grant Review Panel for Care and Services Research. PT is a member of the Advisory Board for the Sativex Registry. The Board provides an independent review of safety data for patients prescribed Sativex. Bournemouth University receives a fee from GW Pharma to cover time spent at meetings, and travel expenses. BODY.AUTHORS’ CONTRIBUTIONS: Contributors: PT: chief investigator, conception, design, analysis, interpretation, drafted article. ST: conception, design, acquisition of data, analysis, interpretation, drafted article. PK and RJ: design, acquisition of data, interpretation, critically reviewed article. AN and VS: design, delivered fatigue management programme, acquisition of data, interpretation, critically reviewed article. ADS: design, delivered fatigue management programme, critically reviewed article. RB and KTG: design, interpretation, critically reviewed article. CH: design, clinical oversight of trial, interpretation, critically reviewed article. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2377/14/109/prepub

TITLE: LNG-IUS treatment of non-atypical endometrial hyperplasia in perimenopausal women: a randomized controlled trial ABSTRACT.OBJECTIVE: To compare the efficacy of the levonorgestrel-releasing intrauterine system (LNG-IUS) and oral norethisterone acetate (NET) for treatment of non-atypical endometrial hyperplasia in perimenopausal women. ABSTRACT.METHODS: One hundred and twenty perimenopausal women with non-atypical endometrial hyperplasia were selected in this randomized controlled trial. Patients received LNG-IUS (n=59) or NET (n=61; 15 mg/day for 3 weeks/cycle) for 3-6 months. Outpatient follow-up with endometrial biopsies were undertaken at 3, 6, and 12 months intervals after treatment. Outcome measures were; the regression rate, the time to regression and hysterectomy rate. ABSTRACT.RESULTS: A significantly higher regression rate was noted in the LNG-IUS group than in NET group at the 3rd, 6th and 12th month follow-up visits using intention-to-treat analysis (67.8% vs. 47.5%, relative risk [RR], 1.42; 79.7% vs. 60.7%, RR, 1.31; and 88.1% vs. 55.7%, RR, 1.58, respectively). However, no significant difference was found regarding the median time to regression (3 months). The hysterectomy rate during the follow-up period was significantly higher in the NET group (57.4% vs.22%, p<0.001). ABSTRACT.CONCLUSION: LNG-IUS treatment of non-atypical endometrial hyperplasia in perimenopausal women is more effective than NET for achieving disease regression for the majority within 1 year. Moreover, it can reduce the number of hysterectomies performed. BODY.INTRODUCTION: Endometrial hyperplasia (EH) is a frequently encountered clinical entity peaking in the early 50s and 60s [1,2]. It presents commonly with abnormal uterine bleeding (AUB) [3,4]. However, its clinical importance largely relates to the risk of progression to endometrial carcinoma which is low for simple or complex non-atypical EH (<5%) compared with atypical EH (approximately 30%) [5,6]. Additionally, a 17%-42.6% risk of concurrent endometrial carcinoma was reported in women with a biopsy diagnosis of atypical EH [7,8]. The optimal management of EH is a subject of considerable debate [4,9]. In presence of atypia, fertility preservation with progestin therapy has been attempted in young motivated patients [10-12], however recommendation of hysterectomy seems appropriate [1,4,9-13]. Meanwhile, EH without atypia have been traditionally treated with high-dose progestin therapy [1,4,13,14]. However, its efficacy is often limited by significant side effects e.g. weight gain, edema and irritability [1,4,9]. Additionally, a resistance rate of 14% and a recurrence rate of 6% were reported following cessation of therapy in cases of EH without atypia [15]. Recently, levonorgestrel-releasing intrauterine system (LNG-IUS) has been reported as a successful treatment option for EH [16,17]. Achievement of higher progestin concentrations in the endometrium by many folds compared with oral administration was reported [18]. Accordingly, these higher local concentrations might end up into more consistent results with lower recurrence rates than temporary treatment with oral progestins. To the best of our knowledge, no randomized controlled trials (RCTs) compared the efficacy of the LNG-IUS and norethisterone acetate (NET) for treatment of non-atypical EH. To examine this issue, we compared both modalities in a prospective RCT in perimenopausal women. BODY.MATERIALS AND METHODS.1. STUDY POPULATION: This RCT enrolled women complaining of AUB among those attending the Outpatient Clinic in Mansoura University Hospitals, Egypt in the period from May 2009 to November 2011. A detailed history, examinations and ultrasound evaluation were carried out. Endometrial biopsy samples were obtained by combination of hysteroscopy with dilatation and curettage (D&C) following inpatient admission. Inclusion criteria were those with histologically confirmed non-atypical simple or complex EH, age between 40 and 50 years with an ongoing menstrual cycle for at least 6 months before the onset of AUB and no contraindication to either LNG-IUS or NET e.g., current or a history of deep venous thrombosis, active thrombophlebitis, thromboembolic disorder, or cerebrovascular accident; myocardial infarction or ischemic heart disease and liver disease. Exclusion criteria were EH with atypia, age >50 years, other pathology e.g., submucosal myomas or polyps, adnexal abnormality, genital infection, hormone therapy or any medication which might affect the menstrual blood loss within the previous 6 months e.g., steroid hormones or anticoagulants, previous endometrial ablation, diabetic and/or hypertensive patients and those unwilling for medical management. The study was approved by Mansoura University Hospital Research Ethics Committee and all participants gave informed consent before inclusion in the trial. The study protocol was registered at the ClinicalTrials.gov (ID: NCT01499602). The trial is reported and analyzed following the Consolidated Standards of Reporting Trials (CONSORT) criteria. BODY.MATERIALS AND METHODS.2. RANDOMIZATION: Women were randomized according to a computer-generated random numeric table prepared by an independent statistician with concealment of treatment allocation by use of sealed opaque envelopes that were given to a third party (nurse) who assigned patients to study arms; group A (LNG-IUS) or group B (NET). Outcome assessors i.e., those performing histological diagnosis (two independent gynecological pathologists) and statistical analysis were blinded to the treatment groups. BODY.MATERIALS AND METHODS.3. PROTOCOL AND TREATMENT: In group A, LNG-IUS (Mirena, Bayer Schering Pharma Oy, Turku, Finland) was inserted and all women underwent follow-up at regular intervals for one year. During follow-up visits, clinic review, transvaginal ultrasonography (TVS) and endometrial histological surveillance by outpatient Pipelle sampling were carried out. The first two visits were at the 3rd month and 6th month after insertion. Successful treatment was defined as histological regression evident by glandular atrophy and stroma decidualization [19,20]. If complete regression was achieved within 6 months, the follow-up interval was extended to 6 months thereafter. For patients with unsuccessful treatment after six-months, histological surveillance after another 3 and 6 months interval was carried out. In group B, NET tablets (Cidolut Nor, Chemical Industries Development, Cairo, Egypt) were prescribed at a dose of 5 mg three times daily (15 mg/day) for 3 weeks over three months. Clinical review, TVS and endometrial histological surveillance by outpatient Pipelle sampling was carried out by the end of the 3rd treatment cycle between the 20th and 23rd day of this artificially created menstrual cycle. Successful treatment was defined as mentioned above [19,20]. Women with successful treatment discontinued progestin therapy and were called for follow-up with TVS and outpatient Pipelle sampling 3 months later. Meanwhile, women with persistant EH after 3 months were prescribed NET for another 3 months and then reevaluated at the end of the 6th month. After that patients were counseled for hysterectomy or further 3 months treatment if there was no histological evidence of complete regression. Meanwhile, women with successful treatment discontinued progestin therapy and were called for follow-up 6 months later. BODY.MATERIALS AND METHODS.4. SAMPLE SIZE: The primary outcome was the proportion of women with complete regression of EH. Secondary outcome measures were; the time to complete regression during the 12 months follow-up period and the rate of hysterectomy. Sample size was calculated based on an expected regression rate of 75%, in cases of non-atypical EH after at least 3 months of any progestin therapy, with at least 3 months follow-up [14]. Accordingly, a total of 98 women was required to show an observed difference of 20% in the regression rate between treatments, with a power of 80% using a two tailed χ2 test with a 5% significance level (type I error). With an assumed attrition rate of 10%, a total of 108 patients were needed (54 in each arm). BODY.MATERIALS AND METHODS.5. STATISTICAL ANALYSIS: Data obtained were statistically analyzed using SPSS ver. 15.0 (SPSS Inc., Chicago, IL, USA). Means were compared using the unpaired Student's t-test while proportions were compared using the χ2 test and relative risk with 95% confidence interval (CI) were calculated. A p-value of less than 0.05 was considered statistically significant. BODY.RESULTS.1. PATIENTS' CHARACTERISTICS: A total of 373 women were assessed for eligibility. One hundred and twenty women were randomly assigned to treatment and comprised the intention-to-treat population. Women received either LNG-IUS (group A, n=59) or NET (group B, n=61). Seven patients with confirmed regression of EH (3 in group A and 4 in group B) were lost to follow up at the end of the 12 months. Fig. 1 shows the flow of participants in the trial. During treatment, 6 patients (9.8%) in group B suffered from some side effects including nausea in 3 patients and weight gain in the other 3 patients, but they continued therapy. There were no significant differences between both groups as regards baseline characteristics, clinical presentation and histological types of EH (Table 1). BODY.RESULTS.2. STUDY OUTCOMES: Table 2 summarizes the effect of therapy in both groups. Following treatment, a significantly higher regression rate was noted in the LNG-IUS group than in NET group at the 3rd and the 6th month follow-up visits (67.8% vs. 47.5%, RR=1.42 [1.04-1.96], p=0.025; 79.7% vs. 60.7%, RR=1.31 [1.03-1.67], p=0.023, respectively). Another 3 women in group A achieved regression at the 9th month follow up and another 2 at 12 months. The remaining seven patients with unsuccessful treatment at 12 months were counseled for hysterectomy. Histological reports of their hysterectomy specimens showed persistent non-atypical complex EH. On the other hand, the remaining 24 women in group B with confirmed persistent non-atypical complex EH at the 6th month follow up visit were counseled for hysterectomy and the same pathology was shown in histological reports of their hysterectomy specimens. Importantly, at the 12th month follow up visit, a significantly higher regression rate was noted in the LNG-IUS group than in NET group using intention-to-treat analysis (ITT) or the per protocol analysis (88.1% vs. 55.7%, RR=1.58 [1.24-2.01], p<0.001; 92.9% vs. 59.6%, RR=1.55 [1.24-1.95], p<0.001, respectively). However, there was no significant difference between both groups regarding the median time to regression (3 months for each; p=0.697). During the study, 6 patients in group A requested hysterectomy to be done due to persistent bleeding with the LNG-IUS in situ for about 10 months. Their final histological reports of hysterectomy specimens showed regression of EH. On the other hand, in group B, none of the 29 cases who achieved regression at 3 months showed recurrent hyperplasia in their second follow up visit. However, 3 of them requested hysterectomy to be done due to persistent irregular bleeding and histological assessment of their hysterectomy specimens confirmed regression of EH. Another 8 out of 30 women in group B who attended the 12th month follow up visit requested hysterectomy to be done due to recurrent episodes of AUB. Endometrial regression was reported upon histological evaluation of the hysterectomy specimens. As a total, so far 13/59 patients (22%) in the LNG-IUS group and 35/61 (57.4%) in the NET treatment group have undergone hysterectomy with highly significant difference; RR=0.38 (0.23-0.65), p<0.001. Moreover, the hysterectomy rate in patients who achieved regression in the LNG-IUS group was significantly less than that in the NET treatment (11.5% vs. 29.7%, RR=0.39 [0.16-0.95] respectively, p=0.031) (Table 2). The operation was extrafascial total abdominal hysterectomy (and bilateral salpingo-oophorectomy in women above 45 years) and no atypia or cancer was reported in the hysterectomy specimens. BODY.DISCUSSION: In this RCT, we have evaluated the efficacy of LNG-IUS in treatment of non-atypical EH in perimenopausal women by comparing it with oral NET. Perimenopausal women were selected as they represent the great sector of outpatient visits presented with AUB necessitating further evaluation and management. NET was used because medroxyprogesterone acetate and megestrol acetate, which represent the most commonly used progestins, are not available at the Egyptian market. Horn et al. [21], treated pre- and perimenopausal patients with complex and atypical EH with NET (5 mg/day) or medroxyprogesterone acetate (10 mg/day) for 3-5 months with an overall remission rate of 61.5%. Bese et al. [22] reported that 3 months of cyclic NET (15 mg/day) treatment reduced both proliferative and apoptotic activities in endometrial tissue with simple non-atypical EH. At the 12th month follow-up visit, a significantly higher regression rate was noted in the LNG-IUS group than in NET group using ITT (88.1% vs. 55.7%). Our findings match those of others who found that 87.5% of patients with non-atypical EH achieved regression by 12 months [17,23]. Furthermore, a recent meta-analysis of 24 observational studies including 1,001 women showed that oral progestins achieved a significantly lower pooled regression rate compared with LNG-IUS for non-atypical complex EH (66% vs. 92%) [16]. In our study, the regression time with the Mirena IUS ranged from 3 to 12 months (median, 3 months) with a significant proportion of the patients (67.8%) achieving regression at the 3rd month. This success rate matches with 66% regression rate within 3 months of Mirena application reported by others with a mean duration of 4.5 months [24]. Different regression times were reported in other studies as follows; range, 4 to 12 months (median, 6 months) [17]; range, 7 to 11.7 months (mean, 9.4 months) [23]. In our study, EH regression rate of 67.8% found after 6 months of Mirena IUS, meanwhile another study [25] reported a 100% regression rate after 6 months. Actually, dissimilarity in the patients' characteristics and other methodical details might explain the differences in achieved regression rate per time in these studies. However, it is noteworthy that beneficial effects were observed within 1 year of therapy in different studies [17,23,25,26] which could be related to complete down-regulation of progesterone receptors in glands coinciding with modulation of apoptosis [27]. During the study, 6 patients requested a hysterectomy due to persistent bleeding with the LNG-IUS in situ for about 10 months. Their final histology result confirmed regression of EH. This finding matches with that reported by others with 2/28 of their patients with non-atypical complex EH requested a hysterectomy to be done due to persistent bleeding with the LNG-IUS in situ for 4 months and 7 months respectively [17]. Importantly, a common side effect of the LNG-IUS is persistent bleeding which may be experienced by up to 35% of its users during the first three treatment months, decreasing to 4% later on [28-30]. This bleeding adds difficulty for reliance on patient symptoms to monitor response in case of EH. The hysterectomy rate during the 12 months follow-up period in our study was significantly higher in the NET group than LNG-IUS group (57.4% vs. 22%). Moreover, the hysterectomy rate in patients with regression was significantly less in the LNG-IUS group compared with NET (11.5% vs. 29.7%). This finding supports the view that the future use of LNG-IUS to treat non-atypical EH can reduce the number of potentially unnecessary hysterectomies [16,17,23]. No atypia or cancer was found in the hysterectomy specimens. This may be attributed to the accuracy of the initial diagnosis of EH which was carried out by combined hysteroscopy with D&C. In conjunction with targeted biopsies or D&C, hysteroscopy was reported to have an excellent sensitivity, specificity, positive, and negative predictive values of 98%, 95%, 96%, and 98%, respectively to diagnose intrauterine pathology when compared with the histological findings of hysterectomy specimens [13,31]. There are some concerns regarding our study. First, it could be argued that conservative management for EH should be limited to young patients who want to preserve their fertility or patients with medical co-morbidities for whom surgery is contraindicated regardless of atypia. However, a recent survey carried out at the UK pointed out that the majority of the UK gynecologists (52.6%) would prefer two conservative choices (oral progestins or LNG-IUS) before deciding a hysterectomy for non-atypical EH. On the other hand, for atypical EH, the majority of them (83.2%) would perform a hysterectomy and would only consider LNG-IUS or oral progestins as a second or third option in women who wish to retain fertility [32]. A second argument is that endometrial curettage performed for initial diagnosis may have a therapeutic effect by removing the hyperplasia lesion from endometrial cavity; thereby the results of comparison in this study might be influenced. However, although curettage was used for removal of endometrial polyps [33], yet the evidence for a possible therapeutic effect in case of EH is lacking. One study evaluated the use of therapeutic curettage for menorrhagia and showed just a temporary effect as menstrual blood loss was reduced for only 1 month after curettage but then returned to previous levels [34]. Thirdly, this study was not triple-blinded because of the different nature of treatments. However, outcome assessors i.e., those performing the histological diagnosis of specimens and statistical analysis were blinded to the treatment groups. A fourth argument is that with the low progression rate of non-atypical EH into endometrial cancer (<5%) [5,6], hysterectomy is not necessary for those women who do not respond to progestin therapy in 6 months and consideration could be given to continued treatment for a longer time. However, following counseling, those women opted for hysterectomy rather than continued oral progestin therapy. Lastly, follow-up evaluation was performed in our study by Pipelle endometrial sampling. Concerns were raised regarding its accuracy as compared with D&C in diagnosing EH. A recent trial evaluated this issue among 673 patients [35]. Notably higher agreements were found in patients having EH with and without atypia. All cases (100%) of non-atypical EH and 90% of atypical EH which were diagnosed by D&C were detected on Pipelle biopsy. Sensitivity of Pipelle biopsy in detection of non-atypical EH was 67% vs. 62% for D&C and 75% for atypical EH vs. 83% for D&C [35]. Another concern is that the presence of the LNG-IUS in the uterine cavity may affect the accuracy of the Pipelle endometrial biopsy obtained during the follow up period. Of note, a prospective multicenter Korean study is currently under-running to estimate the treatment efficacy of LNG-IUS for EH as well as evaluating the consistency of the results of the office endometrial aspiration biopsy performed with the LNG-IUS in situ compared with that obtained by D&C after LNG-IUS removal [36]. Despite the above-mentioned concerns, the strength of our study resides in being the first RCT to date that evaluated the efficacy of the LNG-IUS for treatment of non-atypical EH. In conclusion, our study illustrates the superiority of LNG-IUS than the high dose oral NET therapy for non-atypical EH and should be regarded as a valuable treatment option in these cases being simple, highly effective within one year of treatment in addition to its superior compliance. Moreover, its use can reduce the number of potentially unnecessary hysterectomies performed in this subgroup and hence reduce morbidity and health care costs.

TITLE: Admixture of clonidine and fentanyl to ropivacaine in epidural anesthesia for lower abdominal surgery ABSTRACT.CONTEXT AND BACKGROUND:: Dose sparing action of one adjuvant for another in regional anesthesia. ABSTRACT.AIMS AND OBJECTIVES:: To evaluate and compare the clonidine-ropivacaine combination with fentanyl-ropivacaine in epidural anesthesia and also to find out whether addition of clonidine can reduce the dose of fentanyl in epidural anesthesia. ABSTRACT.MATERIALS AND METHODS:: 60 patients of ASA grade I and II between the ages of 21 and 55 years, who underwent lower abdominal surgeries, were included randomly into three clinically controlled study groups comprising 20 patients in each. They were administered epidural anesthesia with ropivacaine-clonidine (RC), ropivacaine-fentanyl (RF) or ropivacaine-fentanyl-clonidine (RCF). Per-op and post-op block characteristics as well as hemodynamic parameters were observed and recorded. Statistical data were compiled and analyzed using non-parametric tests and P<0.05 was considered as significant value. ABSTRACT.RESULTS:: The demographic profile of the patients in all the three groups was similar as were the various block characteristics. The reduction of clonidine and fentanyl in the RCF group did not make any significant difference (P>0.05) in the analgesic properties of drug combination and hemodynamic parameters as compared to RC and RF groups. However, there was significant reduction of incidence of side effects in the RCF group (P<0.05) and it resulted in increased patient comfort. ABSTRACT.CONCLUSIONS:: The analgesic properties of the clonidine and fentanyl when used as adjuvant to ropivacaine in epidural anesthesia are almost comparable and both can be used in combination at lower dosages without impairing the pharmacodynamic profile of the drugs as well as with a significant reduction in side effects. BODY.INTRODUCTION: Feeling of pain is one of the most important emotional determinants which dominate the perception of patients who undergo the surgical procedures. The thoughts of pain create a lot of unknown fears, anxiety and stress in the minds of such patients despite administration of adequate premedication. Even the post-op period can acquire the dimensions of a nightmare once a patient starts experiencing the agony of excruciating pain if not given proper attention. Epidural anesthesia and analgesia is widely regarded as a boon for such patients as it can provide a relief from pain for a longer duration and the facility of further top-ups and continuous infusion of the analgesic drugs through epidural catheter thus provides an uneventful and smooth recovery. Epidural bupivacaine had been used extensively in the past for providing adequate post-op pain relief in patients undergoing lower abdominal surgeries. However, in recent years, ropivacaine has increasingly replaced bupivacaine for the said purpose because of its similar analgesic properties, lesser motor blockade and decreased propensity of cardiotoxicity.[1] Though a slightly larger dose of ropivacaine is required as compared to bupivacaine to achieve the analgesic and anesthetic effects, the addition of an adjuvant can decrease the dose of ropivacaine required, thereby eliminating quite a few side effects associated with larger doses of ropivacaine.[23] Opioids, given by epidural route to relieve post-op pain, may provide adequate analgesia when given in low doses, but can also cause mental confusion, somnolence, nausea and vomiting, itching and respiratory depression when given in high doses.[45] Local anesthetic and opioid combination was shown to be more effective in epidural analgesia for post-op pain as their effects started rapidly and lasted longer when compared with local anesthetics given alone.[6] Epidural fentanyl has been widely used in neuraxial blockades as a better alternative to morphine as far as opioid-induced complications and side effects are concerned. The main site of action of fentanyl is the substantia gelatinosa in the dorsal horn of spinal cord, where it blocks the neural fibers carrying pain impulses both at pre-synaptic and post synaptic levels.[7] As fentanyl has no effect on sympathetic and motor neurons, it has advantages over local anesthetics. However, when it is used alone, analgesia will not be enough and overdosing is needed, and side effects like itching, nausea, vomiting and urinary retention are observed. Addition of opioid to local anesthetics gives the opportunity to use more diluted local anesthetic solutions for better analgesia, and reduces systemic toxicity risk and motor block incidence of local anesthetics.[8] The addition of an adjuvant has further enhanced the effectiveness of these local anesthetics as they not only help in intensifying and prolonging the blockade effect but also help in the reduction of the dose of local anesthetics. Clonidine is a partial alpha-2 adrenergic agonist that has a variety of different actions including antihypertensive effects as well as the ability to potentiate the effects of local anesthetics. It has been used as an adjuvant to epidural local anesthetics and opioids to improve the quality of analgesia after major abdominal surgeries.[910] It can provide pain relief by an opioid independent mechanism as it directly stimulates pre- and postsynaptic 2-adrenoceptors in the dorsal horn gray matter of the spinal cord, thereby inhibiting the release of nociceptive neurotransmitters.[11] Though clonidine provides intense and long lasting analgesia, these effects are inadequate for surgical anesthesia. Hence, clonidine has been used as an adjunct only to local anesthetics. At low doses, epidural clonidine improves the quality of anesthesia, reduces the dose requirement of the anesthetic agent and provides a more stable cardiovascular course during anesthesia.[12] Though at higher doses, it may further reduce the dose of local anesthetic and prolong the analgesic duration, but at the same time can exert its toxic effects resulting in profound hypotension, bradycardia and deep sedation.[13] Keeping all these pharmacological interactions in consideration, we planned a prospective, randomized, double-blind, clinically controlled trial in our institute. We used a mixture of fentanyl and clonidine as an adjuvant to ropivacaine in epidural block to see whether addition of alpha-2 agonist can help in dose sparing of an opioid while preserving the analgesic quality at the same time. BODY.MATERIALS AND METHODS: The present study was approved by the ethical committee of the institution and a written consent was taken from the patients after explaining to them in detail about the implications of the anesthetic and the surgical procedure. Inclusion criteria comprised 60 patients of ASA grade I and II between the ages of 21 and 55 years, who underwent lower abdominal surgeries (open hernia repair). The patients with hematological disease, bleeding or coagulation test abnormalities, psychiatric diseases, diabetes, history of drug abuse and allergy to local anesthetics of the amide type were excluded from the study. Patients were assigned to one of the following three treatment groups in a double-blinded fashion based on a computer-generated random numbers table: ropivacaine + clonidine (RC), ropivacaine + fentanyl (RF), or ropivacaine + fentanyl + clonidine (RFC). All the patients were administered premedication a night before and on the morning of the surgery which comprised tablet ranitidine 150 mg and tablet alprazolam 0.25 mg. The patients were explained about the sequence of anesthetic procedure in the pre-op room and a good IV access was secured. Thereafter, the patients were shifted to the operation theater and all monitoring devices were attached which included devices measuring heart rate (HR), ECG, SpO2, non invasive blood pressure (NIBP) and respiratory rate. Baseline hemodynamic parameters, respiratory rate, ECG and SpO2 were recorded. The anesthesia technician who prepared the syringes was given a written set of guidelines about the drug preparation and he was unaware of the operation theater team and the patients. Patients were administered epidural block in a sitting position with an 18-gauge Touhy needle and epidural space was localized and confirmed by loss of resistance to saline technique. Epidural catheter was secured 3–5 cm into the epidural space and confirmation for correct placement of the catheter was done by injecting 3 ml of 2% lignocaine HCl solution containing adrenaline 1:200,000. After 4–6 minutes of test dose, patients in group RC received 20 ml of 0.75% ropivacaine and 75 μg of clonidine. Group RF patients were administered 20 ml solution of 0.75% ropivacaine and 75 μg of fentanyl, while group RCF patients received 20 ml of 0.75% ropivacaine, 37.5 μg of clonidine and 37.5 μg of fentanyl. Surgical procedures were initiated only after the establishment of adequate surgical anesthetic effect with minimum level up to T6-7 dermatome. The bilateral pin-prick method was used to evaluate and check the sensory level while a modified Bromage scale (0 < no block, 1 < inability to raise extended leg, 2 < inability to flex knee and 3 < inability to flex ankle and foot) was used to measure the motor blockade effect at 5, 10, 15, 20, 25 and 30 minute intervals after the epidural administration of the drugs. The following block characteristics were observed and recorded: initial period of onset of analgesia, the highest dermatomal level of sensory analgesia, the complete establishment of motor blockade, the time to two segment regression of analgesic level from T6 dermatome, regression of analgesic level to L5 dermatome and time to complete recovery. Hemodynamic parameters, which included HR, ECG, mean arterial pressure (MAP), SpO2 and respiratory rate, were monitored continuously. Initial bolus dose timing was assumed to be the baseline time. Recordings were made every 5 minutes until 30 minutes and at 10-minute intervals, and thereafter up to 60 minutes and then at 15-minute intervals for the next hour and finally at 30 minutes in the third hour. Hypotension (defined as systolic arterial pressure falling more than 20% mm Hg) was treated with inj. mephenteramine 3–6 mg in bolus doses and HR<55 beats/min was treated with 0.3 mg of inj. atropine. Intravenous fluids were given as per the body weight and operative loss requirement, with no patient requiring blood transfusion. The patients were given supplementary O2 with the help of venturi mask. During the surgical procedure, any adverse event like anxiety, nausea, vomiting, pruritis, shivering, bradycardia, or hypotension was recorded. Nausea and vomiting were treated with 4–6 mg of i.v. ondansetron. During and at the end of surgery, the vitals were recorded in the recovery room also at 1, 5, 10, 20 and 30 minute interval. Sedation was evaluated by five-point scale (1 < wide awake; 2 < drowsy; 3 < dozing; 4 < mostly sleeping; 5 < awakening only when aroused). The onset of pain was managed by top-up doses of 8 ml of 0.2% ropivacaine and 50 μg of clonidine in RC group while 50 μg fentanyl was added to the same volume of ropivacaine in RF group. The patients in RCF group received 25 μg each of clonidine and fentanyl along with 8 ml of 0.2% ropivacaine during the first top-up dose. Later on, all the patients received 8 ml of 0.2% ropivacaine for relief of post-op pain. Comparability of the groups was analyzed with analysis of variance test (ANOVA). Student's two tailed "t" test and chi square test were applied to analyze the parametric data (maternal hemodynamic parameters and block characteristics). For all statistical analysis, the value of P<0.05 was considered as significant. BODY.RESULTS: Sixty patients were enrolled in this study and their data were eligible and were processed for statistical analysis. The three groups were comparable with regard to demographic data as shown in table 1. There was no statistically significant variation between the three groups with regard to age, weight, height and body mass index (P>0.05). Duration of surgery was comparable in both the groups and did not show any significant variation. Table 1 Comparison of demographic profile of the RC, RF and RCF groups Onset of anesthesia was faster in group RF as compared to group RC and RCF as shown in table 2. However, once sensory level was established at T6-T7 level, there was no noticeable difference in sensory anesthesia in any of the three groups throughout the surgical procedure. The establishment of complete motor blockade was earlier in the RF group which was statistically significant, when compared to RCF (P<0.05). The hypotension was treated with incremental doses of mephenteramine 3 mg bolus doses, but the total dose did not cross 18 mg in any of the groups. Mephenteramine was used almost 40% lower in the RCF group which was statistically significant (P<0.05) [Table 2]. Table 2 Comparison of initial block characteristics in RC, RF and RCF groups As is clearly evident from the table 3, the regression of block height was slightly prolonged in RF group while the total duration of anesthesia and time for supplementary analgesic doses was prolonged in RF group, which was statistically nonsignificant on analysis (P>0.05). The mean total dose of ropivacaine consumed post-op was highest among the patients of RCF group, which on statistical comparison turned out to be a significant value when compared to RCF group (P<0.05). Table 3 Comparison of per-op and post-op block characteristics in RC, RF and RCF groups Table 4 and the Figures 1–4 shows that MAP and mean HR were comparable in all the three groups during the entire procedure as well as post-op, which was a non-significant value on statistical comparison (P>0.05). Similarly, pulse oximetry trends did not show any significant variation in patients of all the three groups. Table 4 Comparison of vital parameters in patients of RC, RF and RCF groups Figure 1Intra-op MAP comparison in all the groups Figure 2Post-op MAP comparison in all the groups Figure 3Intra-op HR comparison in all the groups Figure 4Showing the post-op HR comparison in all the groups Table 5 outlines the various side effects observed during the entire study with all the three drug groups. In the RF group, 40% of the patients experienced nausea/vomiting as compared to 15% in group RC and 10% in RCF, which was statistically significant (P<0.05). Similarly, we observed sedation in 30% of the patients in group RF as compared to 10% in RC group and 5% in RCF group, which again was a statistically significant value on intergroup comparison (P<0.05).The incidence of dry mouth was statistically higher to significant values in group RC (P<0.05). The incidence of other side effects like shivering, headache, urinary retention and respiratory depression was almost comparable and statistically nonsignificant. Table 5 Incidence of side effects in patients of all the three groups BODY.DISCUSSION: The quest for optimal dose finding of either an opioid or alpha-2 blocker as an adjuvant in regional anesthesia is never ending but there are hardly any studies which have tried to compare the potential benefits of either the two combinations or tried to evaluate the "synergistic" effect of these two in lower concentration.[14–16] Ours is the first study to evaluate rigorously the effects of clonidine on dose reduction concentration of fentanyl, when administered as an adjuvant to ropivacaine in epidural anesthesia for lower abdominal surgeries. Fentanyl has almost completely replaced the traditional opioid morphine as an adjuvant in regional anesthesia, but side effects like nausea and vomiting still persist to an alarming extent of 28–52%, when used in optimal dose, which is quite discomforting for the patients and defeats the purpose of providing a smooth uneventful recovery.[5617] The analgesic effects of the ropivacaine–clonidine combination are equivalent to those of the ropivacaine–fentanyl. The results of the present study demonstrate that it is possible to decrease the unwanted side effects of epidural fentanyl by reducing the dose of fentanyl and adding an equivalent dose of clonidine to the epidural solution, and that too without impairing the analgesic effect. The reduction in opioid requirement does have a significant effect on reduction of incidence of nausea and vomiting, beneficial effects on respiratory functions as well as reduction of other opioid-related side effects.[56] We did not observe a single case of respiratory depression in our study and this probably may be due to smaller dose of fentanyl we used in our study. Alpha-adrenergic agonists produce pain relief through an opioid independent mechanism and may be alternatives to opioid for combination with local anesthetics for analgesia during surgery.[1819] Based on the pharmacokinetic properties like molecular weight, lipid solubility and cerebrospinal fluid level, clonidine can be expected to exert similar onset of its analgesic effect and duration of analgesia as compared to fentanyl, but analgesic effect of fentanyl starts faster and lasts longer.[20] The attempts made earlier for dose determination concluded that 75 μg of clonidine is the optimal epidural dose when added to bupivacaine for analgesia, as smaller doses were not serving the purposes of adequate analgesia while larger doses were associated with bradycardia, hypotension, sedation and other side effects.[21] Therefore, we administered single clonidine dose of 75 μg for operative purpose while top-up doses of 50 μg clonidine were administered with 0.2% ropivacaine for the post-op pain relief. The initial block characteristics in the present study did not show much difference among the three study groups. The onset time and establishment of block was slightly shorter in fentanyl group but clinically nonsignificant, which is almost comparable to other studies.[15] The mephenteramine requirement was significantly lesser in the group receiving a mixture of clonidine and fentanyl, which clearly proves that doses of both fentanyl and clonidine can be reduced, thereby eliminating the incidences of complications and that too without impairing the analgesia. Similarly, peri-op and post-op block characteristics followed the same pattern as initial block effects, which correlate very well with the findings of other studies.[18] The total post-op dose consumption of ropivacaine was significantly lesser in RF group as compared to RCF group, which again helped us to draw the conclusions that epidural doses of clonidine and fentanyl can be reduced in combination and still be able to produce a slightly lesser but acceptable degree of anesthetic and analgesic effects with a very low incidence of side effects. The stable hemodynamic parameters throughout the surgical procedure as well as post-op in all the three groups do point toward the equal effectiveness of all the three combination of drugs in exerting minimal cardiac effects. Both fentanyl and clonidine, when used alone in optimal doses in epidural route with ropivacaine, do produce an array of side effects which include sedation, nausea/vomiting, dry mouth, etc. The side effects become almost nonexistent when these are used in combination with their dose reduced to half of the optimal dose. As a result, the patient in group RCF felt more comfortable in the post-op period and described the anesthesia as the most pleasing experience. BODY.CONCLUSIONS: The study helped us to conclude the established facts about the epidural usage of clonidine and fentanyl, when added as an adjuvant to ropivacaine. Though both the drugs have side effects when used in epidural route in optimal doses, the spectrum of side effects of clonidine is much narrower than that of fentanyl.Combination of clonidine with fentanyl does allow the reduction of individual doses of these drugs through epidural route.The combination of clonidine and fentanyl in half than the optimal doses has almost similar pharmacokinetic and pharmacodynamic profile when used with ropivacaine in epidural anesthesia.The power analysis of the study was estimated at 80%. Therefore, we recommend that the combination of fentanyl and clonidine can be safely used with ropivacaine and that too in half the doses as they have got a synergistic adjuvant effect.

TITLE: Effects of gelatin sponge combined with moist wound-healing nursing intervention in the treatment of phase III bedsore ABSTRACT: Pressure sore pertains to tissue damage or necrosis that occurs due to lack of adequate nutrition following long-term exposure to pressure and decreased blood circulation. The aim of the study was to examine the effects of gelatin sponge combined with moist wound-healing nursing intervention in the treatment of phase III bedsore. In total, 50 patients with phase III bedsore were included in the present study. The patients were randomly divided into the control (n=25) and observation (n=25) groups. Patients in the control group received conventional nursing, while those in the observation group received gelatin sponge combined with moist wound healing nursing. The effects of the two nursing methods were compared and analyzed. The results showed that the improvement rate of the observation group was significantly higher than that of the control group (P<0.05). The Branden score and area of pressure sore of the observation group were significantly lower than those of the control group (P<0.05). The frequency and time of dressing change and the average cost of hospitalization of the observation group were significantly lower than those of the control group (P<0.001). In conclusion, gelatin sponge combined with moist wound-healing nursing intervention may significantly improve the treatment of phase III bedsore. BODY.INTRODUCTION: Pressure sore refers to the tissue damage or necrosis that results from the lack of adequate nutrition caused by the body's long-term exposure to pressure and slacking blood circulation (1). Pressure sore is a common complication in patients with breast cancer (2). Major clinical treatments are traditional medicine and nursing intervention; however, the curative effects are not optimistic (3). In 1962, Dr Winter identified moist wound-healing nursing, which became widely used (4). Type I gelatin sponge has similar components with the connective tissues of the body. It can quickly promote tissue healing, realize rapid degradation, and has good biocompatibility characteristics; thus, it has been widely used in tissue engineering (4). The aim of the present study was to examine the effects of gelatin sponge combined with moist wound-healing nursing intervention in the treatment of phase III bedsore. BODY.MATERIALS AND METHODS.MATERIALS: A total of 50 patients with phase III bedsore, treated at the Liaocheng People's Hospital between March 2013 and July 2014 were included in the study. All the patients conformed to the diagnostic criteria suggested by the National Pressure Ulcer Advisory Panel Society: entire skin defect without muscles, tendons or bones exposed, with or without incrustation and subcutaneous tunnel (6). Approval for the study was obtained from the ethics committee of Liaocheng People's Hospital. Informed consent was obtained from the patients and/or relatives. The patients were randomly divided into the control (n=25) and observation (n=25) groups. The patient age range for the control group was 59–80 years, with an average of 68.0±6.5 years. In control group, 31 pressure sores were located in sacroiliac, 11 pressure sores in hip joint and 8 in ankle and foot. The Branden scores ranged from 10 to 22 points, with an average of 14.0±3.2 points, and the area of pressure sore ranged from (2×1 cm) to (10×8 cm). Concerning the observation group, the patient age range was 56–82 years, with an average age of 67.9±7.2 years. In those patients, 33 pressure sores were located in sacroiliac, 10 pressure sores in hip joint and 6 in ankle and foot. The Branden scores ranged from 11 to 24 points, with an average of 15.3±3.5 points, and the area of pressure sore ranged from (1.5×1.5 cm) to (10.5×7.5 cm). The differences in age, pressure sore sites, Branden score and area of pressure of the two groups were not statistically significant (P>0.05). BODY.MATERIALS AND METHODS.NURSING METHODS: Patients in the control group were provided conventional nursing care. Hydrogen peroxide or iodine was used for local disinfection and mechanical debridement to remove the purulent secretion of necrotic tissues, and ethacridine gauze was used to fill in the potential lacuna of the lacunar pressure sore. Aseptic dressing was used to cover the wound, and the dressing was changed once every 1–2 days. The patients in the observation group received gelatin sponge combined with moist wound-healing nursing. The normal saline or cotton ball was used to clean the wound surface. The yellow wound surface (if any) was cleaned with surgical debridement (blade, or sterile scissors) to excise the necrotic tissues and the gelatin sponge to absorb seepage and cover the wound. In case of infection, gelatin sponge was soaked with silver ion alginate (Kanglebao Company, Guangzhou, China) was used to cover the yellow surface and the skin in or around the dressing was closely examined. If there was seepage and the milk white area was >1/3, the sponge (once/1–2 days) was changed in a timely manner after new granulation tissues appeared on the sponge. The area was washed again to apply the gelatin sponge once per week. Humanized nursing was strengthened gently and the patients were slowly turned over once every 2 h by elevating the bedside to maintain an angle within 30° to prevent exertion of excessive force on the sacral tail. The patients were kept dry and smooth, and an air cushion bed comprehensively improved the nutritional status of the patients. Subsequently, the patients were followed up and provided with health education prior to discharge from the hospital. Additionally, the patients and their families were informed of the risk factors of pressure sores. BODY.MATERIALS AND METHODS.OBSERVATION INDICES AND EVALUATION STANDARD: At 28 days later, the healing state of patients was closely examined including the curative effects, frequency of dressing change and efficiency of end point. The quantitative scoring was performed according to Pressure Ulcer Scale for Healing (7) compiled by the US pressure sore expert group and the curative effects were evaluated (Table I). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis and data were presented as mean ± standard deviation. The comparison between the groups was made using the t-test. The enumeration data were expressed by percentage (%). A comparison between groups was made using the χ2 test. P<0.05 was considered to indicate a statistically significant difference. BODY.RESULTS.COMPARISON OF HEALING EFFECTS: The improvement rate of the observation group (92.0%) was significantly higher than that of the control group (68.0%) (Table I; P<0.05). BODY.RESULTS.COMPARISON OF BRANDEN SCORES AND AREA OF PRESSURE SORE: Prior to treatment, the Branden score and area of pressure sore of the control and observation groups were not statistically different (P>0.05). Following treatment, the Branden score and area of pressure sore of the observation group were significantly lower than those of the control group (P<0.05; Table II). BODY.RESULTS.COMPARISON OF FREQUENCY AND TIME OF DRESSING CHANGE, AND COSTS: The frequency, time of dressing change and average costs of hospitalization of the observation group (7.8±0.9, 20.9±8.4, and 2675.4±234.5, respectively) were significantly lower than those of the control group (16.2±2.7, 31.8±12.6, and 7826.5±1342.1, respectively) (P<0.001) as shown in Table III. BODY.DISCUSSION: Traditional cleaning, disinfection and other commonly used medical gauze, due to their poor moisture absorption, cannot be degraded in vivo and the prescription contains light powder that is toxic to liver and kidney (8). Gelatin sponge effectively absorbs the tissue fluid, and is favorable for local hemostasis. It can absorb water of 50-fold and blood of 48-fold its own weight. After absorbing a large amount of blood, gelatin sponge can promote the rupture of platelet, release a large number of platelet coagulation factors and promote blood coagulation (9). In addition, gelatin sponge supports the blood block to prevent it from decreasing, resulting in anastalsis (10). The gelatin sponge became jelly-like one week following application and was completely absorbed after 1–2 months. It could be left in the body and did not produce any antigenicity, excessive scar tissues or fibrotic reactions (11). Gelatin sponge is a also good drug carrier. Previous findings have shown that it promoted wound surface healing compared to the external application of Chinese medicine or gauze (12). In the present study, the importance of moist healing nursing was also emphasized. Other studies on wound surface have confirmed that dryness can aggravate the degree of tissue damage while moisture was more favorable for the wound surface healing (13). Moisture can promote and accelerate natural healing and the healing speed in the moisture environment is faster than the dry environment. For the pressure sores of patients with breast cancer, the optimal way in which to restore the damaged epithelial tissues is to seal the moist wound, promote the growth of keratinocytes of the wounds and elevate the regenerative velocity by 40% (14). A moist environment is advantageous to the adhesion of dressing and wound surface, and would not damage the granulation tissue or wound epithelial tissue, relieving pain experienced by the patients (15). Patients of the control group received sterile gauze caring. During dressing, gauze dressing was likely to adhere to the surface of the wound, rendering it unfavorable for seepage absorption, and excessive seepage also required frequent change of gauze. Patients of the observation group were provided with gelatin sponge caring, which effectively improved the absorption effects of seepage, kept the area of pressure sore in a closed space and avoid invasion and infection of external microorganisms (16). Patients of the observation group were also assisted by humanized nursing, such as psychological guidance and health education (17), and had nurse assistance in being turned over once every 2 h and keeping their skin smooth and dry. The above measures constitute a good treatment environment for breast cancer patients with pressure sores (18). The results of the present study have shown that the improvement rate of the observation group was significantly higher than that of the control group, The Branden score and the area of pressure sore were significantly lower than those of the control group, and frequency and time of dressing change and average cost of hospitalization were significantly lower than those of the control group. These differences were statistically significant, thereby greatly reducing the workload of nursing personnel (19). In conclusion, gelatin sponge combined with moist wound-healing nursing intervention may significantly improve the healing of phase III bedsore.

TITLE: Comparison between the Effectiveness of Oral Phloroglucin and Cimetropium Bromide as Premedication for Diagnostic Esophagogastroduodenoscopy: An Open-Label, Randomized, Comparative Study ABSTRACT.BACKGROUND/AIMS: Suppression of gastrointestinal (GI) peristalsis during GI endoscopy commonly requires antispasmodic agents such as hyoscine butylbromide, atropine, glucagon, and cimetropium bromide. This study examined the efficacy of oral phloroglucin for the suppression of peristalsis, its impact on patient compliance, and any associated complications, and compared it with intravenous or intramuscular cimetropium bromide administration. ABSTRACT.METHODS: This was a randomized, investigator-blind, prospective comparative study. A total of 172 patients were randomized into two groups according to the following medications administered prior to upper endoscopy: oral phloroglucin (group A, n=86), and cimetropium bromide (group B, n=86). The numbers and the degrees of peristalsis events at the antrum and second duodenal portion were assessed for 30 seconds. ABSTRACT.RESULTS: A significantly higher number of gastric peristalsis events was observed in group A (0.49 vs. 0.08, p<0.001), but the difference was not clinically significant. No significant difference between both groups was found in the occurrence of duodenal peristalsis events (1.79 vs. 1.63, p=0.569). The incidence of dry mouth was significantly higher with cimetropium bromide than with phloroglucin (50% vs. 15.1%, p<0.001). ABSTRACT.CONCLUSIONS: Oral phloroglucin can be used as an antispasmodic agent during upper endoscopy, and shows antispasmodic efficacy and adverse effects similar to those of cimetropium bromide. BODY.INTRODUCTION: Upper endoscopy facilitates the detection and treatment of gastrointestinal (GI) disease. Antispasmodic agents such as hyoscine butylbromide, atropine, glucagon, cimetropium bromide, and L-menthol are often administered prior to GI endoscopy to inhibit peristalsis and improve visualization.1,2 However, these agents must be administered intravenously or intramuscularly and may cause adverse effects such as dry mouth, urinary retention, temporary impairment of visual accommodation, palpitations, anaphylactic shock, and hyperglycemia.3,4,5 Cimetropium bromide (Algiron; Boehringer Ingelheim GmbH, Ingelheim, Germany) is used particularly frequently in South Korea. Cimetropium bromide can cause pain and preprocedural anxiety due to its administration by intravenous or intramuscular injection, the preparation of which is time-consuming.6,7 Phloroglucin (Flospan; Daehwa Pharmaceutical, Seoul, Korea), administered orally, was expected to reduce pain and discomfort more effectively than intravenous or intramuscular injections of other antispasmodic agents. However, few studies of its usefulness as an endoscopic premedication have been performed. In this study, we examined the efficacy of oral phloroglucin for the suppression of peristalsis, its impact on patient compliance, and any associated complications, and compared it with the intravenous or intramuscular administration of cimetropium bromide. BODY.MATERIALS AND METHODS: This was a randomized, investigator-blind, prospective comparative study. From August 2012 through May 2013, we enrolled 174 patients who visited the Ewha Womans University Mokdong Hospital. Eligible patients were aged 18 years or older and scheduled to be examined by esophagogastroduodenoscopy. Patients with a history of upper GI surgery, GI bleeding, pregnancy, or contraindications for anticholinergic agents (glaucoma, myasthenia gravis, and urinary obstruction) were excluded from the study. Written informed consent was obtained from all subjects before enrollment. This study was approved by the Ewha Womans University's Ethics Committee. Patients were randomized into two groups according to the following medications administered prior to upper endoscopy: oral phloroglucin (group A) and cimetropium bromide (group B). All endoscopic procedures were performed by a single experienced endoscopists who was blinded to the patients' group assignments. We evaluated total procedure times (from insertion to removal), total number of peristalsis events (stomach and duodenal motility numbers, counted at the antrum and duodenal second portion for 30 seconds each), and patient responses to questionnaires assessing tolerance and adverse events during the procedure (mouth dryness, nausea, vomiting, dizziness, headache, and abdominal pain). The degree of peristalsis was assessed using visibility scores (range, 0 to 2) at the antrum and duodenal second portion (0, no peristalsis; 1, slight peristalsis but no obscured visibility; 2, severe peristalsis with obscured visibility). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Demographic characteristics and visibility scores were assessed using the chi-square test and Student t-test. A p-value <0.05 was considered statistically significant. BODY.RESULTS: Among the 174 patients enrolled in the study, two were excluded because of severe duodenal stenosis. The remaining 172 patients were randomized into two groups according to medication administered prior to upper endoscopy, namely oral phloroglucin (group A, n=86) or cimetropium bromide (group B, n=86). The demographic and other baseline characteristics of included patients are shown in Table 1. There was no statistically significant difference between the two groups regarding age, sex, medical history (with the exception of thyroid disease), and the proportion of patients taking sedatives for endoscopy. The incidence and degree of peristalsis in each group is presented in Table 2. A significantly higher number of gastric peristalsis events was seen in group A (0.49±0.85 vs. 0.08±0.28, p<0.001), but the number of events was fewer than one in both groups, and the difference was not clinically significant. The degree of peristalsis of the stomach was significantly lower in group B (1.14±0.38 vs. 1.00, p=0.001), but both groups had visibility scores of approximately 1, with a clinically insignificant difference. No significant between-group difference was found for the number of duodenal peristalsis events (1.79±2.0 vs. 1.63±1.8, p=0.569). The degree of peristalsis of the duodenum did not differ significantly between the groups (1.21±0.4 vs. 1.15±0.39, p=0.342). There was no significant difference between the groups in total procedure time (5.28±2.07 minutes vs. 5.10±1.94 minutes, p=0.563). The incidence and degree of peristalsis and procedure time were not different between patients who received a biopsy and those who did not (Table 3). Tolerance of endoscopy was not significantly different between the two groups, and the same number of patients tolerated the procedure well in both groups (group A, n=75, 87.2%; group B, n=75, 87.2%). No serious adverse events occurred in the course of the study. The incidence of adverse effects is presented in Table 4. The incidence of dry mouth was significantly higher with cimetropium bromide than with phloroglucin (50% vs. 15.1%, p<0.001). No significant between-group differences were noted for the incidence of other adverse events such as nausea, vomiting, dizziness, headache, dysuria, and abdominal pain. BODY.DISCUSSION: Cimetropium bromide (Algiron) is often used before GI endoscopy to inhibit peristalsis and improve visualization, and is particularly popular in South Korea. However, cimetropium bromide causes pain and preprocedural anxiety due to its administration by intravenous or intramuscular injection, the preparation of which is time-consuming. This study showed that oral phloroglucin is somewhat inferior to cimetropium bromide in the suppression of gastric peristalsis, but the difference was not clinically significant because the number of peristalsis events was less than one in both groups, and the degree of peristalsis was approximately grade 1 in both groups. In this study, we demonstrated that oral phloroglucin is not inferior to cimetropium bromide in the inhibition of peristalsis during endoscopy. In addition, endoscopic examination using oral phloroglucin was associated with similar procedure times, tolerance of endoscopy, and adverse events profiles. Furthermore, phloroglucin is superior to cimetropium bromide with respect to the incidence of dry mouth. An important advantage of oral phloroglucin is its ease of administration, effective suppression of peristalsis during endoscopy, and reduction in the incidence of dry mouth. Our findings suggest that oral phloroglucin can be used for the suppression of gastroduodenal peristalsis during upper endoscopy. Our study had some limitations. We did not examine the effect of phloroglucin during endoscopic procedures such as endoscopic mucosal resection, endoscopic submucosal dissection, endoscopic retrograde cholangiopancreatography, and colonoscopy. Further studies are needed to examine the effects of oral phloroglucin during a variety of endoscopic therapeutic procedures and colonoscopy. In conclusion, oral phloroglucin can be used as an antispasmodic agent during upper GI endoscopy with similar antispasmodic efficacy and fewer adverse effects when compared with cimetropium bromide.

TITLE: Relationship Between Low‐Density Lipoprotein Cholesterol, Free Proprotein Convertase Subtilisin/Kexin Type 9, and Alirocumab Levels After Different Lipid‐Lowering Strategies ABSTRACT.BACKGROUND: Alirocumab undergoes target‐mediated clearance via binding of proprotein convertase subtilisin/kexin type 9 (PCSK9). Statins increase PCSK9 levels; the effects of nonstatin lipid‐lowering therapies are unclear. Every‐4‐weeks dosing of alirocumab may be appropriate for some patients in absence of background statin but is not yet approved. ABSTRACT.METHODS AND RESULTS: Low‐density lipoprotein cholesterol (LDL‐C), PCSK9, and alirocumab levels were assessed in subjects (LDL‐C >130 mg/dL, n=24/group) after a 4‐week run‐in taking oral ezetimibe, fenofibrate, or ezetimibe placebo, when alirocumab 150 mg every 4 weeks (days 1, 29, and 57) was added. Maximal mean LDL‐C reductions from day −1 baseline (prealirocumab) occurred on day 71 in all groups: alirocumab plus placebo, 47.4%; alirocumab plus ezetimibe, 56.6%; and alirocumab plus fenofibrate, 54.3%. LDL‐C reductions were sustained through day 85 with alirocumab plus placebo (47.0%); the duration of effect was slightly diminished at day 85 versus day 71 with ezetimibe (49.6%) or fenofibrate combinations (43.2%). Free PCSK9 concentrations were lowest at day 71 in all groups, then increased over time; by day 85, free PCSK9 concentrations were higher, and alirocumab levels lower, with alirocumab plus fenofibrate, and to a lesser extent alirocumab plus ezetimibe, versus alirocumab plus placebo. ABSTRACT.CONCLUSIONS: Alirocumab 150 mg every 4 weeks produced maximal LDL‐C reductions of 47% in combination with placebo and 54% to 57% in combination with ezetimibe or fenofibrate. The oral lipid‐lowering therapies appear to increase PCSK9 levels, leading to increased alirocumab clearance. Although the duration of effect was modestly diminished with alirocumab plus ezetimibe/fenofibrate versus placebo, the effect was less than observed in trials with background statins, and it would not preclude the use of alirocumab every 4 weeks in patients taking these nonstatin lipid‐lowering therapies concomitantly. ABSTRACT.CLINICAL TRIAL REGISTRATION: URL: http://www.Clinicaltrials.gov. Unique identifier: NCT01723735. BODY.INTRODUCTION: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds the low‐density lipoprotein receptor (LDLR) on hepatocytes.1 Monoclonal antibodies to PCSK9 block its effect on the LDLR and reduce low‐density lipoprotein cholesterol (LDL‐C) levels. Such antibodies undergo target‐mediated clearance by binding to PCSK9. Although statins reduce LDL‐C, they also increase PCSK9 levels.2 This may limit the extent of LDL‐C reduction possible with statins as well as affecting clearance of PCSK9 antibodies when added to statins. Addition of the PCSK9 antibody alirocumab to statin therapy produces sustained LDL‐C reductions when dosed every 2 weeks (Q2W).3, 4, 5, 6 However, when alirocumab was dosed every 4 weeks (Q4W) in combination with statin, reductions were not fully sustained over the dosing interval.4, 6 This may be because of a statin‐induced increase in PCSK9 levels and target‐mediated clearance of alirocumab. A single 150‐mg dose of alirocumab without background statin appeared to give a more stable reduction over 4 weeks.7 However, multiple dosings of alirocumab Q4W without background statins have not been investigated. Moreover, little is known about potential effects on LDL‐C and PCSK9 from combining alirocumab with nonstatin lipid‐lowering therapies (LLTs) such as ezetimibe and fenofibrate. Ezetimibe inhibits the digestive absorption of cholesterol and related plant sterols and is primarily used for reducing LDL‐C as an adjunct to statin.8 Fenofibrate, an activator of peroxisome proliferator‐activated receptor α, increases lipolysis and clearance of triglyceride‐rich lipoproteins; although fenofibrate reduces LDL‐C (in the context of lower baseline triglycerides), it also has a marked effect on triglycerides.9 The effect of ezetimibe and fenofibrate on PCSK9 is unclear. Animal studies suggest that ezetimibe increases PCSK9 levels,10, 11 but no effect was seen in humans.12 The change in PCSK9 levels with fibrates varies widely among reports.13 This study assessed the pharmacokinetic and pharmacodynamic profile and circulating PCSK9 levels when alirocumab 150 mg is administered Q4W with placebo or in combination with ezetimibe or fenofibrate. BODY.MATERIALS AND METHODS: This randomized partial‐blinded study (clinicaltrials.gov identifier: NCT01723735) was conducted in 2 centers in France in 3 parallel groups. The study protocol was approved by the appropriate institutional review boards and was performed in accordance with the ethical principles of the Declaration of Helsinki and in compliance with good clinical practice. All subjects provided written informed consent. BODY.MATERIALS AND METHODS.SUBJECTS: Male or female subjects were eligible if they were aged 18 to 65 years with a body mass index between 18.0 and 30.0 kg/m2 and had LDL‐C levels >130 mg/dL at screening. Subjects were certified as healthy on the basis of a comprehensive clinical assessment (detailed medical history and complete physical examination) and were not taking any LLTs at the time of enrolment. Any previous LLT had been discontinued at least 4 weeks before screening. Subjects were excluded if they had any clinically significant medical history or if they had taken any medication within the 2 weeks before the study start (with the exception of hormonal contraception or menopausal hormone replacement therapy). BODY.MATERIALS AND METHODS.STUDY DESIGN: After the screening, subjects were randomized into 3 groups, entered a 28‐day initial therapy run‐in period, and received once‐daily oral doses of either ezetimibe placebo, ezetimibe 10 mg, or fenofibrate 160 mg (Figure 1). Subjects with LDL‐C levels ≥100 mg/dL at the end of the run‐in period then entered the alirocumab treatment period, during which they received alirocumab 150 mg Q4W subcutaneously on days 1, 29, and 57, in addition to continuing initial oral therapy. Subjects were instructed to keep to their usual diet throughout the study. The oral ezetimibe placebo matched the ezetimibe tablets; fenofibrate treatment was unblinded because placebo fenofibrate was not available for use in the study. The run‐in period was included to allow steady state of both LLT compounds to be reached and full lipid‐lowering effect to be achieved,14 as well as to assess their effect on PCSK9 concentrations before alirocumab was administered. Figure 1Study design. *Placebo for ezetimibe. Red arrows indicate time of alirocumab injections. At the bottom left of the figure, PK refers to free PCSK9 and total alirocumab. D indicates study day; EZE, ezetimibe; FENO, fenofibrate; PCSK9, proprotein convertase subtilisin/kexin type 9; PO, oral administration; Q4W, every 4 weeks; QD, every day; R, randomization; SC, subcutaneous administration. BODY.MATERIALS AND METHODS.END POINTS: The primary end point was the mean percent change in LDL‐C from day −29 (the day before the initial therapy run‐in). However, because this study was intended to specifically assess the effect of alirocumab, an additional baseline of day −1 (the day before first administration of alirocumab) was considered at the time of the statistical analysis plan. This additional baseline therefore accounted for changes in lipid profiles during the initial therapy run‐in period with ezetimibe and fenofibrate alone and allowed assessment of the specific effect of alirocumab on LDL‐C in combination with placebo (ie, as monotherapy) or in combination with the LLTs. The primary analysis was considered to be percent change from baseline to day 71. Two time points were used as baseline as shown in Figure 1. Secondary end points included the percent change in secondary lipid parameters, including total cholesterol, non–high‐density lipoprotein cholesterol (non–HDL‐C), apolipoprotein B, lipoprotein(a), triglycerides, HDL‐C, and apolipoprotein A1 and changes in free and total PCSK9 levels and total alirocumab levels. Pharmacokinetic parameters calculated for alirocumab included maximum serum concentration (Cmax) following the third injection and the area under the serum concentration–versus–time curve from day 57 to day 85 (AUCD57–85; ie, the 28‐day period after the last alirocumab dose). Safety, including adverse events (AEs), vital signs, electrocardiogram, and laboratory assessments, was also evaluated. BODY.MATERIALS AND METHODS.LABORATORY ASSESSMENTS: Blood samples were taken in the morning after a 10‐hour overnight fast and before study drug administration. Sampling times are shown on Figure 1; note that no measurements were taken after the second alirocumab injection (indicated by a dotted line on the results figures). Total cholesterol, HDL‐C, and triglycerides were directly measured at local laboratories; apolipoprotein B, apolipoprotein A1, and lipoprotein(a) were directly measured at a central laboratory. LDL‐C was calculated by using the Friedewald formula (LDL‐C=total cholesterol−HDL‐C−[triglycerides/5]). Free and total PCSK9 levels and total alirocumab levels in serum were determined by using specific validated enzyme‐linked immunosorbent assays (Regeneron Pharmaceuticals). The lower limits of detection were 31.2 ng/mL for free PCSK9, 156 ng/mL for total PCSK9, and 78 ng/mL for alirocumab; values below these levels are set to 0 for presentation of results. BODY.MATERIALS AND METHODS.STATISTICAL METHODS: Enrollment of 72 subjects into the alirocumab treatment phase was planned to ensure ≥60 subjects (20 per group) could be included in the primary analysis. This sample size was calculated to give a maximal imprecision of ≤18.7% in terms of 95% CIs, for the relative change of LDL‐C means between alirocumab plus ezetimibe or alirocumab plus fenofibrate versus alirocumab plus placebo, with 90% assurance, assuming an SD of 25% based on previous trial results.7 The study was not powered for statistical comparisons of changes in secondary end points. The percent change from baseline in LDL‐C was analyzed by using a linear mixed‐effects model. This model had fixed terms for treatment (alirocumab 150 mg Q4W plus ezetimibe, alirocumab 150 mg Q4W plus fenofibrate, or alirocumab 150 mg Q4W plus placebo), sex, study site, day, and treatment × day interaction term and with "subject (treatment×sex)" as a random effect. A variance component structure was used for the variance–covariance matrix of the random effects. The variance component structure was defined as:σij2=σk21(i=j)and i corresponds to thekth effect Other parameters such as age were not included in the model, because differences between groups were not significant. Estimates and 95% CIs were determined at each time point for the difference between means of alirocumab plus ezetimibe or fenofibrate combinations versus alirocumab plus placebo. This analysis was conducted by using day −29 as baseline and was repeated with day −1 as baseline. Secondary lipid parameters were assessed in a similar way, and P‐values are given for descriptive purposes only. Because all analyses were considered as fully exploratory, multiplicity was not considered. Changes in free and total PCSK9 levels were summarized by using descriptive statistics. Estimates (and 90% CIs) of geometric mean ratios of alirocumab plus ezetimibe versus alirocumab plus placebo, or alirocumab plus fenofibrate versus alirocumab plus placebo, were calculated by using a linear fixed‐effects model. Pharmacokinetic parameters were summarized by using descriptive statistics. The effect on alirocumab serum levels of coadministering alirocumab with ezetimibe or with fenofibrate versus alirocumab plus placebo was assessed by using a linear fixed‐effects model with fixed terms for treatment, sex, and study site and with log of weight as covariate. Estimates and 90% CIs for the ratios of geometric means of Cmax and AUC values between each treatment group were determined. Safety data were summarized by using descriptive statistics. Statistical analyses were conducted with use of SAS Proc Mixed (SAS Institute). BODY.RESULTS: One hundred sixty‐seven subjects were screened, and 79 subjects were randomized and entered the run‐in period. Of these 79 subjects, 72 (24 per group) qualified to enter the alirocumab treatment period and 7 were excluded: 4 because their LDL‐C was <100 mg/dL by the end of the run‐in period, 2 because of AEs (increased alanine transaminase levels while receiving fenofibrate), and 1 withdrew for personal reasons. Baseline characteristics for the 72 subjects who commenced alirocumab treatment and were included in the primary analysis are shown in Table 1. Table 1 Subject Characteristics at Main Baseline (Day −29) Treatment Group Alirocumab 150 mg Q4W+Placebo (n=24) Alirocumab 150 mg Q4W+Ezetimibe (n=24) Alirocumab 150 mg Q4W+Fenofibrate (n=24) Age, y 48.5 (12.8) 49.5 (10.7) 54.6 (7.6) Male, n (%) 11 (45.8) 11 (45.8) 10 (41.7) Race, n (%) Caucasian/white 23 (95.8) 21 (87.5) 24 (100) Black 1 (4.2) 3 (12.5) 0 Body mass index, kg/m 2 23.9 (2.0) 25.5 (2.7) 24.7 (2.5) Calculated LDL‐C, mg/dL 183.3 (38.7) 181.7 (37.1) 180.6 (31.3) Total cholesterol, mg/dL 264.5 (43.7) 260.6 (40.6) 263.7 (40.6) Apolipoprotein B, g/L 1.28 (0.21) 1.28 (0.22) 1.28 (0.17) Non–HDL‐C, mg/dL 198.4 (40.6) 200.3 (39.8) 199.5 (31.7) HDL‐C, mg/dL 65.7 (12.4) 60.3 (13.1) 64.2 (15.5) Apolipoprotein A1, g/L 1.58 (0.16) 1.55 (0.21) 1.57 (0.20) Triglycerides (mg/dL), median (range) 78.8 (44.3–177.1) 95.7 (35.4–168.3) 94.8 (53.1–194.9) Lipoprotein(a) (g/L), median (range) 0.27 (0.0–1.6) 0.33 (0.0–1.6) 0.17 (0.0–1.5) Free PCSK9, ng/mL 146.5 (54.3) 150.7 (48.5) 152.1 (54.1) Values are mean (SD) unless otherwise stated. HDL‐C indicates high‐density lipoprotein cholesterol; LDL‐C, low‐density lipoprotein cholesterol; PCSK9, proprotein convertase subtilisin/kexin type 9; Q4W, every 4 weeks. BODY.RESULTS.EFFECTS ON LDL‐C: During the course of the 28‐day initial therapy run‐in, LDL‐C levels were reduced by a mean (SEM) of 19.8% (2.1%) with ezetimibe and 25.9% (3.2%) with fenofibrate from day −29 baseline values; mean change in the placebo group was +1.6% (3.0%) (Figure 2A). Figure 2Time‐courses of mean LDL‐C percent change from main (day −29) baseline (A) and additional (day −1) baseline (B). Dotted lines between days 29 and 57 indicate no measurement taken for this period. Triangles below the x‐axis indicate timing of alirocumab injections. EZE indicates ezetimibe; FENO, fenofibrate; LDL‐C, low‐density lipoprotein cholesterol; Q4W, every 4 weeks. A, Differences in mean LDL‐C percentage reductions from pre–run‐in baseline values (day −29) were statistically significant between alirocumab+ezetimibe vs alirocumab+placebo at all time points, and between alirocumab+fenofibrate vs alirocumab+placebo at all time points except day 99 (P<0.05). B, Differences in mean LDL‐C percent reductions from prealirocumab baseline values (day −1) were statistically significant between alirocumab+ezetimibe vs alirocumab+placebo on days 8, 15, 22, 64, 71, and 120, and between alirocumab+fenofibrate vs alirocumab+placebo on days 8, 15, and 29 (P<0.05). After the initiation of alirocumab Q4W, the greatest mean percent reduction in LDL‐C levels was observed in all groups on day 71 (Figure 2; Table 2). Coadministration of alirocumab with ezetimibe or fenofibrate produced significantly greater mean LDL‐C reductions from the main (day −29) baseline (ie, pre–run‐in) versus alirocumab plus placebo at almost every time point (P<0.05; primary analysis; Figure 2A). These data represent the combined effect of alirocumab and the concomitant lipid therapy from the original (pre–run‐in) LDL‐C baseline level. Table 2 Percentage Change in Lipid Parameters From the Main Baseline (Day −29) and the Additional Baseline (Day −1) to Day 71 (14 Days After the Third Alirocumab Dose) Treatment Group % Change From Day −29 to 71 (Only Placebo, Ezetimibe or Fenofibrate Were Given From Days −29 to −1) % Change From Day −1 to Day 71 (First Alirocumab Injection Administered on Day 1; Placebo/Ezetimibe/Fenofibrate Treatment Continued) Alirocumab 150 mg Q4W+Placebo (n=24) Alirocumab 150 mg Q4W+Ezetimibe (n=24) Alirocumab 150 mg Q4W+Fenofibrate (n=24) Alirocumab 150 mg Q4W+Placebo (n=24) Alirocumab 150 mg Q4W+Ezetimibe (n=24) Alirocumab 150 mg Q4W+Fenofibrate (n=24) LDL‐C −48.2 (2.3) −65.3 (2.0) † −66.8 (2.7) † −47.4 (3.2) −56.6 (2.5) * −54.3 (3.5) Total cholesterol −31.6 (1.4) −45.7 (1.5) † −46.1 (1.9) † −31.5 (2.6) −36.5 (1.4) −32.4 (2.2) Non–HDL‐C −43.0 (1.7) −60.6 (1.9) † −64.4 (2.5) † −43.0 (2.7) −51.9 (2.1) * −50.5 (3.2) Apo B −39.1 (1.5) −53.5 (1.8) † −58.3 (2.1) † −38.4 (2.4) −44.9 (2.0) −44.6 (2.5) HDL‐C 3.3 (3.4) 5.4 (3.2) 12.3 (3.1) * 3.6 (2.9) 6.4 (3.1) 8.7 (3.0) ApoA1 1.9 (6.3) 1.2 (9.0) 3.4 (7.3) 1.4 (10.1) 1.0 (6.9) 2.9 (8.4) Triglycerides, median (range) 5.7 (−48.5 to 266.7) −13.8 (−53.4 to 53.5) * −36.0 (−57.9 to 11.3) † −3.9 (−41.3 to 77.6) −16.5 (−37.2 to 24.2) −3.5 (−58.0 to 74.1) Lp(a), median (range) −20.3 (−63.2 to 33.3) −27.0 (−71.4 to 35.8) −19.9 (−57.6 to 38.3) −11.7 (−58.8 to 160.9) −9.2 (−67.0 to 66.7) −20.4 (−56.8 to 17.7) * Values are mean (SEM) unless otherwise stated. Apo indicates apolipoprotein; HDL‐C, high‐density lipoprotein cholesterol; LDL‐C, low‐density lipoprotein cholesterol; Lp(a), lipoprotein(a); Q4W, every 4 weeks. P ‐values are linked to tests of the means. * P <0.05, † P <0.0001 vs alirocumab+placebo. The analysis with use of the day −1 baseline (ie, prealirocumab) allows for examination of the specific effects of alirocumab. Mean (SEM) percentage LDL‐C reductions with alirocumab plus placebo showed little change during the 28‐day period after the third dose: day 57, 44.4% (2.8%); day 71, 47.4% (3.2%); and day 85, 47.0% (2.7%) (Figure 2B), suggesting a sustained effect over the Q4W dosing interval. Mean (SEM) LDL‐C reductions from day −1 to day 71 were greater with alirocumab plus ezetimibe versus with alirocumab plus placebo (56.6% [2.5%] versus 47.4% [3.2%], P<0.05); corresponding reductions with alirocumab plus fenofibrate (54.3% [3.5%]) were not significantly different from those with alirocumab plus placebo (P=0.11; Figure 2B, Table 2). In comparison with alirocumab plus placebo, for the alirocumab–LLT combinations, the LDL‐C reduction from day −1 to day 85 was smaller than the reduction to day 71 (49.6% [1.6%] versus 56.6% [2.5%] with ezetimibe and 43.2% [3.8%] versus 54.3% [3.5%] with fenofibrate). This suggests a modest attenuation of the LDL‐C–lowering effect with the alirocumab–LLT combinations during the Q4W dosing interval compared with the placebo combination (Figure 2B). BODY.RESULTS.EFFECTS ON PCSK9 AND RELATIONSHIP WITH LDL‐C: PCSK9 is described here as either free PCSK9, representing unbound PCSK9 available to interact with the LDLR, or total PCSK9, representing all circulating PCSK9 including inactive alirocumab‐bound PCSK9. Mean baseline free PCSK9 concentrations were similar across all groups at the start of the initial therapy run‐in period (day −29; 147–152 ng/mL). By the end of the run‐in and before the first alirocumab dose (day 1), mean free PCSK9 concentrations were 119 ng/mL with placebo, 142 ng/mL with ezetimibe (24% greater than placebo; 90% CI 4–47%), and 217 ng/mL with fenofibrate (92% greater than placebo; 90% CI 62–128%; Figure 3A). Figure 3Mean free PCSK9 levels in all 3 treatment groups (A), and free PCSK9 levels compared with percentage changes in LDL‐C from the main baseline (day −29) for alirocumab plus placebo (B), plus ezetimibe (C), and plus fenofibrate (D). Dotted lines between days 29 and 57 indicate no measurement taken for this period. EZE indicates ezetimibe; FENO, fenofibrate; LDL‐C, low‐density lipoprotein cholesterol; PCSK9, proprotein convertase subtilisin/kexin 9; Q4W, every 4 weeks. Free PCSK9 was reduced to undetectable or very low concentrations (0–5 ng/mL) by day 71 in all groups; then concentrations gradually increased over days 71 to 85 (Figure 3A). In the group receiving alirocumab plus placebo, mean free PCSK9 remained low (17 ng/mL) at day 85, consistent with the relatively stable LDL‐C reductions in this group (Figure 3B). In comparison, mean free PCSK9 concentrations were higher at day 85 with ezetimibe (60 ng/mL) and fenofibrate (102 ng/mL), corresponding with the modest attenuation of the LDL‐C–lowering effect observed when alirocumab was administered with the LLTs (Figure 3C and 3D). Total PCSK9 concentrations increased from baseline in all groups after the first alirocumab dose (Figure 4). Overall, mean total PCSK9 concentrations were highest in the group receiving fenofibrate (Figure 4). Figure 4Total PCSK9 concentrations and LDL‐C percentage changes from the additional baseline (day −1). The continuous line indicates total PCSK9, dashed line indicates LDL‐C. Dotted lines between days 29 and 57 indicate no measurement taken for this period. Triangles below the x‐axis indicate timing of alirocumab injections. EOS indicates end of study; EZE, ezetimibe; FENO, fenofibrate; LDL‐C, low‐density lipoprotein cholesterol; PCSK9, proprotein convertase subtilisin/kexin 9. BODY.RESULTS.EFFECTS ON ALIROCUMAB (PHARMACOKINETICS): When comparing alirocumab plus ezetimibe versus alirocumab plus placebo, total alirocumab Cmax values did not differ statistically (mean [SD] Cmax, 21.9 [8.9] versus 24.3 [8.6] mg/L; point estimate: 0.92 [90% CI 0.78–1.09]), with a nonsignificant trend toward lower AUCD57–85 in the alirocumab plus ezetimibe treatment group (364 [143] versus 445 [189] mg·day/L; point estimate: 0.85 [0.70–1.03]), suggesting slightly faster clearance of alirocumab in this group. Both alirocumab Cmax and AUCD57–85 were reduced with alirocumab plus fenofibrate versus alirocumab plus placebo (Cmax, 17.1 [6.7] versus 24.3 [8.6] mg/L; AUCD57–85, 292 [138] versus 445 [189] mg·day/L; point estimates: 0.71 [0.60–0.84] and 0.64 [0.53–0.77], respectively) indicating a greater rate of clearance for alirocumab when combined with fenofibrate. BODY.RESULTS.RELATIONSHIP BETWEEN LDL‐C, PCSK9, AND ALIROCUMAB CONCENTRATIONS: Figure 5 compares LDL‐C reductions and concentrations of free and total PCSK9 and of alirocumab for days 57 to 85 (the 4‐week period after the third alirocumab dose). Maximal LDL‐C reductions for the LLT combinations were greater than those for alirocumab plus placebo, although PCSK9 levels and the alirocumab clearance rate were increased with the LLTs, and the duration of efficacy was slightly reduced with the LLTs (Figure 5). Comparison of mean free PCSK9 concentrations and total alirocumab levels suggests that if free PCSK9 remains lower than 40 to 50 ng/mL and alirocumab levels remain above 9 to 10 mg/L, then the LDL‐C–lowering effect of alirocumab is stable. Figure 5Mean percentage change in LDL‐C from the additional baseline (day −1) (A) and concentrations of free PCSK9 (B), total PCSK9 (C), and total alirocumab (D) for days 57 to 85. EZE indicates ezetimibe; FENO, fenofibrate; LDL‐C, low‐density lipoprotein cholesterol; PCSK9, proprotein convertase subtilisin/kexin 9. The temporal relationships between free and total PCSK9 concentrations, alirocumab levels, and LDL‐C are plotted in Figure 6. A lag (hysteresis effect) was observed between the time when free PCSK9 concentrations reached the lowest point and when maximal reductions in LDL‐C levels were observed (Figure 6B). Figure 6Hysteresis plots comparing changes over time in LDL‐C vs alirocumab (A) and vs free PCSK9 (B), free PCSK9 vs alirocumab (C), total PCSK9 vs alirocumab (D) and LDL‐C vs total PCSK9 (E). Study variables are plotted on x and y axes with data points representing sample time points (day 0–28 following administration of alirocumab). In the key in (A) "PKD12910" refers to the current study; (A) also includes data from alirocumab Phase I study 1001 for comparison. D indicates study day relative to administration of alirocumab; LDL‐C, low‐density lipoprotein cholesterol; non‐FH, non‐familial hypercholesterolemia; PCSK9, proprotein convertase subtilisin/kexin 9; Q4W, every 4 weeks; SC, subcutaneous. BODY.RESULTS.EFFECTS ON OTHER LIPIDS AND LIPOPROTEINS: Effects on other lipids and lipoproteins are summarized in Table 2. Similar to the effects on LDL‐C, reductions in total cholesterol, apolipoprotein B, non–HDL‐C, and triglycerides (from day −29 to day 71) were greater when alirocumab was coadministered with the LLTs compared with placebo. The fenofibrate combination, but not the ezetimibe combination, resulted in larger increases in HDL‐C compared with alirocumab plus placebo, reflecting the independent effects of fenofibrate on raising HDL‐C that are not seen with ezetimibe. BODY.RESULTS.SAFETY: Treatment‐emergent AEs are summarized in Table 3. There were no serious AEs and no subjects discontinued because of AEs. No AEs were considered by the investigator to be related to the study treatments. There were no clinically significant changes in vital signs or electrocardiograms or in hematologic or biochemical parameters. Table 3 Safety Summary From the Main Baseline (Day −29) to End of Study at Day 120 (All Randomized Patients) Treatment Group Alirocumab 150 mg Q4W+Placebo (n=24) Alirocumab 150 mg Q4W+Ezetimibe (n=24) Alirocumab 150 mg Q4W+Fenofibrate (n=24) Subjects with any TEAEs, n (%) 12 (50.0) 14 (58.3) 12 (50.0) Most frequent TEAEs (recorded in ≥2 subjects in any group), n (%) Headache 3 (12.5) 5 (20.8) 2 (8.3) Nasopharyngitis 3 (12.5) 4 (16.7) 4 (16.7) Influenza 2 (8.3) 0 (0) 1 (4.2) Gastroenteritis viral 0 (0) 0 (0) 2 (8.3) Influenza‐like illness 1 (4.2) 3 (12.5) 1 (4.2) Abdominal pain 2 (8.3) 1 (4.2) 1 (4.2) Q4W indicates every 4 weeks; TEAE, treatment‐emergent adverse event. BODY.DISCUSSION: This study was designed to explore changes in LDL‐C, PCSK9, and alirocumab concentrations when alirocumab was administered Q4W without background statins, either with placebo or in combination with nonstatin LLTs (ezetimibe and fenofibrate). LDL‐C reductions were maintained duringthe 4‐week dosing interval with alirocumab 150 mg Q4W plus placebo. Maximal LDL‐C reductions for the LLT combinations (65.3% and 66.8% with ezetimibe and fenofibrate, respectively) were greater those than for alirocumab plus placebo (48.2% versus day −29). In contrast, PCSK9 levels and the alirocumab clearance rate were increased and the duration of efficacy was slightly reduced with the LLTs. These findings are summarized in Figure 7. This contrasts with results from previous studies in which alirocumab was dosed Q4W on background statins, showing a notably reduced duration of effect compared with Q2W dosing.4, 6 Figure 7Relationship between LDL‐C, free PCSK9, and alirocumab levels when alirocumab 150 mg Q4W is administered in combination with ezetimibe or fenofibrate without background statin. LDL‐C indicates low‐density lipoprotein cholesterol; LLT, lipid‐lowering therapy; PCSK9, proprotein convertase subtilisin/kexin 9. Treatment with ezetimibe or fenofibrate during the course of a 28‐day run‐in resulted in reductions in LDL‐C of 19.8% and 25.9%, respectively, and caused increases in free PCSK9 levels of 24% and 92% (versus placebo). Although free PCSK9 concentrations before the third alirocumab dose were higher with fenofibrate cotherapy (versus ezetimibe or placebo co‐therapy), at day 71 they were reduced to similar levels in all groups. At the same time, total PCSK9 concentrations were highest with fenofibrate cotherapy. By comparing free and total PSCK9 concentrations, we speculate that total PCSK9 mainly represents inactivated PCSK9 complexed to alirocumab. Following the last alirocumab dose and up to end of study, a more rapid rate of return of free PCSK9 was observed with fenofibrate versus either ezetimibe or placebo. We hypothesize that the increases in PCSK9 levels with the LLTs are the result of increased PCSK9 production, with fenofibrate resulting in a greater increase in PCSK9 production compared with ezetimibe. Further, the actual increase in PCSK9 production would be underestimated by simply measuring free PCSK9 levels, because some are eliminated from the system as a result of the interaction of PCSK9 with the LDLR. Because alirocumab can be thought of as a trap for newly produced PCSK9, we propose that the difference in peak total PCSK9 values after alirocumab administration may serve as a better indicator of the effect on PCSK9 production. It has been proposed that the effect of fenofibrate on PCSK9 is indirect,13, 15 rather than a direct effect on gene transcription as is observed with statins.2 There is some evidence from animal studies that ezetimibe upregulates the PCSK9 and LDLR genes11; however, a previous study in humans did not find an effect of ezetimibe on PCSK9.12 Increases in PCSK9 concentrations of 13% to 41% (versus placebo) have been reported after treatment with simvastatin or atorvastatin.15 These increases will, however, underestimate the actual increased PCSK9 production for reasons noted earlier, because statins also induce an increase in LDLR levels. Monoclonal antibodies that bind PCSK9 undergo target‐mediated clearance.16 Therefore, we hypothesize that the increased rate of clearance of total alirocumab with the LLT combinations versus the placebo combination resulted from the increased PCSK9 concentrations (and thus increased target‐mediated clearance). Measurement of total alirocumab concentrations will underestimate clearance of the biologically active drug ("free" alirocumab—ie, that which is not bound to PCSK9). Based on the binding stoichiometry of PCSK9 to alirocumab (2:1) in the target‐mediated phase and on the total and free PCSK9 concentrations, we estimate that the proportion of total alirocumab present as free alirocumab becomes negligible toward the end of the dosing interval when free PCSK9 is again detectable and begins to increase. However, this will need to be confirmed in future studies. The modest attenuation of LDL‐C–lowering efficacy during the Q4W dosing interval with the alirocumab–LLT combinations versus alirocumab plus placebo can be explained by the increased PCSK9 concentrations and clearance of alirocumab with these combinations. Results from the current study suggest that if free PCSK9 concentrations remain lower than 40 to 50 ng/mL and alirocumab levels remain above 9 to 10 mg/L, then the LDL‐C–lowering effect of alirocumab is stable. Based on the results from the current study and previous work,4, 6 we hypothesize that there is a gradient in terms of the magnitude of peak LDL‐C reductions and duration of effect after alirocumab administration, from alirocumab added to statins, to coadministration with fenofibrate and ezetimibe, and finally as monotherapy (ie, alirocumab plus placebo in the current study). This gradient reflects the extent of PCSK9 induction by each LLT that is coadministered with alirocumab. In a reciprocal manner, we also hypothesize that by raising PCSK9 levels, the LLTs (statin, ezetimibe, or fenofibrate) may exert a self‐imposed limit on the maximal LDL‐C reduction possible. Adding alirocumab to the LLT and thus bringing down PCSK9 levels may blunt the limitation in LDL‐C reduction, leading to an additive LDL‐C–lowering effect. With regard to safety, alirocumab in combination with placebo, ezetimibe, or fenofibrate was generally well tolerated, with no serious AEs throughout the study. There was a similar incidence of AEs reported in each group, and none of the reported AEs were considered by the investigator to be related to the study drugs. However, this was a small study and not powered for safety. Safety findings will be extended with data from the large cardiovascular outcomes study.17 Observational studies suggest that a large proportion of patients do not tolerate statins.18 Ezetimibe and fenofibrate are often used as alternative drugs for these patients. The present study indicates that alirocumab 150 mg Q4W—representing a single, once‐monthly injection—could be an option for such patients not currently receiving a statin. The results from the present study informed the designs of the phase 3 alirocumab trials ODYSSEY CHOICE I and CHOICE II (clinicaltrials.gov identifiers: NCT01926782 and NCT02023879, respectively). In CHOICE I, alirocumab 300 mg Q4W is administered either with or without a concomitant statin; in CHOICE II, alirocumab 150 mg Q4W is administered without a statin. Both studies allow for a dose regimen change to 150 mg every 2 weeks if LDL‐C targets are not reached after 8 weeks of treatment. BODY.CONCLUSIONS: Alirocumab 150 mg Q4W produced robust LDL‐C reductions when administered either with placebo or in combination with ezetimibe or fenofibrate. Maximal LDL‐C reductions were greater when in combination with the LLTs. The oral LLTs appear to increase free PCSK9 levels, leading to increased clearance of alirocumab; thus, LDL‐C reductions with the LLT combinations were slightly less sustained between injections versus the placebo combination. However, the slight loss of duration of efficacy was not as pronounced as seen in trials performed with Q4W dosing with background statin treatment. Therefore, alirocumab 150 mg Q4W may provide an additional treatment option when used as monotherapy or when administered on a background of ezetimibe or fenofibrate therapy in patients in need of LDL‐C lowering. The ODYSSEY CHOICE studies are investigating the efficacy and safety of alirocumab Q4W dosing. BODY.SOURCES OF FUNDING: This work was funded by Sanofi and Regeneron Pharmaceuticals, Inc. BODY.DISCLOSURES: Jacques Rey and Jean‐Louis Pinquier were both employees of and stockholders in Sanofi during conduct of this study. Franck Poitiers, Tobias Paehler, Aurélie Brunet, Howard Surks, and Corinne Hanotin are all employees of and stockholders in Sanofi. A. Thomas DiCioccio and William Sasiela are employees of and stockholders in Regeneron Pharmaceuticals. Christopher Cannon has received grants from Accumetrics, Arisaph, AstraZeneca, Boehringer‐Ingelheim, CSL Behring, Essentialis, GlaxoSmithKline, Janssen, Merck, Regeneron, Sanofi, and Takeda and consultant/advisory board fees from Bristol‐Myers Squibb, Lipimedix, and Pfizer.

TITLE: Implementation findings from a hybrid III implementation-effectiveness trial of the Diabetes Prevention Program (DPP) in the Veterans Health Administration (VHA) ABSTRACT.BACKGROUND: The Diabetes Prevention Program (DPP) is an effective lifestyle intervention to reduce incidence of type 2 diabetes. However, there are gaps in knowledge about how to implement DPP. The aim of this study was to evaluate implementation of DPP via assessment of a clinical demonstration in the Veterans Health Administration (VHA). ABSTRACT.METHODS: A 12-month pragmatic clinical trial compared weight outcomes between the Veterans Affairs Diabetes Prevention Program (VA-DPP) and the usual care MOVE!® weight management program (MOVE!). Eligible participants had a body mass index (BMI) ≥30 kg/m2 (or BMI ≥ 25 kg/m2 with one obesity-related condition), prediabetes (glycosylated hemoglobin (HbA1c) 5.7–6.5% or fasting plasma glucose (FPG) 100–125 mg/dL), lived within 60 min of their VA site, and had not participated in a weight management program within the last year. Established evaluation and implementation frameworks were used to guide the implementation evaluation. Implementation barriers and facilitators, delivery fidelity, participant satisfaction, and implementation costs were assessed. Using micro-costing methods, costs for assessment of eligibility and scheduling and maintaining adherence per participant, as well as cost of delivery per session, were also assessed. ABSTRACT.RESULTS: Several barriers and facilitators to Reach, Adoption, Implementation, Effectiveness and Maintenance were identified; barriers related to Reach were the largest challenge encountered by site teams. Fidelity was higher for VA-DPP delivery compared to MOVE! for five of seven domains assessed. Participant satisfaction was high in both programs, but higher in VA-DPP for most items. Based on micro-costing methods, cost of assessment for eligibility was $68/individual assessed, cost of scheduling and maintaining adherence was $328/participant, and cost of delivery was $101/session. ABSTRACT.CONCLUSIONS: Multi-faceted strategies are needed to reach targeted participants and successfully implement DPP. Costs for assessing patients for eligibility need to be carefully considered while still maximizing reach to the targeted population. BODY.BACKGROUND: Incidence of type 2 diabetes (referred to as diabetes throughout) was reduced so dramatically (by 58%) in a landmark trial of the Diabetes Prevention Program (DPP) in the USA that the trial was stopped early in 2001 [1–4]. Since the original trial, many large-scale translations around the world, including Finland, Australia, China, and India, have successfully reduced onset of diabetes among patients with prediabetes and/or predictors like weight loss [5]. To gain an overall perspective of the effectiveness of diabetes prevention studies, Balk et al. conducted a review of 53 studies (72% of which were randomized controlled trials), evaluating 66 combined diet and physical activity programs (41% of which were based on DPP), and concluded that such programs are effective in reducing incidence of diabetes, body weight, and FPG [6]. Programs varied widely in design: ranging from 3 to 72 months in duration and from no contacts (virtual contacts only) up to 72 contacts with participants. The review concluded that more intensive programs appeared to yield more positive outcomes, but there was little insight into how other program characteristics may contribute to outcomes. Since these studies focused on patient outcomes, they also provided no insight into how to successfully implement these interventions. Aziz et al. partially filled this gap with their systematic review of 38 studies with the goal of identifying factors leading to successful implementation of DPP in "real-world" settings [7] using the penetration, implementation, participation, and effectiveness (PIPE) impact metric. Figure 1 shows the distribution of program characteristics for eight dimensions and two outcomes: a wide range of program designs and durations have been implemented within clinical and community settings around the world. Across this diverse array of studies, two thirds reported low participation and 42% reported low weight loss (<4.6 kg) [7]. Both reviews by Aziz et al. and Balk et al. concluded that more intensive programs may enhance weight loss outcomes. However, Aziz et al. stress that even modest weight loss can have significant population-level impact if a high proportion of high-risk individuals participate in the program [7]. This broader view of impact is essential for policy- and other decision-making.Fig. 1DPP characteristics reported by Aziz et al.'s systematic review. A red box indicates category for VA-DPP. aWorkplace and primary care settings. bCommunity, church, YMCA, various venues, leisure, and community settings. cHealth care facilities, outpatient settings, hospitals. dOther modes include telephone, fax, text, email, online. eThirty-nine studies reported because one study reported low and high sites. fStandard curriculum = delivery of DPP following a standard curriculum. gQA = quality assurance = use of measures to monitor implementation Kahn and Davidson [8] highlighted the dearth of real-world confirmation of the remarkable clinical outcomes reported by the original DPP study [4]. The Balk et al. review of DPP-like programs reported significant reduction in diabetes incidence (0.59; 95% CI, 0.52–0.66, based on 16 (30%) of 53 studies that reported this outcome) [6]. Aziz et al. reported "moderate" or "high" risk reduction for 7 (18%) of 38 studies; this outcome was "unknown" for the remaining 31 (82%) of studies [7]. The low proportion of studies reporting these clinical outcomes may indicate potential reporting bias toward positive results; on the other hand, these downstream effects are more challenging to assess within clinical settings outside of highly controlled lengthy clinical trials. Despite the "voltage drop" [9] often seen in outcomes within real-world settings, definitive guideline statements have been disseminated recommending DPP around the world [10–12]. Despite large-scale population-based availability of DPP in some countries (Finland being a notable leader [13]), within other countries, access to DPP is limited, in part because of the expense, lack of reimbursement by insurance or funding entities, and challenges of effectively implementing DPP across diverse uncontrolled settings and populations [14–17]. Within the USA, interest in implementing DPP is increasing because the US Centers of Medicare and Medicaid Services (CMS) plans to reimburse participation expenses for eligible individuals starting in 2018 [18]; over 1000 programs were listed in the Centers for Disease Control and Prevention (CDC) registry of certified programs [19] as of November 2016 compared to 500 in May 2014 [12]. In the USA, recognition status by the CDC Diabetes Prevention Recognition Status (DPRP) requires at least 50% of participants to have a diagnosis of prediabetes based on blood testing (or have documented history of gestational diabetes) [20], though risk assessments, administered by short surveys, are also available [21–30]. When eligible individuals are identified, outreach is needed to encourage participation in DPP and often clinical testing is used to verify diagnosis. Thus, significant challenges remain: designing and reliably executing robust approaches to identify, enroll, and engage individuals at high risk for diabetes. Within VA, the national policy office responsible for prevention efforts, the National Center for Health Promotion and Disease Prevention (NCP), commissioned a pragmatic trial to demonstrate impact and feasibility of implementing DPP (VA-DPP) in VHA, in the context of the already existing MOVE!® weight management program (MOVE!) [31]. This clinical demonstration was conducted in three geographically diverse medical centers [31]. Candidate participants included patients with prediabetes (HbA1c 5.7–6.4% or FPG 100–125 mg/dL) who lived within 60 min of a demonstration site, were obese (BMI > 30 kg/m2) or overweight (BMI 25–30 kg/m2) with diagnosis of an obesity-related condition (e.g., hypertension, diabetes), and attended a MOVE! orientation session. Eligibility criteria were confirmed clinically; participants who did not have a HbA1c or FPG within prior 6 months were invited to have HbA1c screening, which is aligned with CDC DPP and national care guidelines [32, 33]. Patients for whom anti-glycemic medication (including metformin) was documented within their electronic health record in the last 6 months and those with contraindications to uptake of intensive lifestyle change were excluded. VA-DPP characteristics are indicated in comparison to the Aziz et al. review in Fig. 1. In an intention-to-treat analysis, participants in two study arms (VA-DPP, usual care weight management program) experienced significant weight loss (p < 0.001). Participants in VA-DPP lost significantly more weight at 6 months compared to those in MOVE! (4.1 vs. 1.9 kg; p < 0.001) but the difference between programs was no longer statistically significant (3.4 vs. 2.0 kg, p = 0.16) at 12 months [34]. Reach was higher for VA-DPP compared to MOVE!: more participants assigned to VA-DPP completed at least one session (73%) compared to those assigned to MOVE! (58%; p = 0.002) [34]. VA-DPP also had higher rates of participation, e.g., 42.5% completed at least eight sessions compared to 31% for MOVE!. Neither VA-DPP nor MOVE! resulted in changed HbA1c compared to baseline [34]; this trial was not powered to examine differences in diabetes incidence. This evaluation was guided by the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework, which was designed to inform translation of evidence-based programs into practice and heighten public health impact by examining impact on domains beyond clinical outcomes [35, 36]. RE-AIM includes five domains: (1) Reach, the proportion of the targeted population willing to participate in the intervention; (2) Effectiveness, the impact of the intervention on key outcomes; (3) Adoption, the proportion of organizations and individuals within organizations who are willing to initiate the intervention within their setting; (4) Implementation, the fidelity and cost of delivery at the setting level; and (5) Maintenance, the extent to which the intervention is sustained over the longer term at the setting and individual levels. The aim of this study was to identify prevalent contextual factors that may have influenced outcomes within each RE-AIM domain. BODY.METHODS.STUDY DESIGN: The current study focuses specifically on implementation experiences within a pragmatic hybrid III effectiveness-implementation trial of VA-DPP compared to MOVE! within three medical centers [31]. This type of study has a primary focus on implementation but also evaluates clinical outcomes [37] that were reported elsewhere [34]. This paper reports quantitative and qualitative findings related to implementation. This evaluation was approved by five Institutional Review Boards (IRBs) for each of the five research institutions involved in the evaluation (three demonstration sites, one national coordinating center, and one research collaboration site). BODY.METHODS.INTERVENTIONS: Details about the programs evaluated are provided elsewhere [31, 34]. Briefly, VA-DPP and MOVE! were group-based lifestyle interventions. Each VA-DPP was a 12-month program with 22 planned sessions delivered by a single CDC-certified coach in closed cohorts (i.e., participants started and finished the program with the same group). MOVE! was an 8-10-week program followed by monthly maintenance sessions delivered in open cohorts (i.e., participants started the program at any time in two of three sites) by a multi-disciplinary team. BODY.METHODS.IMPLEMENTATION APPROACH: The VA-DPP implementation strategy was guided by the Simpson et al. program change model [31, 38]. First, each site's clinical champion worked to elicit site leadership commitment, documented by a formal memorandum of understanding (MOU). Second, VA-DPP coaches and team members were trained by the Diabetes Prevention Support Center (DPSC) [39, 40]. Third, each site adapted a protocol to implement VA-DPP, with special focus on how it would interface with MOVE! [13, 31]. Similar to other published implementation studies [41], coordinating center staff ensured a uniform approach was followed across the sites, assisted with planning and problem-solving, and helped to obtain and manage MOUs and IRB approvals. BODY.METHODS.ASSESSMENT OF OUTCOMES IN RE-AIM DOMAINS: Multiple data sources (e.g., staff interviews, site visits, participant questionnaires) and data types (qualitative, quantitative) were used to assess barriers and facilitators that affected RE-AIM domains, as well as fidelity, participant satisfaction, and cost. Additional details are provided in the following sections. BODY.METHODS.ASSESSMENT OF OUTCOMES IN RE-AIM DOMAINS.BARRIERS AND FACILITATORS: REACH, EFFECTIVENESS, ADOPTION, IMPLEMENTATION, AND MAINTENANCE: Semi-structured interviews were conducted with VA-DPP team members during the early stages of implementation (n = 15) and after the enrollment ended (n = 23) either by phone or face to face during site visits. A purposive sample of other site staff and clinicians was also asked to participate in interviews. Interview guides were based on the Consolidated Framework for Implementation Research (CFIR) and are published elsewhere [31]. The CFIR was operationalized as a codebook for qualitative analysis to understand how contextual factors influence RE-AIM domains [31]. The CFIR describes 39 constructs across five domains that can be used to systematically assess and articulate contextual factors that may influence program implementation: (1) intervention characteristics (e.g., adaptability); (2) outer setting (e.g., external policies and incentives); (3) inner setting (e.g., leadership engagement); (4) individual characteristics (e.g., self-efficacy); and (5) process (e.g., planning) [42]. All interviews were audiorecorded and transcribed verbatim. In addition, site visit notes, meeting minutes, and emails between the coordinating center and sites were included in qualitative analyses to provide additional information about ongoing implementation processes. Using published methods [43], two analysts independently coded and assigned ratings based on qualitative data. Differences in coding and rating were resolved through consensus. To enable comparison within and between sites, a comprehensive matrix of ratings by construct and site was developed [44]. This approach identified constructs that, from the perspective of the program teams, influenced implementation outcomes. NVivo® Version 10 software was utilized to aid coding and analysis [45]. BODY.METHODS.ASSESSMENT OF OUTCOMES IN RE-AIM DOMAINS.DELIVERY FIDELITY: Corresponding sessions of VA-DPP and MOVE! (two sessions from each program with similar content) were assessed for delivery fidelity. At each site, the VA-DPP coordinator or other team member attended five to seven sessions and used a pre-specified checklist to rate various fidelity domains [46]. This analysis includes fidelity ratings of delivery of educational content, review of goal progress, goal setting, group cohesion, and coaching characteristics (managed the session, stayed on track, and created a supportive and empathetic environment (Table 1)). Previous work indicated that items related to goal progress review and coach delivery characteristics (Table 1) were associated with weight loss [46, 47]. Raters used a Likert scale (1 = strongly disagree to 7 = strongly agree) and provided optional open-ended comments. Factor analyses were conducted to confirm the appropriate groupings of items within domains, and Cronbach's alpha was calculated for each domain to determine internal reliability. T tests were used to compare mean fidelity ratings between VA-DPP and MOVE!.Table 1VA-DPP coordinator ratings of fidelity for delivery of VA-DPP and MOVE!VA-DPP(n = 37)MOVE!(n = 34) p b Questiona Mean (SD)Delivery of educational content Coach elicited discussion of the educational content in order to help participants develop a self-management skill or change cognitions6.62 (0.55)6.00 (1.22)0.0094Goal setting, Cronbach's alpha = 0.66c Type 2 diabetes prevention was discussed as a goal of the group4.59 (2.20)1.97 (1.70)0.0000 Coach presented standardized goals (i.e., everyone had the same goal to complete the following week) to the participants and asked them to commit to a goal5.59 (1.64)3.62 (2.12)0.0001Review of goal progress, Cronbach's alpha = 0.94 Coach prompted review of goal progress and attainmentd 6.41 (0.86)4.76 (2.31)0.0002 Coach elicited discussion of successes and challenges since the last sessiond 6.38 (0.82)4.62 (2.32)0.0001 Coach initiated problem-solving when necessary to address challenges since the last sessiond 6.44 (0.70)5.21 (1.92)0.0008Group cohesion, Cronbach's alpha = 0.81 Group identity includes having a diagnosis of prediabetes5.21 (1.63)1.47 (0.90)0.0000 Group members communicated easily with one another6.10 (1.22)5.35 (1.57)0.0385 There were positive relationships among the group members6.16 (1.10)5.35 (1.54)0.0184 Group members had a positive attitude toward the coach6.74 (0.51)6.06 (1.28)0.0056 Group members participated actively in the group6.41 (0.74)5.59 (1.58)0.0076Coach characteristics, Cronbach's alpha = 0.90Managed the session Coach came prepared and organizedd 6.85 (0.36)6.65 (0.65)0.1091 Coach elicited clarification of participant engagement by seeking feedback about didactic contentd 6.65 (0.60)6.36 (0.96)0.1510 Coach delivered didactic material in an engaging, matter of fact, and respectful wayd 6.79 (0.41)6.65 (0.69)0.2900 Coach facilitated discussion and interaction using open-ended questions, affirmations, reflections, and summariesd 6.53 (0.61)6.26 (0.96)0.1814 Coach allocated time appropriately in order to cover the appropriate content focus points for the sessiond 6.62 (0.65)6.47 (0.99)0.4726 Coach supplied the necessary materials for the participantsd 6.91 (0.29)6.76 (0.43)0.1026Stayed on track Coach addressed process (tangential) issues but did not allow them to disrupt content agendad 6.53 (0.71)6.18 (1.06)0.1105 Coach avoided delving too deeply into psychological issuesd 6.74 (0.51)6.50 (0.71)0.1206Created a supportive and empathetic environment Coach responded empathetically and accurately to participant behavior (verbal, nonverbal)d 6.85 (0.36)6.59 (0.61)0.0326 a1 = strongly disagree to 7 = strongly agree b t test cFactor analyses were conducted to confirm the appropriate groupings of items within domains; Cronbach's alpha was calculated for each domain to determine internal reliability dItem was taken from published fidelity checklist [46] BODY.METHODS.ASSESSMENT OF OUTCOMES IN RE-AIM DOMAINS.PARTICIPANT SATISFACTION: After 12 months, participant perspectives of their program were elicited through an administered survey that included questions about program satisfaction (Table 2). T tests compared mean ratings between VA-DPP and MOVE!. All analyses were conducted using Stata 13 [48].Table 2Participant program satisfaction at 12 months (N = 260)Survey questionVA-DPP(n = 183)Mean (SD)MOVE!(n = 77)Mean (SD) p a Group preference If you had had the chance to switch into a different group working on diet and exercise, how would you have felt about switching?(1 = very much want to switch, 5 = very much want to stay)3.75 (1.11)3.19 (0.85)0.0005Group cohesion How well did you bond with your group members?(1 = did not bond, 2 = bonded a little, 3 = bonded very well)2.45 (0.65)2.20 (0.80)0.0181Participant satisfaction with coach When you had important questions to ask your coach, did you get answers you could understand?(1 = no, 2 = sometimes, 3 = always)2.82 (0.44)2.80 (0.41)0.7002 Did you feel you were treated with respect and dignity during your group sessions?(1 = no, 2 = sometimes, 3 = always)2.91 (0.33)2.93 (0.31)0.6653 Did you have confidence and trust in your coach?(1 = no, 2 = sometimes, 3 = always)2.85 (0.43)2.68 (0.60)0.0233 Did your coach provide useful suggestions to help you overcome barriers in meeting your PA goals?(1 = no, 2 = sometimes, 3 = always)2.81 (0.46)2.57 (0.67)0.0036 Did your coach provider meaningful feedback regarding your progress toward meeting your goals?(1 = no, 2 = sometimes, 3 = always)2.80 (0.47)2.48 (0.69)0.0002 My coach motivated me to do my very best.(1 = strongly disagree, 5 = strongly agree)4.48 (0.77)4.0 (0.84)0.0001The overall response rate (67%) was similar to the response rate from VA-DPP (67%, n = 183) and MOVE! (68%, n = 77). aBased on t tests for differences between MOVE! and VA- DPP fidelity ratings for sample of sessions delivered BODY.METHODS.ASSESSMENT OF OUTCOMES IN RE-AIM DOMAINS.IMPLEMENTATION COSTS: Details of the micro-costing approach used are described elsewhere [32]. Briefly, costs of implementing VA-DPP were estimated for two categories: (1) recruiting participants and implementing VA-DPP and (2) conducting VA-DPP sessions. VA-DPP coordinators and coaches at each site recorded time spent on a range of tasks: recruitment, administrative tasks, team meetings, session preparation, and session delivery. Total recruitment time was multiplied with per-minute wage rates to derive site-specific total cost. Total costs were divided by the number assessed and enrolled per site to derive per participant enrollment cost. VA-DPP sessions included costs of the labor inputs and participation rates associated with each group session. Appropriate per-minute wage rates were applied to the aggregated activity times. For delivering sessions, labor cost was divided by the number of participants who completed each session. BODY.RESULTS: The following sections describe facets of the RE-AIM domains that were evaluated, including quantitative measures of delivery fidelity, participant satisfaction, and implementation costs. Common barriers and facilitators were evaluated for their influence on each of the RE-AIM domains. Each barrier and facilitator is demarcated with the associated CFIR construct (in parentheses) in order for readers to easily associate findings with the underlying theoretical framework. Because findings build on earlier published findings, there are additional citations to integrate the rich array of findings. BODY.RESULTS.REACH.BARRIERS AND FACILITATORS: RECRUITMENT FOR VA-DPP: As reported elsewhere, 1830 individuals attended a MOVE! orientation session, and 21% were eligible for the study [13, 34]. Overall, 11% were women and 48% were a racial/ethnic minority. The only demographic differences between VA-DPP and MOVE! participants were related to race/ethnicity; there were higher proportions of non-Hispanic black and non-Hispanic white VA-DPP participants, but a lower proportion of Hispanic participants (p = 0.04). Recruitment fell far short of the targeted sample size (N = 720 targeted; N = 387 enrolled; N = 386 in final analytic sample, due to a missing weight); there were 273 participants assigned to VA-DPP and 114 assigned to MOVE!. None of the sites achieved their recruitment goal due to several barriers. A process to systematically identify individuals with prediabetes did not exist prior to VA-DPP implementation (Negative Compatibility). However, MOVE! had an existing obesity screening and referral process in place; VA-DPP recruitment relied on the MOVE! referral process. However, clinician referrals to MOVE! were lower than expected in all three sites. First, although health promotion and disease prevention was a high priority in VHA, clinicians felt they had limited time to discuss weight management and diabetes prevention with their patients. Patients frequently presented with multiple chronic and acute conditions which were a more immediate medical priority (Negative Relative Priority).They don't have time for [prediabetes], you know, they're having a hard-enough time just dealing with the [patients with] out of control diabetes] [...], it's the old story about when you're killing alligators, it's hard to drain the swamp (Provider). Second, some clinicians did not believe behavior modification programs were effective for their patients and were reluctant to refer patients (Negative Evidence Strength and Quality).I've heard one physician who's pretty vocal at the meetings say, "[MOVE!] doesn't work, they re-gain the weight anyway." A couple of physicians have said that actually. "Bariatric surgery's the only thing that works." So I don't know that they even believe that a lifestyle program can work, and frankly some of our participants do well but a lot don't (MOVE! Coordinator). Third, primary care clinics at the three sites were overwhelmed by the major reorganization to align with VA's version of PCMH (Negative Relative Priority). Fourth, one site had no formal MOVE! referral process and instead, patients were advised by their primary care and specialty providers to schedule a MOVE! orientation visit during the check-out process. This may have reduced one barrier (not requiring a provider referral) but introduced another when patients did not follow up (Negative Compatibility). In addition, determining eligibility to participate in VA-DPP required a lab or point-of-care (POC) HbA1c or FPG test. One site had a POC testing procedure in place while another changed their electronic health record (EHR) to prompt clinicians to order an HbA1c test if they referred a participant to MOVE! (Positive Compatibility). The third site did not have either process in place; VA-DPP team members used a relatively labor-intensive process that involved evaluation based on EHR data or ordering a new HbA1c lab test for willing individuals (Negative Compatibility). Lastly, the percentage of individuals referred to MOVE! with prediabetes was lower than expected; prevalence rates for normal glycemic status, prediabetes, and diabetes were 43% (N = 796), 22% (N = 404), and 28% (N = 504), respectively [34]. In effect, most patients referred to MOVE! were ineligible because they did not meet prediabetes criteria, which further contributed to unexpectedly low enrollment. BODY.RESULTS.EFFECTIVENESS.BARRIERS AND FACILITATORS: PARTICIPATION AND ATTRITION IN VA-DPP: The percentage of participants who attended at least one session of VA-DPP was higher than for MOVE! (73.3 vs. 57.5%; p = 0.002). Although eligibility for VA-DPP was restricted to individuals who lived within 60 min of their sites, some participants still had transportation barriers (Negative Patient Needs and Resources):I think it's hard to come to a program every week in the middle of the day. [...] [Site Three] is 5 miles away, that can be half an hour or 45 minutes and that's not a doable thing for most people (Site Investigator). Staff interviews revealed additional participation challenges such as time conflicts with work and family schedules or more seriously, insecure housing, unstable employment, and low income; these issues were more common for participants at two of the sites and affected participation in both MOVE! and VA-DPP. VA-DPP participants also remained more engaged: more VA-DPP participants completed at least four sessions compared to MOVE! participants (57.5% VA-DPP; 42.5% MOVE!, p = 0.007) and more VA-DPP participants completed at least eight sessions compared to MOVE! participants (42.5% VA-DPP; 31% MOVE!, p = 0.035) [34]. VA-DPP team members described their VA-DPP participants having a positive experience (Positive Patient Needs and Resources):They love coming to class, they like being around each other, they like hearing from each other, they like encouraging each other. [...] I feel like overall the feedback has been pretty positive (VA-DPP Coach). If asked, the MOVE! coordinators may have offered similar statements about their patients having positive experiences, but participant-reported satisfaction shows differences in satisfaction by program. Of the 387 participants across the two programs, 286 (74%) completed satisfaction questions 12 months after their baseline. Participants in VA-DPP reported higher levels of group preference (p = 0.0005) and group cohesion (p = 0.0181) than MOVE! participants (Table 2). They also reported modestly higher satisfaction with their coach than MOVE! participants for four of six items (Table 2). BODY.RESULTS.ADOPTION.BARRIERS AND FACILITATORS: FACILITY ADOPTION OF VA-DPP: VA-DPP site leaders were familiar with the evidence base for VA-DPP (Positive Evidence Strength and Quality), which helped spark initial enthusiasm for having the program at their sites. In addition, these leaders acknowledged the benefits of targeting and engaging high-risk individuals in VA-DPP (Positive Relative Advantage), which they felt their existing MOVE! program did/could not do. However, one site leader expressed concerns about implementing the program, fearing bureaucratic challenges such as hiring new staff (Negative Structural Characteristics). Ultimately, all site directors signed an MOU as described above, visibly demonstrating their commitment to implementing VA-DPP (Positive Leadership Engagement). In addition, one site was motivated by the prospect of achieving CDC recognition for VA-DPP [32], while another was motivated by the enthusiastic endorsement of a national-level political leader (Positive External Policy and Incentives). BODY.RESULTS.IMPLEMENTATION.FIDELITY AND PARTICIPANT SATISFACTION: VA-DPP teams rated fidelity of program delivery for 71 sessions (37 sessions for VA-DPP, 34 sessions for MOVE!). Fidelity ratings were relatively high for both programs (Table 1) but were significantly higher for VA-DPP, except for comparable ratings for characteristics of the coaches leading the sessions. BODY.RESULTS.IMPLEMENTATION.COST: Costs for labor to recruit participants and implement VA-DPP averaged $40,348 across the sites ($35,283–$54,877), which translated to $68 ($46–$97) per participant assessed and $330 ($210–$481) per participant identified to be eligible for VA-DPP. Costs related to scheduling VA-DPP group sessions, sending out reminders to participants, and preparing for sessions averaged $28,462 across the sites ($16,889–$49,680), which translated to $328 ($156-$591) per participant. Costs related to conducting VA-DPP sessions averaged $101 per group session conducted ($64–$177), which translated to a total cost of $2220 ($1410–$3889) for the planned 22 sessions for VA-DPP. Participants generated, on average, 57 completed sessions per site (means ranged from 19 to 119 across the sites); it cost $46 (range $41–$60) per participant per session. BODY.RESULTS.IMPLEMENTATION.BARRIERS AND FACILITATORS: IMPLEMENTATION OF VA-DPP: A strong VA-DPP team at each site supported by a central coordinating center was the single most important facilitator for successful implementation (Positive Engaging: VA-DPP Teams; Positive Engaging: External Change Agent). Team members strongly believed in VA-DPP to prevent diabetes, effectively solved problems, and felt a strong affinity with participants (Positive Engaging: VA-DPP Teams). In addition, they were familiar with previous DPP study findings (Positive Evidence Strength and Quality) and believed that VA-DPP had advantages over MOVE! (Positive Relative Advantage).[VA-DPP] really focuses specifically on diabetes prevention, and not just weight loss in general, and I think those are [...] two separate things, two separate goals (Site Investigator). VA-DPP teams attended a 2-day training delivered by the DPSC (Positive Access to Knowledge and Information) that was well received.I thought [the DPSC staff members] were excellent presenters, just very impressed...I was hoping to get them [...] out here to do a workshop for us. That's how effective I thought they were (VA-DPP Coordinator). The DPSC also provided high-quality materials for VA-DPP teams and participants (Positive Design Quality and Packaging). Teams were further supported by the coordinating center (Positive Engaging: External Change Agent), which hosted bi-weekly meetings to provide new information and reflect on progress and problem-solve (Positive Access to Knowledge and Information; Positive Reflecting and Evaluating). However, teams encountered several challenges. Some staff outside the VA-DPP team perceived VA-DPP as a competitor to MOVE! and questioned whether a new program was necessary (Negative Compatibility, Negative Relative Advantage).If people are prediabetic, losing weight will help decrease their risk of becoming diabetic, so MOVE! in and of itself could serve as a Diabetes Prevention Program, although it's not called that (MOVE! Coordinator). In addition, sites were provided funding to hire staff, but hiring took months longer than expected and led to program delays (Negative Structural Characteristics). However, as one VA-DPP Coach noted, space was "...really far and away the biggest issue." Sessions were rescheduled or canceled when the rooms were needed for other uses. Furthermore, VA-DPP session times were determined by available space, not by convenience for participants (Negative Available Resources). Although site directors signed an MOU, mid-level managers were largely disengaged and did not help teams resolve hiring or space issues (Negative Leadership Engagement). Lastly, VA-DPP coaches often struggled to cover all the content specified in the DPSC facilitation guides within hour-long sessions (Negative Design Quality and Packaging). However, the DPSC team clarified that coaches had latitude to adapt content to the needs of their groups (Positive Access to Knowledge and Information; Positive Reflecting and Evaluating). BODY.RESULTS.MAINTENANCE.BARRIERS AND FACILITATORS: MAINTENANCE OF VA-DPP: Two of the three sites continued to deliver VA-DPP as a separate program after the end of their VA-DPP demonstration period. At one site, monthly maintenance sessions were held after participants completed 12 months of VA-DPP followed by two separate projects focusing on peer-led and gender-specific VA-DPP implementation. One site did not sustain VA-DPP after funding ended due to insufficient resources to deliver VA-DPP and MOVE! concurrently (Negative Available Resources). At the national level, however, NCP leaders recognized the early improvements in outcomes from VA-DPP compared to MOVE! [34] (Positive Evidence Strength and Quality). Subsequently, NCP updated guidance for MOVE! with VA-DPP features thought to contribute to greater weight loss (Positive Relative Advantage), including (1) having one consistent coach to lead all sessions; (2) offering closed cohorts; (3) providing 16 sessions within 6 months; and (4) aligning session topics more closely with VA-DPP topics (see www.move.va.gov/grpSessions.asp). BODY.DISCUSSION: The challenge of implementing complex behavioral programs is well-recognized [6, 7, 49, 50]. The current findings point to the need for multi-level, multi-component strategies that include, for example, maximizing reach by carefully integrating processes to identify high-risk patients and engaging primary care providers. Conducting educational outreach and attempting to heighten clinical priority for preventing diabetes in the face of other competing demands is necessary to engage providers, the main source of referrals in the current study. Many efforts to vastly scale up identification of high-risk individuals and encourage them to participate in DPP-like programs are underway in other countries [12, 51, 52]. Lindstrom and colleagues published the IMAGE toolkit, motivated by the premise that "Small changes in lifestyle will bring big changes in health...The time to act is now. ([53]; p. 537)" These motivating words are aimed at aligning partners toward a common high-priority goal around diabetes prevention. The toolkit describes foundational principles (e.g., engage partners from multiple strata including communities) and functions (e.g., reach out to high-risk individuals). The CDC also developed a toolkit for implementation [54]. Table 3 extends these recommendations by providing potential strategies aimed at optimizing outcomes within each of the five domains of RE-AIM based on barriers and facilitators shown in Fig. 2, which in turn reflect the experiences at the three study sites. These strategies are offered as hypotheses to be tested in a larger trial.Table 3Recommended strategies to address organizational level barriers as described by RE-AIMDomainRecommendationsReach targeted participant population• Design referral processes that are (1) compatible and integrated with existing clinical processes; (2) effective in identifying and engaging high-risk participants; and (3) easy to use• Engage clinicians who are the primary source of referrals through personal outreach and by providing easy-to-access, targeted information about DPP highlighting its (1) evidence base, (2) compatibility with local clinical processes, (3) advantages compared to status quo, and (4) organizational and clinical priority for diabetes preventionEffectiveness of program• Schedule sessions at a time and place convenient for participantsAdoption by clinical settings; evidenced by visible demonstration of commitment by executive leaders• Target education and information to executive leadership about DPP including its (1) evidence base, (2) compatibility with clinical processes, (3) advantages compared to the status quo, and (4) organizational and clinical priority to inspire them to adopt the program• Obtain a formal agreement (e.g., memorandum of understanding), signed by executive leadership, to commit to implement DPPImplementation with consistency (track costs and adaptations)• Target education and information to mid-level and clinical managers about DPP including its (1) evidence base, (2) compatibility with clinical processes, (3) advantages compared to the status quo, and (4) organizational and clinical priority to inspire them to help implementation teams solve problems and review progress• Ensure adequate time to hire and train skilled and enthusiastic implementation leaders and coaches to deliver DPP• Provide high-quality materials to coaches and participants that can be used effectively to support delivery of effective coaching during sessions (e.g., see http://www.diabetesprevention.pitt.edu/).• Ensure adequate space availability for sessions• Schedule sessions at locations and times that are convenient to participants, e.g., in community settings outside of normal clinic hoursMaintenance of DPP in the clinical setting over time• Effectively report on outcomes and other key benefits from the local DPP to executive leadership, managers, and clinicians (especially those who may refer their patients to DPP) to gain support for the program and build a robust referral network Fig. 2Conceptual framework: integration of CFIR contextual factors and RE-AIM domains Within VA, the usual care MOVE! weight management program can prevent incidence of diabetes [5, 55]. It is a relatively low-intensity program with no diabetes risk assessment. The present study evaluated a higher intensity intervention based on a CDC-recognized structured curriculum targeted to individuals with clinically verified prediabetes. This trial shows that having national and local leaders who are committed to diabetes prevention is a necessary, though far from sufficient, condition for successful implementation within a large healthcare system. Reaching and engaging target populations is a universal challenge for comprehensive lifestyle programs across settings [7, 56]. There was relatively high participation in this trial (Fig. 1); more participants assigned to VA-DPP attended at least one session compared to participants assigned to MOVE!. In addition, unlike most behavioral intervention studies, which tend to over-represent non-Hispanic whites [57], 48% of VA-DPP participants identified as a racial/ethnic minority (compared to 25% of national VHA users) [58] and 11% of VA-DPP participants were women (compared to 8% of national VHA users [59]). Robust referral processes and networks to identify and engage participants at high risk are essential [13]. Once high-risk individuals have been referred, scheduling sessions at locations and times that are convenient to participants [60, 61] or offering online programming [62] may help bolster participation. The single largest investment incurred for the VA-DPP demonstration was the total average $40,348 per site to assess eligibility and implement the program. In the current study, new clinical processes had to be designed and adapted to each local setting to accomplish the required clinical testing of HbA1c or FPG levels. Several countries have administered short self-assessment risk instruments [63, 64]. Individuals at high risk are then encouraged to enroll in a nearby community-based DPP-like program. The later the risk is identified, the more intensive the intervention should be [65]. The current study used more intensive risk identification based on clinical testing that relied on preexisting obesity treatment referral processes already in place. This approach may have resulted in later risk identification and contributed to the smaller than planned sample size but may justify the more intensive DPP. Number of referrals was lower during the demonstration period than in the years leading up to the demonstration, perhaps because of other organizational priorities during that time (e.g., implementation of PCMH during the demonstration) and there was an unexpectedly high prevalence of patients who already progressed to diabetes, especially compared to the general VHA patient population (43 vs. 25%, respectively) [34]. This illustrates the potential missed opportunities to identify and engage high-risk individuals before they progress to diabetes. Additionally, VA-DPP relied on primary care physicians to refer patients; however, there were cases where physicians did not believe in the effectiveness of lifestyle change programs for their patients. Few studies report the extent to which DPP is delivered as designed, though stated use of a standard curriculum is a recognized contributor to greater weight loss [7, 66, 67]. The current study assessed fidelity of delivery. Though ratings of characteristics of the coaches delivering each of the programs (VA-DPP and MOVE!) were comparable (Table 1), VA-DPP had higher fidelity ratings for delivery of educational content, goal setting, review of goal progress, and group cohesion. Reinforcing this finding, VA-DPP participants reported higher satisfaction for six of eight program characteristics compared to MOVE!. Together, program differences in fidelity ratings [46] and participant satisfaction helped to identify factors that may explain higher participation rates for VA-DPP compared to MOVE!, as well as more weight loss for VA-DPP, [6, 7] at least in the short term. It is not clear what contributed to higher delivery fidelity for VA- DPP compared to MOVE!; specific characteristics of DPP as well as more recent training of DPP coaches may have contributed to this difference. Although there were no statistical differences in effectiveness of VA-DPP versus MOVE! at 12 months (3.4 vs. 2.0 kg lost, respectively; p = 0.16 [34]), the lack of statistical difference between groups may be in part due to the smaller than planned sample size. The challenge of evaluating implementations of complex behavior change programs like DPP is well-recognized [68]. Use of a theoretical framework provides concepts and language that can be expressed consistently across diverse studies to aid comparisons and build knowledge about complex implementation processes [42, 69]. Based on current findings, Fig. 2 posits relationships between contextual factors and domains of RE-AIM based on common barriers and facilitators to VA-DPP implementation. These relationships are offered as hypotheses that require testing in other settings and within larger-scale implementations. Multi-faceted strategies that address multiple domains of context are needed to implement DPP in a way that maximizes outcomes within each RE-AIM domain (Table 3). Though DPP has been shown to be cost-effective, including when delivered in a community setting [70, 71], health system and policy decision-makers will not implement programs like DPP without first knowing the upfront investment and ongoing delivery costs. Costs to deliver VA-DPP sessions averaged $101 per session. In the current study, attendance was highly variable across sites, across cohorts within each site, and across sessions within each cohort. While the average cost per participant per session was $46 ($1012 for 22 sessions) for VA-DPP, this cost could be as low as $12 per participant per session ($264 for 22 sessions), if the lowest-cost site ($1410) had the highest observed level of participation (119). This cost range was lower than cost reported for the original DPP (estimated costs of $1399 per person in the first year) [71] and are similar to those of a YMCA group-based DPP (estimated costs between $275 and $325 per person in the first year) [61]. In addition, administrative tasks related to preparing for each VA-DPP session, such as reminding participants to attend and planning the session, which averaged $328 per participant in the current study, must also be considered when allocating staff resources. It is important to note limitations in this evaluation. First, only three academically affiliated VHA sites participated; therefore, implementation experiences reported may be unique to their respective settings. In addition, the study occurred during an organizational transformation to the VA's model of PCMH within the primary care clinics, which may have contributed to lower than expected number of referrals. Second, fidelity ratings may be biased positively because they were determined by VA-DPP coordinators/coaches who rated their peers. However, this bias may be present for both programs because VA-DPP teams worked closely with MOVE! teams. It is important to note that comparison of ratings for VA-DPP and MOVE! was limited to a sample of program sessions and delivery components at these sites and not a comprehensive rating for all aspects of all sessions. Third, this effort was done as part of an effectiveness-implementation hybrid trial. National partners contributed funding for the clinical teams at each of the three facilities and did not assess diabetes incidence or clinical measures of cardiovascular disease or risks. When funding ended, two of the three sites were not able to maintain the program or continue screening for prediabetes. A strength of this study was that partners and local leaders deemed local activities, including eligibility screening, delivery of VA- DPP, and outcome assessments as part of a clinical quality improvement (QI) initiative that required no additional assessments for research purposes. Patient satisfaction was elicited via surveys funded by research. IRB approvals included use of data collected through local clinical QI activities. Fourth, an economic evaluation of usual care was not completed and intervention impacts on blood pressure and blood cholesterol were not assessed. Costs were estimated for eligibility screening, implementation, and delivery of only DPP. BODY.CONCLUSIONS: The comprehensive qualitative and quantitative findings from this pragmatic trial of VA-DPP reveal barriers and facilitators that influence overall program success. Findings suggest a number of strategies that may help support future real-world implementations of DPP, including gaining visible support from system and local leaders, highlighting the evidence base and benefits of DPP for key stakeholders including referring clinicians, and providing sufficient time and resources for high-quality staff training. As the need and demand for DPP increases, it is important to recognize, address, and leverage implementation process and contextual factors that contribute to maximum success of DPP.

TITLE: The effect of humidified heated breathing circuit on core body temperature in perioperative hypothermia during thyroid surgery ABSTRACT: Purpose: During general anesthesia, human body easily reaches a hypothermic state, which is mainly caused by heat redistribution. Most studies suggested that humidified heated breathing circuits (HHBC) have little influence on maintenance of the core temperature during early phase of anesthesia. This study was aimed at examining heat preservation effect with HHBC in case of undergoing surgery with less exposure of surgical fields and short surgical duration. Methods: Patients aged 19 to 70 yr - old, ASA-PS I or II who were scheduled for elective thyroidectomy were assigned and divided to the group using HHBC (G1) and the group using conventional circuit (G2) by random allocation. During operation, core, skin, and room temperatures were measured every 5minutes by specific thermometer. Results: G1 was decreased by a lesser extent than G2 in core temperature, apparently higher at 30 and 60 minutes after induction. Skin and room temperatures showed no differences between the two groups (p>0.05). Consequently, we confirmed HHBC efficiently prevented a decrease in core temperature during early period in small operation which has difficulty in preparing warming devices or environments were not usually considered. Conclusions: This study showed that HHBC influences heat redistribution in early period of operation and can lessen the magnitude of the decrease in core body temperature. Therefore, it can be applied efficiently for other active warming devices in mild hypothermia. BODY.INTRODUCTION: The human body is composed of two compartments for maintaining temperature. One is the central compartment, which is responsible for core temperature. The other is the peripheral compartment, such as musculoskeletal system, which plays a role as the buffer in the thermoregulatory system 1. Generally, body temperature changes due to circadian rhythm, sex, patient's disease or various environment factors. In general, anesthesia, interthreshold range, which is only 0.2-0.4°C in normal body temperature initially, widens up to ten-fold depending on the anesthetic drugs, allowing a hypothermic state to occur easily 2. Therefore, although mild hypothermia, defined on 34-35.9°C is a common phenomenon 3 which leads to complications such as surgical wound infection 4. The further progressed the state of hypothermia, the greater the chance of side effects. In prevention of these complications, many kinds of methods are used, such as a forced air warming blanket, fluid warming devices, heat-pads, or heated humidified breathing circuit (HHBC) intra-operatively in addition to preoperative warming 5. A decrease in core temperature is apparent when the operation time is longer than two hours and usually the first decrease appears about 30 minutes after starting the operation. Therefore, many investigators consider body temperature measurement to usually not be necessary during Monitored Anesthetic Care or regional anesthesia, minor procedures, or surgeries completed in less than 30 minutes. In addition, the report said that temperature should be monitored at no more than 15- minute intervals during all general anesthesia lasting longer than 60 minutes 6. Previous studies showed that air blanket and warming air were effective in reducing a decrease in core temperature 7. However, humidified warming circuit did not show any large difference, especially in wide body exposure and operations lasting longer than two hours 8. We were interested in the core temperature changes in 60- to 120- minute operations (neither too short nor too long), and considered how we should manage it during this period. In the case of thyroid surgery, hyperthermia is usually overlooked because of its limited surgical conditions; the operation time takes less than two hours, doesn't need a wide skin exposure, and has smaller incision sites than other surgeries. Therefore, we can ignore the changes in body temperature and prepare no warming devices. However, no matter what the condition is like above, it is important to maintain intraoperative normothermia to prevent postoperative inadvertent complications 9. We studied the core temperature change in thyroid surgery with humidified heated breathing circuit (HHBC). From this study, we aimed to validate the efficacy of HHBC in minor surgery, and to place emphasis on a more practical application of it for preserving core temperature in normothermia. BODY.METHODS: Prospective, single-blind, randomized study was done with the 230 patients, ASA-PS (American Society of Anesthesiologist Physical Status) I or II, scheduled on elective thyroidectomy from August 2010 to June 2011. All of them were benign or malignant neoplasm in thyroid with normal thyroid function and no endocrine abnormality. We excluded the patients with thyroid disease, such as Grave's disease, thyroiditis, toxic nodular goitor, preoperative body temperature above 38°C, extreme age (under 18 and over 70), infectious disease, emotional abnormality like anxiety, etc. Before starting this study, according to a computer-generated list of random numbers, patients were allocated randomly to one of the two groups. The two groups were: One (G1) used HHBC (Mega Acer Kit, Ace medical, Seoul, South Korea) and the other (G2), control group which used usual conventional breathing circuit (Disposable breathing circuit, King Systems corp.IN, USA) with electrostatic filter (DARTM, Covidien, MA, USA). In the study period, 230 patients in total were enrolled. We got Informed Consent from each patient preoperatively. This study was approved by the Institutional Review Board of Yeouido St. Mary's Hospital, The Catholic University of Korea (approval number: SC11OIS10265) and registered with Clinical Research Information Service of Korea National Institute of Health (CRIS, identification number: KCT0001459). To assess initial temperatures, core temperature in tympanic membrane was measured just before entering the operating room. We attached a skin temperature probe (Therma-Temp® Probes, 409, Cincinnati Sub Zero Products Inc., Ohio, USA) onto patient's lateral skin-surface of left lower leg. Anesthesia was induced with intravenous thiopental sodium (5 mg/kg) and succinylcholine (1mg/kg). After patients lost self-respiration, we intubated endotracheal tube then inserted 12Fr. esophageal stethoscope (Esophageal Stethoscope, DeRoyal Industries Inc., Powell, TN, USA) for measuring core temperature and its tip was placed on lower third of esophagus where strong heart sounds with breath sounds are detected 10 before removing the laryngoscope from oral cavity. Each group was connected to the designated breathing circuit. We tried to maintain constant room temperature between 20°Cand 22°C. Room temperature was measured by electric thermometer placed onto the side wall of the operating room. After finishing the anesthetic setting, the patient was covered surgical draps with an exposure of operation site only. At the beginning of operation, we did not use other active warming devices for excluding sources which influence temperature. We estimated core temperature, itself. For severe hypothermia (less than 34°C) during the operation, we planned to apply active warming methods such as forced-air warming blankets and fluid-warming devices for elevating temperature directly. Because we could not rule out the effect of potential sources of interference, these cases were excluded. All temperatures (core, skin, room) were measured every 5 minutes until the end of the surgery. Total fresh gas flow was maintained at 3 L/min throughout the operation. An initial set temperature of heated circuit inlet was 36.5 - 37.0°C depending on the initial core temperature and the circuit temperature controlled to set point by outlet temperature automatically. Sample size was calculated to detect a 25% increase in core temperature for the HHBC compared with conventional breathing circuit (2-sided test with α=0.05, β=0.2, allowing dropout 20%).We compared demographic distribution in two groups using paired t-test and McNemar's test by matching patients' age. We could obtain 85 patients in each group. Statistical analysis was performed with the use of SAS software, version 9.2 (SAS institute, Cary, NC, USA). Baseline characteristics conducted by paired t-test and longitudinal changes between groups were tested with repeated measured ANOVA. For correcting continuous variables, we analyzed by Bonferroni's correction to adjust by time sequence. Statistic significant P value was less than 0.05. The primary outcome of this study was intraoperative core temperature between two groups at each time point. Secondary outcomes were the degree of decrease in core and skin temperature from initial to the end of operation in each group. BODY.RESULTS: When we started the study, there were 230 patients assessed for eligibility. There were no dropouts from intraoperative severe hypothermia less than 34°C. 26 patients who were dropped out were due to missing datum after study, incorrect temperature records and preoperative abnormal temperature of over 37.5°C or less than 36°C. All drop-outs (26 patients) were included in control group. Finally, 204 patients (HHBC group (G1); 119 and control group with conventional circuit (G2); 85) were analyzed. In demographic data, we need to match with age when taking into consideration statistical significance. Each group was composed of 85. After matching, demographic datum showed no differences (age; P =0.2941, sex; P =0.2693) (Table 1). And, average core, skin and room temperature during study period didn't show the differences between two groups (core T.; P = 0.0568, skin T.; P= 0.6648, room T.; P= 0.5917) (Table 1). In the analysis based on total operation time, each thyroidectomy showed variable surgical duration and all of them represented more than 30 minutes. The largest distribution of it was between 60 and 75 minutes and none were over 120 minutes (Figure 1). When we compared at five-minute intervals, there represented no differences in two groups skin and operating room temperature as well as core temperature in all time sequences. Therefore, after the difference at each time point was applied to the analysis every 30-minutes considering previous study 8, the trends of temperature change in Figure 2 seemed to be different in both groups. However, statistical results didn't show any differences, except core temperature. G1 showed lesser decrease in core temperature than G2 (P = 0.0233). Particularly, at 30 and 60 minutes showed an apparent smaller decrease in G1 (30 min; P = 0.0001, 60 min; P = 0.0026) in each time point (Table 2, Figure 2A). However, skin and room temperature still did not show significant differences (skin; P = 0.5293, room; P = 0.5997) (Figure 2B and 2C). During the operation, core temperature was above 35°C in mild hypothermic category in both groups and mostly less 1.0°C decrease than baseline temperature. There were no extreme hypothermic states and adverse events related with hypothermia during operation. BODY.DISCUSSION: Generally, humans try to maintain internal body condition from decreasing body temperature. Under normal conditions, central temperature is maintained within narrow range of 37 ± 0.2°C 11. There are some risks which will decrease the efficacy of thermoregulatory response and increase in prevalence of hypothermia, such as advanced age, infirmity, medication. Despite circadian rhythm in body temperature, the core temperature is very tightly controlled and regulated by a highly effective system that balances heat production and heat loss. Heat is produced as a consequence of cellular metabolism and lost by radiation, conduction, convection, and evaporation 12. Core temperature normally ranges from 36.5°C to 37.5°C. The definition of hypothermia is variable. When we look over many reviews, hypothermia is divided into three categories, and the usual concept of hypothermia is less than 36°C 13. Especially perioperative hypothermia is defined as a core temperature of less than 36°C 14. From 35.9°C to 34°C, we call it mild hypothermia 3. Therefore, it is reasonable to maintain core temperature greater than 36°C in surgical patients unless hypothermia is therapeutically indicated 12. Mostly after surgery, the amount of decrease on core body temperature depends on operation time, range of exposure, operation site, etc. Temperature in the peripheral compartment is usually 2°C to 4°C lower than the core temperature, and a gradient becomes higher when the anesthesia is induced and vasodilation occurs despite individual differences 1, 14. How to monitor body temperature is a standard of anesthesia care. The continual observation of temperature changes in anesthetized patients allow for the detection of accidental heat loss or malignant hyperthermia 12. Core temperature can be estimated using probes that can be placed into the distal esophagus, ear canal, trachea and nasopharynx 15. In addition, pulmonary artery blood temperature is also a good indication of core body temperature 12. But, we selected the esophageal probe because of inaccuracy, inconvenience or invasiveness of other devices 15-18. During general anesthesia, intraoperative hypothermia is a known consequence 19 and the combination of anesthetic-induced impairment of thermoregulatory control and exposure to a cool operating room environment are the main causes 3. Core temperature shows a three phase temperature pattern: First, during one hour after the induction, it falls rapidly from 0.5 to 1°C 7, 8. Second, this is followed by slow, linear decrease. Eventually, it finally plateaus after 2 to 4 hours of anesthesia 1; this initial rapid onset of hypothermia results from redistribution of heat from the warmer core to the cooler periphery 20. On the basis of intraoperative core temperature characteristics, we used HHBC to derive the usefulness and proprieties as described below. In a recent article, they emphasized the fact that more than half the patients had core temperatures below 36°C within the first hour of anesthesia. After then, core temperature progressively increased 5. This means the decrease in core temperature during the first hour results from the particular action (such as heat distribution) rather than external influences mentioned above. In addition, core-to-peripheral redistribution, the main cause of hypothermia during this phase, can remain the dominant cause, even after 3 hours 1. Therefore, we think that preventing early hypothermia as much as we can is important for lowering adverse outcomes. Thus, if we want to lessen the gap of interthreshold, we have to minimize the core-to-peripheral redistribution. In our study, operation times did not exceed two hours and mostly were less than 90 minutes. Most of them were finished before third phase of temperature drop - between first and second phase of surgical duration. In this condition, the main cause of temperature decline resulted from heat redistribution and a small part from other circumstances 21. On the basis of this result, we applied HHBC to reduce the decrease in core temperature. However, several studies showed that active airway heating and humidification slightly contribute to the maintenance of central normothermia. Therefore, its efficacy is controversial on either the decreasing of heat loss or the active warming of hypothermia 22-24. Some studies also state that the main purpose of this warm and humidification is for the optimum level of humidity necessary to prevent drying of secretions and deleterious effects on ciliary function 25. Consequently, airway heating and humidification are less effective in patients most in need of effective warming 26. In addition, other reported respiratory heat loss is smaller than radiative heat loss, heating and humidification cannot prevent the temperature drop 7. Patients undergoing a procedure with general anesthesia lasting longer than 30 minutes are easily exposed to the risk of hypothermia. Therefore, active warming devices such as forced-air warmer, minimum skin exposure and maintenance of optimal room temperature are required 27. In spite of many kinds of methods, core temperature decreased during first 60-minutes, noticeably after one hour 8. And forced-air warmer is introduced more effective than circulating water blanket or heated humidifier 6, 28, 29 in addition to some negative opinions about a heated humidifier in which core temperature became more hypothermic throughout the operation 8. And, even though it prevents the temperature drop that occurs 30-minutes after induction, it cannot prevent the subsequent drop 30. In contrast with above results, we controvert these opinions of HHBC. A smaller decrease in core temperature happened apparently between 30 and 60 minutes and it showed a different pattern to existing one, which focused on the last half of initial phase. It suggested that core-to-peripheral temperature change by redistribution can be regulated by HHBC. Even though we cannot confirm how much this circuit influenced heat redistribution, the main cause of initial phase of temperature drop is heat redistribution and we can measure the role of HHBC as a device of temperature modulation. As a result, in consideration of our study, it is improper to ignore use of HHBC at this first phase. In spite of known evidence, HHBC reduced the decrease of core temperature without other devices in our results. The role of HHBC in small operations such as thyroidectomy can be mentioned. Thyroidectomy has some limiting conditions: warming devices cannot be used freely because of narrow operation site, typical surgical position of patients and surgeons, drap coverage pattern and scrub nurse's position. The advantage of application on HHBC is easier access than other thermoregulation devices. Others suggested that Bair Hugger forced air warming with a surgical access blanket can be used for preventing a decrease in core temperature during anesthesia 31. However, these forced air warming devices are not cost-effective and in fact difficult to use routinely in our hospital system. Generally, the differences in compensation against the decrease of patient's core temperature depend on preoperative physical status, disease, temperature in operating room, and no innerwear. In this study, we tried to maintain the same conditions for excluding other factors; for example, room temperature maintained at 20-22°C. In most of the surgeries, many conditions can interfere with maintaining the appropriate temperature: difficulty to access the active warming devices in operation field, sudden drop of room temperature by malfunction, low temperature irrigation fluid or main fluid, unopposed wide skin surface exposure, etc. Considering inadvertent conditions like above, we considered the easiest and most effective devices without taking up too much space. The HHBC is suitable for these kinds of surgical conditions. There is another report that intraoperative hypothermia is minimized by 2-hours of active skin-surface warming before starting general anesthesia as well. However, such prolonged pre-warming is impractical in most surgical conditions and hospital systems 32. From this reason, application of this is also difficult to maintain normal temperature. When we proceeded with the study, skin and room temperatures had also showed some changes. In particular, there were similar increase patterns until 30 minutes, after then showed slight increase patterns in HHBC groups between 30 to 60 minutes without clinical significances. In any case, this explains that HHBC is advantageous and influences on the peripheral temperature in spite of any statistically proven result. However, further studies are necessary to reveal the dual (core and peripheral temperature) effect of HHBC. As mentioned above, the conclusions from this study were as follows: Firstly, HHBC has some advantages: less pace occupied, easy access, and smaller decrease in core temperature, as well as protection of dryness and diluting secretion. Secondly, though Hynson et al. 8 described this humidified heated circuit as useless during any time point of operation, the efficacy of this circuit on protecting a decrease in core temperature is apparent in minor operations which take more than 30-minutes, in contrast to previous suggestions that there is no effect of HHBC compared with other devices 12. Third, HHBC has an influence on heat redistribution between initial and early second phase temperature drop. Therefore, core temperature can be preserved longer than usual by lowering the effects of vasodilation. From these results, we recommend HHBC for preserving core temperature on early perioperative period or especially in cases lasting less than 60 minutes if other devices are difficult to prepare and have limited conditions. The intension to try this is considered to change the usual concept as a warming device and show positive effect on temperature preservation. In addition, it can be used widely and contribute to the reduction of postoperative complications in unexpected hypothermia. In this study, there were some limitations to take into consideration. First is the lack of comparative methods. We did not compare with other active warming devices. We intended to suggest the benefit of HHBS itself. Therefore, further investigation should be tried between humidified heating circuits and other active warming devices. Also, we need to find out whether or not other devices will show similar efficacy compared with heating circuits. Second is range of patient selection. We did not consider the postoperative complications related to hypothermia because of the exclusion of extreme temperature and operative characteristics. In the future, in-depth studies should be done toward the efficacy of HHBC on high-risk patients who are prone to complications. Third is lack of observation period about adverse outcomes related to hypothermia. We observed temperature only in the operating room. Therefore, we could not find any complications related to hypothermia even though we already mentioned the possibility of complications. More specific studies were needed to detect the adverse events such as comparing complications between HHBC and conventional BC. Fourth is no basic temperature conserving method is used in the control arm. Thyroidectomy is included in low-risk, minor surgery in surgical grade classification. As previously mentioned, it has an operation time that is not too long and small incision site which could not influence on heat loss by evaporation. In addition, the recruited patients were involved in ASA-PS class I or II without respiratory or airway problems. After considering these conditions, at first we obtained written informed consent from control group patients of not using HME (Heat and Moisture Exchanger). We just prepared the warming method to manage accidental hypothermia. The last is lack of precision of measurement of core temperature. We used only esophageal stethoscope and low fresh gas flow only 2 L/hr. Heated air can influence esophageal temperature. In consideration of surgical limitations, we have to use esophageal stethoscope. However, we also have to consider applying other measurements for core temperature and further studies should be done about comparison with other measurements for core temperature.

TITLE: Stentless laparoscopic pyeloplasty: A single center experience ABSTRACT.AIM:: To assess the effectiveness of laparoscopic stentless pyeloplasty for congenital ureteropelvic junction obstruction. ABSTRACT.MATERIALS AND METHODS:: This was a prospective comparative study conducted over a period of 5 years. The study included 35 cases of primary ureteropelvic junction obstruction (UPJO) with mean age of 29.5 years, divided in two groups- Group A (stent-less, 18 patients) and Group B (stented, 17 patients). Follow up ranged from one to 4years (mean 2 years). Transperitoneal laparoscopic Anderson- Hyene's pyeloplasty was standard for both the groups. Perioperative and postoperative complications were prospectively collected and analyzed by Statistical Package for Social Sciences (SPSS) 17 version using Pearson chi square test. ABSTRACT.RESULTS:: Both the groups were comparable with respect to preoperative differential renal function (DRF) and time required for maximum activity in minutes (tmax.min). Average post operative DRF was significantly higher than preoperative DRF in both the groups. Average tmax was significantly lower after pyeloplasty than pre operative tmax. Mean operative time, mean duration of urethral catheter, and mean duration of drain removal were comparable in both the groups. However bothersome irritative lower urinary tract symptoms (LUTS) and hematuria were significantly more in group B patients (P < 0.0001 and <0.013 respectively). ABSTRACT.CONCLUSION:: In experienced hands, laparoscopic stentless pyeloplasty is as effective method for treating UPJO as its stented counterpart. It is cost effective, avoids stent-related morbidity, and could be performed without compromising the success rate. However, more randomized studies are needed to evaluate the safety of stentless pyeloplasty. BODY.INTRODUCTION: Pelvi-ureteric junction (PUJ) is the most common site of obstruction in upper urinary tract. It occurs nearly 1 in 500 to 1:1250 live births.[12] There are two types of ureteropelvic junction obstruction (UPJO) – more common intrinsic and the extrinsic variety.[3] The main indications for active interventions are to relieve obstruction, relieve pain, to preserve renal function and/or treat pathologies secondary to such obstruction like calculi and infections.[4] Recent improvements in pre-natal ultrasonography now allow most of the cases to be diagnosed in utero.[4] Obviously, for patients of any age, a reconstructive procedure is always indicated whenever overall renal function is compromised because of involvement in a solitary kidney or bilateral disease.[5] UPJO may not become apparent until middle age or later.[6] However, the majority of affected patients can in fact benefit from reconstructive intervention.[7] When intervention is indicated, the procedure of choice is the repair of PUJ i.e. pyeloplasty. Surgical management of UPJO has recently been revolutionized by the introduction and widespread adoption of minimally invasive techniques as alternative to standard open reconstructive procedures in an effort to reduce the morbidity of the treatment. Initially, minimally invasive approaches included antegrade and retrograde endoscopic endopyelotomy. Although, these procedures are associated with relatively few complications, brief hospitalization and little disability, the reported success rates are lower (71_ 88%) as compared to open approach, and it has limited indications. Also these procedures have an increased risk of hemorrhage.[8] BODY.MATERIALS AND METHODS: This prospective study was conducted in Sir Ganga Ram Hospital, New Delhi, India, in the Department of Urology from October 2007 to December 2011. Total no of patients were 35. Patients were divided in two groups as Group A (stent less, 18 patients) and Group B (stented, 17 patients). Age, gender, and preoperative differential renal function were recorded in all patients. Group A patients were explained the procedure to undergo stentless transperitoneal laparoscopic pyeloplasty and the need for stent placement, if any, in postoperative period, and the Group B patients were explained the procedure to undergo laparoscopic stented pyeloplasty. Indications for surgery were decrease in differential renal function (DRF) in presence of symptoms, decrease in DRF of more than 10% than previous reading in an asymptomatic patient, or decrease in DRF of ≥25% at any point in time, in presence or absence of symptoms, supported by diuretic renographic evidence of UPJO. The decision for not using the stent was based on patient's preference after explaining the pros and cons of the procedure, and surgeon's decision. All patients were preoperatively assessed with history, physical examination, abdominal ultrasound and X--ray kidney, ureter, and bladder (KUB), Intravenous ureterogram (I.V.U), and diuretic renal scan. All patients had radiographic evidence of UPJO and hydronephrosis on diuretic renography and I.V.U. Laboratory tests like complete blood picture, kidney function test, and urine routine and culture were done in all patients. Exclusion criteria were patients with stone, active infection, children below 6 years of age, and renal units of PUJ obstruction with glomerular filtration rate (GFR) less than 15 ml/minute. All patients were kept on liquid diet 1_day prior to surgery. Patients were given parentral cephalosporins and Amikacin at the time of induction of anesthesia and these antibiotics were continued postoperatively till the time of drain removal. All procedures were performed under general anesthesia. Foleys catheter was placed in the bladder in all patients before positioning. All procedures were carried out via laparoscopic transperitoneal approach and by a single surgeon. Pneumoperitoneum was created by closed technique by Veres needle and carbon dioxide gas. Three ports were placed in a standard way with variation in position as per the body habitus. Approach to the PUJ area was made by mobilizing the colon by incising the peritoneum laterally. The ureter was identified and dissected in cephalad direction to achieve mobilization of proximal ureter, ureteropelvic junction (UPJ) and renal pelvis. The pelvis was divided, diseased segment excised, watertight, spatulated uretero-pelvic anastomosis performed with a wide funnel shaped dependent new PUJ, taking care of any rotation/ twisting of the ureter. In cases of difficulty in spatulation, the ureter was brought outside body through lower working port and spatulated extra corporally (in three and two cases in Group A and B respectively). Pyeloureteral anastomosis was done with vicryl 4/0 continuous suture. We found no difference in beginning with either posteriorly or anteriorly except that starting from anterior margin was more convenient. After completion of anterior suture line at UPJ, double J stent was indwelled in group B patients via 10 mm port, loading the stent on guide wire, and with the help of pusher. An infant feeding tube (5Fr or 6 Fr ) was placed in the ureter as a temporary splint to facilitate anastomosis. Water tight, funnel shaped, dependent anastomosis was made. A tube drain size 20 Fr was placed near the anastomosis site. Post operative complications (pain, high and or prolonged drain output, urinoma and fever), hospital stay and improvement in renal functional outcomes on technetium 99m diethylenetriaminepentaacetic acid (Tc 99 DTPA) renal scan were recorded. Drain was removed in the post operative period when its output decreases to lessthan 50 ml in 24hr. patients with continuous drainage >600 ml on 3rd/4th pod were discharged along with drain and were explained in detail about daily recording of drain output for 24 hrs and communicated on phone. Foley's catheter was removed first followed by drain removal. In case of stented group Foley's catheter was removed after 2 days and drain was removed once output <50 ml. (after catheter removal) BODY.MATERIALS AND METHODS.FOLLOW UP: The decision for postoperative DJ stenting in stentless pyeloplasty was made if there was persistent high drain output for more than 10 days. The follow up schedule included a visit after 7 post operative day for port site clip removal and clinical examination followed by three week visit with urine analysis and detailed symptomatology and clinical examination. Diuretic renogram was done at third and or sixth month of surgery to compare with pre-operative renal scan in both Group A and Group B. BODY.RESULTS: In our study all patients had primary UPJO. In all patients surgery was successfully completed laparoscopically. The demographic profile, pre op/ post op tmax and DRF is given in Tables 1 and 2 for group A and B patients, respectively. Mean age was 28 and 31 years in group A and B, respectively. All patients had proven significant obstruction on Tc 99 DTPA renal scan. Mean operative time was 180 mts (range155-210 mts) and mean blood loss was 50 ml (range 20-100 ml) in both the groups. In Group A patients mean duration of post operative Foley's catheter was 2 days. Drain output ranged from 500-1200 ml on first postoperative day that decreased gradually in 16 patients and drain was removed from 3rd to 5th postoperative day (mean 4 days). Two patients had persistent drainage >600 ml/24 hrs on 5th postoperative day and were discharged along with drain which was removed on 7th pod in one and 8th pod in another patient. One patient had high grade fever and vomiting on 10th pod. As the drain was removed on 3rd pod, possibly drain was blocked in immediate postoperative period. Ultrasound KUB showed features of perirenal urinoma. Subsequently DJ stent was inserted by the same surgeon with position of DJ stent checked on fluoroscopy. He was put on injectable antibiotics and hospitalized for 2-days till fever subsided. None of the patients had any signs of peritonitis or evidence of bowel injury in the post operative period. Table 1 Demography, t max and DRF, both pre operatively and post operatively in Group A patients Table 2 Demography, t max and DRF, both pre operatively and post operatively in Group B patients In group B patients, Foley's catheter was removed after a mean of 2 days and drain was removed on 3rd pod (when 24 hr drain output was <50 ml). On follow up these patients were noted to have significant bothersome urinary tract symptoms as compared to non stented counterpart (P value- < 0.0001) [Table 3, Figure 1]. Two patients experienced stent related pain affecting daily activities. Table 3 Complications in the two groups Figure 1Bar diagram showing comparison between Group A and B patients with respect to their pre op/post op DRF and tmax In stented Group 1, one patient after removal of DJ stent had no significant improvement in post operative DTPA renal scan done at 3month and on 6 month repeated DTPA renal scan showed obstruction (and deterioration of renal function). On retrograde pyelogram there was stricture distal to UPJ area. On repeat diuretic DTPA renal scan at 3months of endopyelotomy, DRF improved. In both groups the mean difference in the pre op and post op renal function is statistically significant. Average post op DRF was significantly higher than pre op DRF in both the groups. Likewise, average tmax was significantly lower than tmax pre operatively in both the groups. In all patients diuretic renogram at 3 month interval after surgery showed significantly improved function as compared to preoperative function (P value < 0.0001) [Table 4, Figure 1], similar to that of stented pyeloplasty. However there was no significant difference in tmax and DRF between the two groups after pyeloplasty [Table 5, Figure 2]. There was no statistically significant difference between the two groups post operatively with respect to renal function improvement, complications like urinoma formation, urinary tract infection (UTI), prolonged drainage, fever and flank pain. However bothersome irritative LUTS and hematuria were significantly more in group B patients (P < 0.0001 and <0.013 respectively [Table 3, Figure 1]. Table 4 Comparison between pre operative and post operative DRF and t max in both the groups Table 5 Pre and post operative comparison between the two groups showing that both the procedures were equally effective Figure 2Bar diagram showing comparison of complications between the two groups BODY.DISCUSSIONS: Laparoscopic pyeloplasty has evolved worldwide as the first minimally invasive option to match the success of open pyeloplasty, while achieving the added goals of low morbidity, improved cosmesis, short hospital stay and convalescence. Laparoscopic pyeloplasty is continuously evolving with various modifications to simplify the technique to make it a truly minimally invasive approach. There has been an ongoing debate on the merits of intubated versus non-intubated (stentless) repair of UPJO done either by laparoscopic or open technique. Despite high success rate, open pyeloplasty has the disadvantage of a loin wound and consequent increased morbidity and long convalescence. Dismembered pyeloplasty is the popular operation when there is dyskinetic segment or proximal ureter is hooked over lower pole blood vessels.[9] The use of stenting catheters and proximal diversion at the time of pyeloplasty has been the subject of debate. Excellent results have been reported both with and without stents and diversion.[9] Stents and nephrostomy tubes, once considered integral part of pediatric reconstructive surgery are now rarely placed.[9] Most pediatric urologists now believe that routine use of stents and nephrostomy tubes is no longer indicated and is reserved for complicated cases.[10] Laparoscopic pyeloplasty was originally developed by Schuessler et al.[11] in an attempt to duplicate the results of open pyeloplasty while simultaneously decreasing postoperative morbidity. Since then several groups have reported its successful use.[12] Although associated with greater technical complexity and a steeper learning curve, in the hands of the experienced laparoscopic surgeons it has been shown to provide lower patient morbidity, shorter hospitalization, and faster convalescence with the reported success rate matching those of open pyeloplasty (90% or higher).[12] More recently, with advancing laparoscopic skills and the introduction of robotic assisted surgery, many centers have moved to laparoscopic pyeloplasty as first-line therapy.[13] Improved suturing skills and the use of robotic assistance have greatly facilitated laparoscopic dismembered pyeloplasty for primary and secondary repairs.[13] The use of ureteral stents following pyeloplasty ensures adequate drainage, particularly in the presence of postoperative edema.[14] The advantage of stent placement following pyeloplasty include: Lowering the risk of urinoma formation in the event of leak, thereby reducing periureteric fibrosis and restenosis,[15] and providing support and alignment of the fresh suture line.[16] The importance of the stent is highlighted when the anastomosis is not watertight, or after endopyelotomy, allowing healing of the defect while urine is diverted by the stent. However, ureteral stents are not free from risk, and potential problems include irritative urinary symptoms, flank pain and increased risk of infection,[17] migration, encrustation, retained or forgotten fragments,[18] exposure of the upper urinary tract to high pressure during urination, and need for additional procedure for removal. Although, the role of stents has been well described after endopyelotomy, its role after laparoscopic pyeloplasty, where water tight closure can be achieved remains to be evaluated. More recently, there seems to have been a trend towards non stented repairs.[1920] It is of interest to mention the comment from Anderson and Hynes on their technique, "We are convinced that the so called splinting of any anastomosis is not only unnecessary but it is against all the principles of plastic procedure, as it leads to fibrosis at the suture and subsequent stricture. The line of anastomosis should be wide enough and so fashioned as to render any subsequent contraction innocuous". They did not drain the pelvis or use a trans-anastomotic tube/stent.[21] Stent has been found to be associated with stent syndrome (defined as dysuria, frequency, flank pain and hematuria commonly seen with short term placement of ureteral stents), interfere with daily activities and result in reduced quality of life.[922] Stents act as foreign bodies causing compromised vascularity and fibrosis at anastomotic site[922] Joshi et al.[17] in their study on indwelling ureteral stents reported that 78% patients had bothersome urinary symptoms that included storage symptoms, incontinence and hematuria.[12] More than 80% of patients experienced stent related pain affecting daily activities, 32% reported sexual dysfunction, and 58% reported reduced work capacity and negative economic impact. In our study in stented pyeloplasty group, 11 (65%) patients had irritative lower urinary tract symptoms needing treatment. Though stent can be removed under local anesthesia in adults, its removal requires general anesthesia in children. The benefits of stentless pyeloplasty are reduced risk of infection, avoiding the risk of developing stent syndrome, and avoiding need for cystoscopic removal after 1 month.[922] The complications noted in our study in stentless pyeloplasty group were urinoma in one patient (5.5%), two patients (11%) had persistent drainage on 5th pod and were discharged along with drain, similar to the observations made by Bilen CY et al.[20] They concluded that laparoscopic stentless pyeloplasty is as feasible technique as its stented counterpart. Although, it has relatively high prolonged leakage risk, it could be performed without compromising the success rate in experienced hands. In stented group, one patient (5.8%) developed secondary UPJO on follow up after stent removal. Retrograde endopyelotomy was done in him. It seems that stent does not replace the need of good technique of surgery and it cannot prevent re-stenosis if surgical principles are not adhered to. In our study, there was failure of laparoscopic pyeloplasty (5.8%) even when stent was placed. Smith KE et al.[15] in a study in the pediatric population concluded that stent less pyeloplasty is a safe procedure. In a study by Pasquale casale et al. (2010)[23] they concluded that stentless robotic assisted pyeloplasty is a safe and effective option for surgical treatment of UPJO. A larger prospective long-term cohort is needed to confirm the safety and efficacy of the stent less approach. In a similar study conducted by Sethi AS et al. (2010)[24] it was suggested that non stented robotic assisted pyeloplasty is a safe and feasible procedure for the treatment of ureteropelvic junction obstruction. There were no clinically significant differences between the stented and unstented groups in their study. BODY.CONCLUSION: In experienced hand laparoscopic stentless pyeloplasty is as effective method for treating UPJO as its stented counterpart. Although, it has a relatively high initial prolonged urine leakage and prolonged hospital stay, it avoids stent related morbidity, is cost effective, and could be performed without compromising the success rate. However, since the number of patients in our study is limited, more work has to be done to evaluate the safety of stentless pyeloplasty. In our preliminary experience, we find that stent is not a mandatory requirement in pyeloplasty and there is no compromise in the outcome, while avoiding stent related problems at the same time. BODY.CONCLUSION.LIMITATIONS OF THE [STUDY]: Our study has a small size (under powered), and it is non-randomized (selection bias).

TITLE: Evaluating Complementary Therapies for Canine Osteoarthritis—Part II: A Homeopathic Combination Preparation (Zeel ABSTRACT: A homeopathic combination preparation (HCP) for canine osteoarthritic pain was evaluated in a randomized, double-controlled and double-blinded clinical trial. Forty-four dogs with osteoarthritis (OA) that were randomly allocated into one of three groups completed the study. All dogs were fed test products or placebo for 8 weeks. The dogs were evaluated at the clinic four times, with 4-week intervals. Six different variables were assessed: veterinary-assessed mobility, two force plate variables, an owner-evaluated chronic pain index and pain and locomotion visual analogue scales (VASs). Intake of extra non-steroidal anti-inflammatory drugs was also evaluated. A Chi-squared test and a Mann–Whitney test were used to determine significant improvement between groups. When changed into dichotomous responses of 'improved' or 'not improved' three out of the six variables showed a significant difference (P = 0.016, P = 0.008, P = 0.039) in improved dogs per group, between the HCP group and the placebo group. The odds ratios were over one for the same variables. As extent of improvement in the variables from start to end of treatment, the HCP product was significantly more improved in four (P = 0.015, P = 0.028, P = 0.049, P = 0.020) of the six variables, compared with the placebo. Our results indicated that the HCP Zeel® was beneficial in alleviating chronic orthopedic pain in dogs although it was not as effective as carprofen. BODY.INTRODUCTION: We analyzed the use of a low-dilution complex formulation, the homeopathic combination preparation (HCP) Zeel® (1) for the treatment of osteoarthritis (OA) in dogs. OA is today the most common joint disease affecting dogs (2), with canine hip dysplasia (CHD) and elbow dysplasia (ED) being two common variants. In spite of the prevalence and seriousness of canine OA, to date, no research has been published on the effects of homeopathy on OA in dogs (3). In contrast, there is a growing body of research in human medicine on the utilization of homeopathy in general and for OA in particular [for an excellent lecture series on homeopathy and inflammatory diseases, see (4–9) and their references]. Comprehensive meta-analyses of the use of homeopathy on humans have indicated that homeopathy indeed has a positive input on a range of diseases (10,11), whereas others have shown the opposite (12). Two double-blind studies have been conducted specifically on HCP Zeel® in human patients suffering from OA. A therapeutic equivalence was found between Zeel® and the non-steroidal anti-inflammatory drug (NSAID) Diclofenac (13). In another comparative trial on human OA, Zeel comp.® was found to be as good as hyaluronic acid (14). Neither of these trials included placebo groups. It has been recommended for human use (15) and also used on rabbits (16) and horses (17). Our aim was to evaluate the Zeel® HCP as a therapy for canine OA, using a randomized, double-controlled and double-blinded clinical trial (18–22). Based on previous research, we hypothesized a positive effect of HCP. In addition to a placebo group as a negative control, we included an established canine analgesic as a positive control so as to further explore the impact of the treatment. It should be stressed that this is not a classical homeopathic treatment either in the sense of using one drug at a time or in that it is extremely diluted; the product used here was a complex combination and only diluted until molar concentrations of 10−5–10−12 mol/l. BODY.SUBJECTS AND METHODS.DOGS: Inclusion criteria were that dogs have clinical signs and a radiographic diagnosis of OA in either a hip joint or an elbow joint (23). The owner had to have described at least two of the following signs as being chronic or frequent: difficulty in lying down and/or in getting up from a lying position, difficulty in jumping or refusing to jump, difficulty in walking up or down stairs, or definite lameness. Dogs were excluded from the study if they had had prior surgery on the evaluated joint, inadequate clinical symptoms, systemic or infectious disease, neurological deficits, lameness from articular infection, or recent trauma. In this study, 51 dogs were included and 44 concluded the study. Baseline data in Table 1. Table 1.Baseline data for all three groupsPossible confounding factors at baselineCarprofenHCPPlacebon151415hip OA/elbow OA (n)12/312/212/3Sex: male/female7/88/710/5Median age (years),57,56 Min–Max1–91–111–11Median duration of signs (years)>21–21–2Median weight (kg),3827,534 Min–Max31–5622–5418–54Mean ± SD of continuous variables at start of trialVeterinary-assessed mobility indexa5.00 ± 4.616.79 ± 6.465.20 ± 4.26Force plate–PVFb75.92 ± 23.4870.96 ± 22.5878.46 ± 22.23Force plate–impulseb10.92 ± 4.028.64 ± 3.069.72 ± 3.43Chronic pain indexa16.47 ± 6.2115.86 ± 6.2014.87 ± 4.79Pain VAS (cm)a3.55 ± 2.174.24 ± 2.163.70 ± 1.77Locomotion VAS (cm)a4.57 ± 2.034.87 ± 2.264.61 ± 2.12Median, Min–Max of variable at 4 weeks prior to trial (W−4)NSAID doses per monthnone, none to 3–5/weeknone, none to daily/almost dailynone, none to about 1/weekDistribution of possible confounding factors between groups at time W0 (for extra NSAIDs at W−4). HCP, homeopathic combination preparation; NSAID, non-steroidal anti-inflammatory drugs; OA, osteoarthritis; PVF, peak vertical force; VAS, visual analogue scale.aA higher value means more pain.bA lower value means more pain. BODY.SUBJECTS AND METHODS.TEST PRODUCTS AND TREATMENT REGIME: The groups of dogs were medicated as follows: the active treatment group was given the HCP (Zeel® ad us. vet. 5 ml, Biologishce Heilmittel Heel GmbH, Baden-Baden, Germany, see Table 2) at a dose of 1⁄2–1 ampoule/day [<25 kg or >25 kg body weight (BW)] given once daily. The positive control was given a canine NSAID, carprofen (Rimadyl® 50 mg, Pfizer, Helsinki, Finland), at a dose of 2 mg/kg twice daily. The products differed from each other in form; the carprofen and its placebo (lactose) came as a white pill, the HCP and its placebo (an isotonic sodium chloride solution) as an ampoule of clear liquid. Further, all dogs received a slightly green (lactose) capsule, that acted placebo for a parallel study (24). All dogs in the treatment groups were administered three products; one real and two placebos. In the negative control (placebo) group, all products administered were placebos. The products were coded and assembled by a research assistant who was not involved in the rest of the study. For ethical reasons, all owners were given a supplementary package of the same NSAID carprofen (Rimadyl®) in normal packaging at the start of the trial. This could be used as additional pain relief (dose of 1 tablet for a dog of 20–30 kg, 2 tablets for a dog of 31–40 kg and 3 tablets for a dog of 41–60 kg) if the dog was in considerable pain, but this had to be recorded in the questionnaires. Table 2.Content of Zeel® ad us. vet.Zeel® ad us. vet.DilmgCartilago suisD65.0Funiculus umbilicalis suisD65.0Embryon totalis suisD65.0Placenta totalis suisD65.0Solanum dulcamaraD325.0Symphytum officinale e radiceD625.0NadidumD85.0Coenzyme AD85.0Sanguinaria canadensisD47.5Arnica montanaD350.0SulfurD69.0Natrium diethyloxalaceticumD85.0Acidum alpha-liponicumD85.0Toxicodendron quercifolium e summitatibus recD225.0The content of one 5.0 ml ampoule of the low-dilution homeopathic combination preparation (HCP): Zeel®–injection solution. Mg of the different dilutions in the ampoule (Dil = D2–D8 (often also marked as 2×–8×) = diluted 1:10 two to eight times). From the Heel company veterinary guide, Baden-Baden, Germany 1997. BODY.SUBJECTS AND METHODS.STUDY PROTOCOL: Prior to the study, candidates were screened through telephone interviews. Owners were asked not to give the dogs NSAIDs or corticosteroids for at least 30 days and no Na-pentosan polysulfate (Carthrophen®, Biopharm Australia Pty. Ltd., Australia) for at least 90 days prior to the initiation of the study. The first questionnaire was answered at home 4 weeks before the trial started (W−4). The following four questionnaires where answered at the follow ups, at 4-week intervals (W0, W4, W8 and W12). An assistant made the first appointments for the dogs, which were then allocated, in order of arrival, into the groups using a computer-generated random list. Only the location of diagnosed OA (hip or elbow) was stratified in the randomization. At W0 initial clinical, orthopedic and neurological examinations were performed and diagnostic criteria included decreased range of motion and pain on stretching the hip or flexing the elbow. The hip and elbow, as well as all other limb joints, were evaluated for pain, swelling and/or crepitus. Radiographs of the dogs' hips and/or elbows, and other joints if needed, were taken only at time W0. The W0 evaluation and W0 questionnaire was set as baseline, except for extra carprofen where W−4 was used as baseline. The dogs were then given the products orally from W0 to W8. At W12, the dogs had been washed off from all medication for 4 weeks and were evaluated to determine possible long-term effects of the different treatments. All evaluators (veterinarians and owners) and trial personnel were blinded. Owners of the dogs were required to sign informed consent forms. The study protocol was approved by the Ethics Committee of the University of Helsinki. BODY.SUBJECTS AND METHODS.VETERINARY EVALUATION: Two veterinarians subjectively assessed locomotion, jumping and walking stairs at W0, W4, W8 and W12, using 5-point scales where 0 = normal and 4 = dog is totally lame/does not jump at all/does not walk stairs at all (24). The evaluations of the two veterinarians correlated well (R = 0.853, P < 0.01). The three scores assigned by the two veterinarians were summed to form a veterinary-assessed mobility index [2 × 3 × (0 to 4)]. BODY.SUBJECTS AND METHODS.OWNER EVALUATION: The questionnaire consisted of three parts. First, the owners responded to 18 questions about attitude, behavior and locomotion using a descriptive scale from 0 to 4. Of these, 11 questions were combined to form a validated owner-assessed chronic pain index (25). The second part contained two 10-cm visual analogue scales (VASs): one for pain and the other for locomotion. The end of the lines to the left (0 cm) represented no pain or difficulties in locomotion and to the right (10 cm), the worst possible pain or the most severe difficulties in locomotion. The third part consisted of five questions about possible adverse reactions to treatment, including change in appetite, vomiting, diarrhoea, atopic skin reactions and the need for extra carprofen. The question about extra analgesics used the following scale: during the last 4 weeks, additional carprofen was given; 1 = not at all, 2 = 1–2 times, 3 = about once a week, 4 = about 3–5 times a week and 5 = daily/almost daily. BODY.SUBJECTS AND METHODS.OBJECTIVE EVALUATION OF GAIT: Gait was analyzed by force plate gait analysis (Kistler force plate Type 9286, Kistler Instrumente AG Winterhur, CH-8408, Switzerland), which objectively assesses weight bearing of limbs. The signal from the plate was processed and stored using a computer-based software program, and velocities and acceleration were determined by three photoelectric cells placed 1 m apart and a start-interrupt timer system (Aquire 6.0, Sharon Software Inc., DeWitt, USA). The dogs were trotted from left to right by their owners. The speed had to be in the same range (±0.5 m/s) for the dog each time the test was performed (at W0, W4, W8 and W12). The acceleration was <0.5 m/s/s and there had to be contact with the plate first by the forelimb and shortly after with the hind limb of the same side for the evaluation to be valid. The test was repeated until sufficient valid results were obtained for both left and right limbs. Three valid measurements for each side and for each visit were then chosen by a blinded assistant, who was not otherwise participating in the study, according to speed, acceleration and no interferences, such as gait abnormalities or extra body movements. The mean of these three measurements was used for analysis. The ground reaction forces were normalized for each dog's BW and mean peak vertical force (PVF) and mean vertical impulse were used as variables. Only measurements from the more severely affected limb at time W0 were used in the analysis. BODY.SUBJECTS AND METHODS.BLOOD SAMPLES: Blood samples were collected from the dogs at each visit. Kidney, liver and protein values [blood urea nitrogen (BUN), creatinine, serum alanine aminotransferase (ALAT), alkaline phosphatase (AFOS), total proteins and albumin] were analyzed. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSIS: The number of dogs needed in each group was calculated with a two-tailed test. The sample size was sufficiently large to detect a 47% difference in treatment outcome (effective versus non-effective) with a statistical power of 0.8 and allowing for a 5% alpha error. For the dogs that had used extra carprofen more than three times per week at W8, we changed all their variables values at evaluation W8 into the most negative value measured at that time, separately for each variable. This was done to counteract the effect of the taken NSAID and enabled us to use the whole data in the first statistical analyses. For calculating the percentage of dogs per group that improved between baseline and W8 and the odds ratio, the results of each variable were converted into dichotomous responses of 'improved' and 'not improved'. Dogs that deteriorated and dogs with no change in the evaluated variable were considered 'not improved'. The difference between the treatment groups and the placebo where calculated using a Chi-Squared test. The odds ratio was calculated using the common Mantel Haenszel odds ratio estimate and the confidence interval (CI) was set to 95%. An odds ratio over 1.0 indicated a beneficial effect of the test treatments. The change from baseline to W8 was also calculated for each variable. The difference between the GLM and placebo group was analyzed using the Mann–Whitney test. The change from W0 to W8 in the force plate variables was similar for the front and hind legs, although the values were different. Therefore, force plate data of all four legs were analyzed together. Dogs that did not manage to get force plate results due to major lameness were considered 'not improved' in the dichotomous analyses and were excluded from the median change data. A correlation test was used to evaluate the association between the assessments of the two veterinarians. Statistical significance was set at P < 0.05. Statistical tests were preformed using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA). BODY.RESULTS.BASELINE: Seven of these 51 dogs were excluded from the material at some time during the study because they did not meet the medical inclusion criteria (operation on the affected hip joint, a transverse vertebra (n = 2), a cruciate ligament injury, degenerative myelopathy, polyarthritis of the phalanges and castration just prior to the third visit). The baseline differences between groups were not statistically significant (Table 1). BODY.RESULTS.DICHOTOMOUS RESPONSES: All major data are presented in Table 3. Four dogs (all in the placebo group) used extra carprofen more than three times per week at W8 and two dogs were not able to trot over the force plate. When the data of all variables were changed into dichotomous responses of either 'improved' or 'not improved', three of the variables [veterinary-assessed mobility index (P = 0.018), force plate PVF (P = 0.006) and pain VAS (P = 0.043)] indicated significantly more improved dogs in the HCP group compared with the placebo group. The odds ratio for the veterinary-assessed mobility index was 6.88 (95% CI 1.35–35.06) indicating that a dog that had received the HCP was 6.88 times more likely to have a positive response than a dog that had received the placebo. The odds ratio for the force plate PVF was 9.17 (95% CI 1.63–51.43), for the force plate impulse 3.24 (95% CI 0.69–15.20), for the owner-assessed chronic pain index 2.00 (95% CI 0.46–8.78), for the pain VAS 5.33 (95% CI 1.02–27.76) and for the locomotion VAS 3.67 (95% CI 0.77–17.43). Table 3.Percentage of improved dogs and median (+range) of improvement from W0 to W8, for evaluated variables, per groupcarprofen (n = 15)HCP (n = 14)Placebo (n = 15)Improved P=Improvement Median (range), P=Improved P=Improvement Median (range), P=Improved (%)Improvement Median (range)Veterinary mobility index66.7% 0.0313 (0 to –8) 0.00171.4% 0.0181.5 (−5 to 7) 0.01526.7−3 (−14 to 3)Force plate PVF66.7% 0.0313.2 (−8.2 to 1.8) 0.07978.6% 0.0062.3 (−3.4 to 10.2) 0.02826.7−0.9 (−33.6 to 10)Force plate Impulse80.0% 0.0110.4 (−0.5 to 1.3) 0.00964.3% 0.1010.2 (−1.3 to 1.3) 0.09333.3−0.0 (−3.3 to 0.8)Chronic pain index80.0% 0.0289 (−9 to 19) <0.00157.1% 0.3642 (−6 to 9) 0.04940.0−3 (−25 to 8)Pain VAS85.7% 0.0011.4 (−6 to 8.4) <0.00157.1% 0.0430.2 (−3.5 to 4.9) 0.02020.0−1.7 (−7 to 3.2)Locomotion VAS85.7% 0.0023.1 (−1.9 to 6.2) 0.00157.1% 0.1020.7 (−5 to 4.8) 0.20526.7−1 (−6.6 to 5)For each treatment group: First column: Percentage of dogs in the group that improved. Second column: Median (with range) of change from W0 to W8 ((+) = improvement, (−) = deterioration) in evaluated variables for the carprofen-, HCP- and placebo groups. P = Difference in improvement between treatment groups and placebo (the force plate values here do not include two dogs for whom no results were obtained). (n, number of patients per group; HCP, homeopathic combination product; Improved, percentage of dogs that improved per group; PVF, peak vertical force; VAS, visual analogue scale). BODY.RESULTS.MEDIANS (+ RANGE) OF THE CHANGE FROM : The medians and ranges of the changes from W0 to W8 in each variable are shown in Table 3. Two dogs from the placebo group are excluded from the two force plate evaluations. All variables showed a similar trend of improvement, with carprofen being most efficient, placebo least, and HCP being between these two. In the negative control group (placebo), all change medians were on or below the 'no change' line, indicating deterioration. There was a significant difference in four of six variables in the extent of improvement between the HCP and the placebo group; the veterinary-assessed mobility index (P = 0.015), the PVF (P = 0.028), the chronic pain index (P = 0.049) and the pain VAS (P = 0.020). Data from W4 are not shown, as all values were between the values of W0 and W8. Statistical analyses were not performed on the results from W12, as so many dogs were using extra carprofen at this time. BODY.RESULTS.EXTRA CARPROFEN: The use of the additional NSAIDs varied as follows: at W−4, 14% of the carprofen group, 28% of the HCP group and 8% of the placebo group used extra NSAIDs once a week or more often. At W8, the respective values were 0, 14 and 27%, and at the follow up at W12, 33, 21 and 29%. Only the difference between the carprofen group and the placebo group was significant (P = 0.012) at W8. BODY.RESULTS.COMPLICATIONS AND SIDE-EFFECTS: All of the altered blood values and the clinical side-effects were considered mild or within normal range. No difference between groups was seen. One dog in the placebo group was euthanized due to severe pain during the follow-up period. BODY.DISCUSSION: We examined the effect of the HCP Zeel® on OA in dogs. Compared with the placebo group, the HCP treatment gave significantly better results both as number of cases that improved per group and as efficacy or extent of improvement. Thus, the results of what apparently is the first randomized, controlled, double-blinded study on the use of a HCP on OA in dogs indicate that the treatment indeed tends to have significant positive impact. The results add to previous positive results on the use of this HCP on OA in studies on humans (13,14), rabbits (16) and horses (17), suggesting that the treatment may be beneficial also for other species. To be noted is that, compared with the placebo group, the group of dogs being treated with carprofen improved even more. This is in accordance with previous studies that have demonstrated a treatment effect of 56–81% for carprofen and only 23–38% for placebo (26,27). But, as carprofen and other NSAIDs can potentially have severe side-effects such as hepatic disease, renal toxicosis and irritation of the gastrointestinal tract, ultimately leading to hemorrhagic ulcers and even death (28,29), it is of utmost importance that we continue doing clinical research on all alternative treatments for OA. Other data also support our results: it is generally accepted that seasonal differences influence OA, with patients being worse in cold, damp and unstable weather (in our study = W4–8) and better in dry (W0) and warm (W12) weather (30,31). In the placebo group, a trend was detectable; in the change of means between W0 and W8, we could see deterioration in nearly all variables (negative values in Table 3) and use of more extra NSAID; and improving at follow up (W12) as the weather warmed up. In the two treatment groups an opposite pattern was detected. They demonstrated a positive effect during the test period (W0–8) but worsened at the follow up (W12), which can be speculated to indicate the positive effect of the drugs and a worsening after discontinuation at W8. The increase in the intake of extra NSAIDs at W12 in the carprofen group further supports this. However, although significant differences between groups were found in this study, it suffers from some limitations that at the same time point to possibilities for future research. Future studies should ideally ascertain that the treatment group, as well as the positive and negative control groups, is large enough to detect difference even when data are lost. Having two dogs in the placebo group that were not able to perform the runs over the force plate and were therefore excluded from the data, slightly ameliorated the median values for this group, perhaps resulting in less significance in the treated groups. Second, the location (hip or elbow), the multitude (uni or bilateral) and severity of OA varied across the dogs, leading to assessment problems e.g. using the force plate due to transferring the weight to other legs. Heterogeneity also leads to high variance; a problem in a small study like ours, with a relatively moderate sample size, and subsequently results in lower significance. Third, the dose and mode of application of HCP in our study was 1⁄2−1 ampoule/day (depending on BW) in one oral dose. However, the dosing and presentation of the Zeel® product has since changed; it is now given as a tablet and at 300% of the dose used in this study. Furthermore, for optimal results, Zeel® is often combined with other homeopathic products (such as Traumeel®) in clinical practice (1), but combining products was not suitable for our protocol. Future research might want to assess effect using different doses, methods of application or combinations. Finally, as very little research on chronic pain assessment for dogs still is available, it will make future research easier when we have more validated scales to use. Measuring ground reaction forces using a force plate (32–35) were the most objective measurement used, but they are not so reliable to use on a very variable cohort. Validated owner-based scales are now also becoming available and will maybe be used more in the future (25,36–38). Thus we must conclude that the results are intriguing, as all positive results in homeopathic medicine. As the study was rigorously carried out as a randomized, double-controlled, double-blind trial and as the placebo effect anyway should be smaller when evaluating dogs and not humans, we can elaborate on a possible working mechanism for this product. It seems plausible that the treatment effects reported previously could have been seen in this trial as well: in a randomized, sham-controlled placebo study on rabbits with experimentally induced knee OA, a significant difference in gross morphology and in histopathological score was found between the joints treated with HCP Zeel comp.® and those untreated (16). Also, cartilage slices incubated for 6 days in a medium containing Zeel® showed better preservation of structures than controls in an in vitro study using methods of interference polarization microscopy and x-ray difractometry for analysis (39). An in vitro study (40) demonstrated that two of the ingredients in our test product were able to inhibit leucocyte elastase activity; Arnica montana D4 up to 70% and Rhus toxicodendron (same as Toxicodendron quercifolium in Table 2.) D3 up to 77% (elastase is an enzyme that is released during inflammatory reactions and attacks the articular cartilage which is rich in proteoglycans). In a second study (41), Rhus toxicodendron at D1 and D2 potencies, as well as 10 other plant extracts, was shown to inhibit cell growth of human cutaneous F54 fibroblasts. Recently, a reconstituted Zeel comp. N® combination (as well as its constituent mother tinctures) were reported to show distinct inhibitory effects on the production of Leukotriene B4 by 5-lipoxygenase (5-LOX) and on the synthesis of prostaglandin PGE2 by COX-1 and 2 enzymes. Together with the other inhibiting activities, this dual inhibition of both LOX- and COX-metabolic pathways may offer an explanation for the reported clinical efficacy and favorable gastrointestinal tolerability of the original Zeel comp. N® remedy (42) and of the veterinarian product we used in this study, that was very similar. In conclusion, the results of this relatively small study of dogs with moderate to severe OA showed that dogs receiving the HCP Zeel® for 8 weeks had significantly less pain than their placebo peers. Homeopathy as a treatment is often seen as controversial, so this positive treatment result for dogs for this low-dilution HCP should be of major interest for human OA researchers and clinicians, alike. As chronic pain due to OA is a major reason for decreased quality of life nowadays, both for humans and dogs, we should proceed with more studies in this direction. BODY.FUNDING: The Finnish Foundation of Veterinary Research, Helvi Knuuttila Foundation.

TITLE: Laparoscopic Versus Open Appendectomy: A Comparison of Primary Outcome Measures ABSTRACT.BACKGROUND/AIM:: The aim of the study was to compare laparoscopic and open appendectomy (OA) in terms of primary outcome measures. Study design: A randomized controlled trial. Place and duration of the study: Khyber Teaching Hospital, Peshawar, Pakistan, February 2008 to December 2009. ABSTRACT.PATIENTS AND METHODS:: A total of 160 patients were divided into two groups, A and B. Group A patients were subjected to laparoscopic appendectomy (LA), whereas Group B patients were subjected to OA. Data regarding age, gender, and primary outcome measures, such as hospital stay, operative duration, and postoperative complication, were recorded and analyzed. Percentages were calculated for categorical data, whereas numerical data were represented as mean ± SD. Chi-square test and t test were used to compare categorical and numerical variables, respectively. Probability ≤ 0.05 (P ≤ 0.05) was considered significant. ABSTRACT.RESULTS:: After randomization, 72 patients in group A and 75 patients in group B were analyzed. The mean age of patients in groups A and B was 23.09 ± 8.51 and 23.12 ± 10.42 years, respectively, (P = 0.981). The mean hospital stay was 1.52 ± 0.76 days in group A and 1.70 ± 1.06 days in group B (P = 0.294). The mean operative duration in group A and B were 47.54 ± 12.82 min and 31.36 ± 11.43 min, respectively (P < 0.001). Pain (overall level) was significantly less in group A compared with group B (P = 0.004). The two groups were comparable in terms of other postoperative complications, such as hematoma (P = 0.87), paralytic ileus (P = 0.086), urinary retention (P = 0.504), and wound infection (P = 0.134). ABSTRACT.CONCLUSION:: LA is an equivalent procedure and not superior to OA in terms of primary outcome measures. BODY: Appendicitis is the most common cause of surgical abdomen in all age groups.[12] Approximately 7%–10% of the general population develops acute appendicitis with the maximal incidence being in the second and third decades of life.[34] Since 1894, the first description of open appendectomy (OA) by McBurney, it was the gold standard for treating patients with acute appendicitis for more than a century.[5] In contrast, the first laparoscopic appendectomy (LA) was performed in 1983 by Semm, a German gynecologist.[6] LA, unlike laparoscopic cholecystectomy, has not gained much popularity since its introduction.[7] Despite numerous randomized controlled trials published so far, comparing OA and LA, the relative advantages of the two procedures are still to be established.[8–11] Furthermore, it is argued that the advantages of LA over OA, such as short hospital stay, less analgesia requirement, rapid postoperative recovery, and better cosmetic outcome, are not significant.[12] In the developing countries only a few studies have been conducted comparing the two modalities in the treatment of acute appendicitis.[13] There is thus a need to carry out further trials. Our hospital is a government hospital and majority of the patients attending it belong to lower socioeconomic group. LA is frequently practiced in our department. With not much regional studies comparing the two procedures, we endeavored to analyze and compare the LA and OA in terms of operative and postoperative outcomes. BODY.PATIENTS AND METHODS: This study was conducted at Khyber Teaching Hospital, Peshawar, from February 2008 to December 2009, as part of a single-center randomized clinical trial. The objective of the study was to compare LA and OA in terms of primary outcome measures, such as operative duration, length of hospital stay, and postoperative complications, as these are the ultimate determining factors in deciding between LA and OA. All other outcome measures, such as cost of the treatment of the two techniques, are regarded as secondary, and were not considered. Approval for the study was obtained from the Ethics Committee of the hospital. In this study, 160 patients, presenting to outpatients department (OPD) with clinical diagnosis of acute appendicitis were included. The diagnosis was made clinically with history (right iliac fossa [RIF] or periumbilical pain shifting to RIF, nausea/vomiting), physical examination (tenderness or guarding in RIF), and TLC (white blood cell (WBC) count > 10,000/dL). The clinical and laboratory data were used to calculate the Alvarado score of patients with MANTRELS (migratory pain, anorexia, nausea/vomiting, tenderness in RIF, rebound tenderness, elevated temperature, leukocytosis, and shift of WBCs). Any patient with a score of 7 or more was operated on as having acute appendicitis. Appendicitis was confirmed on histopathology, which also showed different grades of inflammation. The patients were selected through consecutive nonprobability sampling and were randomly allocated to two groups, A and B, using lottery method. Group A patients were subjected to LA and group B patients to OA. The inclusion criteria were patients with clinical diagnosis of acute appendicitis, age 12–60 years, American Society of Anesthesiologists Class I, informed consent, and willing to abide by the follow-up protocol. Patients with previous abdominal surgery, large ventral hernias, mass RIF, and history of symptoms for more than 5 days were excluded from the study. Patients with converted LA were included in the LA group (intention to treat principle). All the patients included in the study were admitted a day before surgery, as part of the routine protocol of our unit. History, physical examination and investigations, such as full blood count, urine routine examination, ultrasonography (US) of abdomen and pelvis, were performed for diagnosis. Once a diagnosis of acute appendicitis was made, investigations for anesthesia fitness, such as blood urea and sugar, serum electrolytes, chest radiography, Hepatitis B and C screening, and electrocardiography, were also performed. The patients were explained the risks and benefits of the two procedures and an informed consent was obtained. The patients were operated by a single consultant surgeon, under general anesthesia, with sufficient capability of performing the two procedures (LA and OA). The patients were given, in both the groups, a prophylactic dose of third-generation cephalosporin and metronidazole at induction as part of the protocol, whereas two doses of the same were repeated postoperatively at 8 and 16 h. LA was performed through a 3-port technique with carbon dioxide used for the creation of pneumoperitoneum through a 5-mm infraumbilical port up to a pressure of 12 mmHg. The other two ports were placed in the lower abdomen according to the individual surgeon's choice. After identification of the appendix, the mesoappendix was ligated, with Vicryl 1 after creation of a window in its base, and cut. The base of the appendix was crushed and ligated using Vicryl 1 endoloop. The appendiceal specimen was retrieved through a 10-mm infraumbilical port. Endodiathermy was used for hemostasis. OA was performed through standard Lanz incision. After the incision, peritoneum was accessed and opened to deliver the appendix, which was removed in the usual manner. Skin incision in both the procedures was closed with subcuticular prolene 2/0 suture. The patients were not given oral feed until they were fully recovered from anesthesia and had their bowel sounds returned when clear fluids were started. Soft diet followed by regular diet was introduced when the patients tolerated the liquid diet and had passed flatus. Patients were discharged once they were able to take regular diet, afebrile, and had good pain control. A standardized questionnaire was used to record the data. All the operative details were recorded. The operative time (minutes) for both the procedures was counted from the skin incision to the last skin stitch applied. Pain was measured qualitatively (subjectively) using visual analog scale. The length of hospital stay was determined as the number of nights spent at the hospital postoperatively. Postoperative complications were recorded in the proforma during the hospital stay and till 1 month (follow-up visit fortnightly in OPD). Wound infection was defined as redness or purulent or seropurulent discharge from the incision site observed within 30 days postoperatively. Seroma was defined as localized swelling without redness with ooze of clear fluid. Paralytic ileus was defined as failure of bowel sounds to return within 12 h postoperatively. Confounding variables were controlled through strictly following the exclusion criteria. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: The data were analyzed using statistical package for social sciences (SPSS, version 11.0; Chicago, IL, USA). Continuous variables, such as age, hospital stay, and operative duration, were presented as mean ± SD, while categorical variables, such as gender and postoperative complication, were expressed with frequency and percentages using 95% confidence interval. Student's t test was used to compare the means of continuous variables, while categorical variables were compared using Chi-square or Fisher's exact test, as appropriate. Probability equal to or less than 0.05 (P ≤ 0.05) was considered significant. BODY.RESULTS: A total of 160 patients were included in the study, 80 each in LA and OA groups, respectively. Eight patients from group A and 5 patients from group B were lost to follow-up because they did not abide by the protocol. Therefore, a total of 72 patients in group A and 75 patients in group B were analyzed, as shown in Figure 1. The mean age of the patients was 23.09 ± 8.51 years in group A and 23.12 ± 10.42 years in group B. In group A there were 40 males and 32 females with male to female ratio (1.2:1), whereas in group B there were 44 males and 31 females (male to female ratio 1.4:1) as shown in Table 1. The two groups were comparable in terms of body mass index (BMI) and WBC count, as depicted in Table 1. Figure 1Patient allocation Table 1 Demographic features (n=147) Negative appendectomy rate, as confirmed on histopathology, was 4 (5.3%) and 2 (2.6%) in LA and OA group, respectively. The final diagnosis in these patients being, Meckel's diverticulitis (n=1), ureteric colic (n=2), and ruptured ovarian cyst (n=1) in the LA group, and ureteric colic (n=2) in the OA group. Peroperatively, appendix in LA was phlegmonous 6.6% (n=5), perforated 9.3% (n=7), gangrenous 1.3% (n=1), and acutely inflamed in 77.3% (n=58) patients. The corresponding figures in the OA group were 2.6% (n=2), 10.6% (n=8), 0% (n=0), and 84% (n=63), respectively. One patient in the LA group was converted to OA due to mass formation. As shown in Table 2, the mean postoperative hospital stay was 1.52 ± 0.76 days in LA compared with 1.70 ± 1.06 days in OA, which was statistically not significant (P = 0.287). The mean operative duration was 48.26 ± 12.82 min in LA and 31.36 ± 11.43 min in OA (P < 0.001). Table 2 Comparison of primary outcome measures Postoperative complications were compared and they did not reveal any statistically significant difference between the two modalities as shown in Table 2. There was, however, an increased proportion of patients experiencing some degree of pain in OA vs LA group, 90.6% and 72.2%, respectively (P = 0.004). Pain was qualitatively stratified into mild, moderate, and severe, according to visual analog scale, with decreased incidence of severe pain in the OA group compared with the LA group, 24% vs 38% patients, respectively, which proved statistically significant (P = 0.023), as shown in Table 2. BODY.DISCUSSION: In recent years, a great majority of surgeons have embarked on LA, which is mainly attributable to excellent results gained in laparoscopic cholecystectomy. However, OA is not lagging much behind LA and has flourished as a minimally invasive procedure due to the shorter and cosmetically acceptable incision. The advantages of LA over OA are thus, if any, only marginal and difficult to confirm.[14] The clinical presentation of acute appendicitis can overlap significantly with other clinical conditions with only 50%–60% of patients having the classical presentation.[15] In our study, a majority of the patients with negative appendectomies, in both the groups, were females, which is corroborated by another study.[15] The higher rate of misdiagnosis in females may be due to the gynecologic pelvic diseases and female functional abnormalities.[4] Therefore, in patients with atypical presentation, diagnostic laparoscopy instead of US, computed tomography, or serial WBC counts, should be considered early due to its easy availability, nil mortality, excellent diagnostic yield, and low morbidity.[16] This is one aspect where laparoscopic approach outshines the open approach. This study shows that there was not a significant difference in the hospital stay between the two modalities of treatments. This is in corroboration with other studies.[1214] Studies published in the early 1990s showed significantly shorter hospital stay in favor of LA.[17] Similarly, a local study also demonstrated a significantly shorter hospital stay in the LA group.[18] Milewczyk et al. demonstrated a longer hospital stay in LA vs OA group.[19] The question whether LA is associated with a shorter hospital stay has been a matter of controversy and the current literature yields conflicting data.[20] The difference in the hospital stay between the two procedures may be due to the difference in the health care system rather than the difference in the two procedures.[21] This appears to be one area where OA has fast caught up with LA. Operative duration remains a much talked about aspect among experts whenever LA and OA are compared.[21] This study shows that the operative duration was significantly longer in LA compared with OA, which is consistent with other studies.[38121822] Peiser et al. reported no significant difference in the operative duration comparing the two procedures.[14] All the procedures in our study were performed by a consultant surgeon with sufficient minimal invasive surgery load and the difference can be explained by the fact that LA involves the additional steps of gas insufflation, trocar entry, and diagnostic laparoscopy. According to Johnson, a new procedure must have, in addition to other benefits, such as less operative time and ease in performance, the advantage of safety (less complications and morbidity).[23] In our study, the overall complication rate was significantly lower in LA compared with OA. This is in agreement with other studies.[24] Guller et al. did not find any statistically significant difference in the overall morbidity between the two procedures.[20] Wound infections may not be a serious complication as such but can cause inconvenience to the patient, impacting convalescence and quality of life.[8] In this study, fewer patients developed wound infection in the LA group, but the difference was not statistically significant. Similar observations are reported from other national and international studies.[381214] In contrast to our results, a prospective study showed that a significant proportion of patients, undergoing OA, developed wound infection as opposed to LA.[24] In OA, direct delivery of the appendix through the wound may risk contamination, whereas utilization of laparoscopic port or bag for appendix retrieval may favor reduced frequency of wound infection in LA.[25] In contrast, intraabdominal abscess formation is almost 3 times more common in LA compared with the open counterpart.[12] This can be attributed to the fact that CO2 insufflation in LA may facilitate spreading of microorganisms in the peritoneal cavity, especially in perforated appendicitis.[24] In the present study, however, no patient developed intraabdominal abscess. In this study, 6.9% patients in the LA group and 1.3% patients in the OA group developed paralytic ileus, which did not reach statistical significance. This finding is mirrored in other studies.[2426] Some studies have reported statistically significant postoperative ileus in LA arm compared with OA.[325] Postoperative ileus along with pain and wound infection may hamper the mobility of the patient, in turn prolonging the hospital stay and increasing the cost of treatment. Reduced postoperative ileus and wound infection can be beneficial in so many ways: less pain, early oral intake, early mobilization, all resulting ultimately in reduced hospital stay.[25] In our study, we found that pain (overall level as measured on visual analog scale) was significantly less in LA, which is in accordance with other studies.[1221] In contradistinction with our results, other studies failed to show significant difference in pain perception between the two treatment options.[38] Smaller incisions and minimal tissue handling may be the reason for decreased postoperative pain perception in LA. It would be befitting to acknowledge the limitations of our study. First, we did not include residents as surgeons in the study, although appendectomy is commonly performed by residents in our setup. Second, our follow-up was limited to 1 month postoperatively. Our aim was to look for early postoperative complications postdischarge. Third, this study was not blinded (treatment allocation and clinical outcome assessment not being blinded). BODY.CONCLUSION: In our study, postoperative pain was significantly less in the LA group. In contrast, operative duration was longer in LA, which touched statistical significance. There was, however, no difference in other primary outcome measures. In conclusion, LA is an equivalent procedure and not superior to OA in terms of primary outcome measures, as the benefit gained through reduced postoperative pain was balanced by significantly longer operative duration. Based on the results of this study, firstly, we would like to recommend further trials comparing LA and OA not only for primary outcome measures assessment, but also for secondary outcome measures. Secondly, where the facilities and expertise are available for LA, the choice for treating acute appendicitis with any of the two modalities should finally be decided by the patient or the operating surgeon.

TITLE: Effects of tadalafil once daily or on demand versus placebo on time to recovery of erectile function in patients after bilateral nerve-sparing radical prostatectomy ABSTRACT.PURPOSE: We report time to erectile function (EF)-recovery data from a multicenter, randomized, double-blind, double-dummy, placebo-controlled trial evaluating tadalafil started after bilateral nerve-sparing radical prostatectomy (nsRP). ABSTRACT.METHODS: Patients ≤68 years were randomized post-nsRP 1:1:1 to 9-month double-blind treatment (DBT) with tadalafil 5 mg once daily (OaD), 20 mg tadalafil on demand ("pro-re-nata"; PRN), or placebo, followed by 6-week drug-free washout (DFW) and 3-month open-label OaD treatment. Secondary outcome measures included Kaplan–Meier estimates of time to EF-recovery (IIEF-EF ≥ 22) during DBT (Cox proportional hazard model adjusting for treatment, age, and country). ABSTRACT.RESULTS: A total of 423 patients were randomized to tadalafil OaD (N = 139), PRN (N = 143), and placebo (N = 141); 114/122/155 completed DBT. The proportion of patients achieving IIEF-EF ≥22 at some point during DBT with OaD, PRN, and placebo was 29.5, 23.9, and 18.4 %, respectively. DBT was too short to achieve EF-recovery (IIEF-EF ≥ 22) in >50 % of patients; median time to EF-recovery was non-estimable. Time for 25 % of patients to achieve EF-recovery (95 % CI) was 5.8 (4.9, 9.2) months for OaD versus 9.0 (5.5, 9.2) and 9.3 (9.0, 9.9) months for PRN and placebo, respectively. Showing a significant overall treatment effect (p = 0.038), the probability for EF-recovery was significantly higher for OaD versus placebo [hazard ratio (HR); 95 % CI 1.9; 1.2, 3.1; p = 0.011], but not for PRN versus placebo (p = 0.140). Of 57 OaD patients (41.0 %) with ED improved (by ≥1 IIEF-EF severity grade) at the end of DBT, 16 (28.1 % of 57) maintained this improvement through DFW and 27 (47.4 %) declined but maintained improvement from baseline after DFW. ABSTRACT.CONCLUSIONS: Data suggest that the use of tadalafil OaD can significantly shorten the time to EF-recovery post-nsRP compared with placebo. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00345-014-1377-3) contains supplementary material, which is available to authorized users. BODY.INTRODUCTION: Erectile dysfunction (ED) can be a relatively common sequela after radical prostatectomy for localized prostate cancer [1–3], despite the use of nerve-sparing techniques (nsRP). Many men may not recover erectile function (EF) for more than 18 months post-nsRP [4, 5]. Rarely, time to EF-recovery can extend well beyond 2 years [6]. The rate of and time to EF-recovery post-nsRP can vary widely and may be influenced by a number of factors, including patient age, type of surgery, and treatment during the recovery phase [5–7]. Phosphodiesterase type 5 (PDE5)-inhibitors are generally well-tolerated and effective in the treatment of ED post-nsRP [1, 2, 8]. However, they are less effective in the post-nsRP population when compared to the general population, and the optimal time point for starting PDE5-inhibitor treatment is still undetermined [9]. To date, four randomized clinical trials (RCTs) have evaluated the impact of the early use of short-acting PDE5-inhibitors on EF-recovery in men post-nsRP. Sildenafil, vardenafil, and avanafil have all been shown to improve drug-assisted EF when given on demand ("pro-re-nata," PRN) [10–12]. However, EF-recovery up to 1 year post-nsRP did not differ between sildenafil given once daily (OaD) or PRN [13]. For the long-acting PDE5-inhibitor tadalafil, an initial retrospective study in 92 patients showed that tadalafil OaD started early after robot-assisted laparoscopic nsRP was well tolerated and significantly improved EF compared to patients without PDE5-inhibitor treatment [14]. Montorsi et al. [15] have published a randomized, placebo-controlled trial evaluating the early use of tadalafil, given OaD or PRN, on both drug-assisted EF after 9 months of double-blind treatment (DBT) with tadalafil OaD or PRN and on unassisted EF (without PDE5-inhibitor support) after 6 weeks of drug-free washout (DFW) in men who developed ED post-nsRP. Tadalafil OaD improved drug-assisted EF-recovery, as measured by the proportion of patients achieving an International Index of Erectile Function-Erectile Function domain score (IIEF-EF) ≥22 at the end of DBT, while unassisted EF-recovery after DFW was not improved by tadalafil OaD or PRN [15]. This paper specifically addresses the effects of tadalafil OaD and PRN treatment on the time to recovery of EF (IIEF-EF ≥ 22) during the DBT period of this trial and the maintenance of treatment response after DFW. BODY.MATERIALS AND METHODS.PATIENTS: Adult men aged <68 years at the time of nsRP with normal preoperative EF who underwent nsRP for organ-confined, non-metastatic prostate cancer (Gleason score ≤7, prostate specific antigen <10 ng/mL) were enrolled between November 2009 and August 2011 in 50 centers from nine European countries and Canada (NCT01026818). Post-surgical inclusion criteria included the development of ED, as measured by a patient-reported Residual Erection Function (REF) score of ≤3 ("penis is hard enough for penetration but not completely hard"). This criterion was used because of the limited validity of IIEF-EF domain scores for direct EF assessment post-nsRP [15, 16]. Detailed eligibility criteria have been published [16]. BODY.MATERIALS AND METHODS.TRIAL DESIGN: This multicenter, Phase IV, randomized, double-blind, 3-arm, placebo-controlled parallel-group trial consisted of the following periods, as previously described (Supplementary Figure S1) [15]: screening (including nsRP surgery), 9-month randomized, double-blind, double-dummy treatment with 5 mg tadalafil OaD, 20 mg tadalafil PRN, or placebo (DBT); 6-week DFW; and 3-month open-label treatment with 5 mg/day tadalafil OaD (OLT, all patients). BODY.MATERIALS AND METHODS.OUTCOME MEASURES.IIEF-EF SCORES: The primary objective was to evaluate the efficacy of tadalafil OaD and tadalafil PRN, compared with placebo, in improving unassisted EF (EF after 6 weeks of DFW), as measured by the proportion of patients achieving an IIEF-EF score ≥22 at the end of DFW (primary outcome) [15]. An IIEF-EF ≥22 was required at screening (after cancer diagnosis, ≤6 weeks pre-nsRP). This cutoff was considered appropriate because many men with newly diagnosed prostate cancer claim to have unimpaired EF, but have IIEF-EF scores of 22–25 (mild ED) [15, 17]. Time to EF-recovery (additional secondary analysis pre-specified in the statistical analysis plan, finalized and approved before database lock) was defined as the time from baseline to reach an IIEF-EF ≥22 during DBT. BODY.MATERIALS AND METHODS.OUTCOME MEASURES.ED SEVERITY: IIEF-EF scores were categorized into the following ED severity categories: severe (0–10), moderate (11–16), mild (17–25), and normal (26–30) [18]. ED severity was assessed at baseline, end of DBT, and end of DFW. Improvement was defined as an IIEF-EF score of ≥1 category higher than baseline (or maintaining normal EF) at the end of DBT. Maintenance of treatment response, assessed for patients who improved ≥1 category after DBT, was defined as either maintaining this improved category until the end of DFW or declining after DBT but still maintaining a higher category at the end of DFW than at baseline. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The planned sample size of 412 patients was based on the primary outcome (proportion of patients achieving IIEF-EF ≥22) [15]. All analyses were based on the intent-to-treat (ITT) population, including all randomized patients with baseline data and at least one post-baseline visit. Pre-specified treatment group comparisons were tadalafil OaD versus placebo and tadalafil PRN versus placebo. The Kaplan–Meier product-limit method was used to estimate rates for the time to EF-recovery (IIEF-EF ≥ 22) including 95 % confidence intervals (CI). Only patients with IIEF-EF <22 at screening were included; patients not reaching IIEF-EF ≥22 were censored at the end of DBT. Hazard ratios (HRs) and p values were derived from a Cox proportional hazard model adjusting for treatment, age (<61 years vs. 61–68 years), and country. IIEF-EF score changes from baseline were analyzed using a mixed model for repeated measures (MMRM) analysis, assuming an unstructured covariance structure and including visit, treatment, treatment-by-visit interaction, country, age group, and baseline as fixed effects, and patient and error as random effects. Adjusted least square means (LSmeans) and 95 % CIs were calculated from the model. A minimally clinically important difference (MCID), defined as ≥4 points difference in IIEF-EF [19], was used to determine the average needed treatment effect that has clinical relevance for patients. For p values, a 5 % level of significance was used. Data were analyzed using the SAS 9.2 software (SAS Institute Inc., Cary, USA). BODY.RESULTS.PATIENT DISPOSITION AND BASELINE CHARACTERISTICS: Of 583 patients screened, 423 were randomized: 139 (32.9 %) to tadalafil OaD, 143 (33.8 %) to tadalafil PRN, and 141 (33.3 %) to placebo (Supplementary Figure S2). Patients in the PRN group took a mean (SD) of 1.5 (0.95) tadalafil 20 mg tablets per week. Patient disposition, baseline demographics, and relevant disease characteristics were balanced in all 3 treatment groups (Table 1) [15]. As per inclusion criteria, all patients had to have IIEF-EF ≥22 pre-nsRP. Post-nsRP at baseline, 83.9 % of patients reported severe ED based on IIEF-EF scores (mean [standard deviation; SD] score 6.4 [5.81]) and >98 % reported an REF ≤3.Table 1Baseline characteristics and status post nsRPVariableTadalafil OaD (N = 139)Tadalafil PRN (N = 143)Placebo (N = 141) Age (years)Mean (SD)58.6 (5.07)57.5 (5.91)57.6 (5.69)<61 (n, %)82 (59.0)85 (59.4)91 (64.5)61–68 (n, %)57 (41.0)58 (40.6)50 (35.5) Ethnicity (n, %)Caucasian137 (98.6)141 (98.6)138 (97.9) BMI (kg/m2)Mean (SD)26.6 (2.97)26.9 (2.93)27.1 (3.08) IIEF-EF N with data137140137Mean (SD)6.0 (5.80)6.7 (5.57)6.5 (6.08) ED severity (IIEF-EF categories) (n, %)a Missing2 (1.4)2 (1.4)4 (2.8)Normal (26–30)4 (2.9)2 (1.4)2 (1.4)Mild (17–25)5 (3.6)8 (5.6)9 (6.4)Moderate (11–16)9 (6.5)10 (7.0)11 (7.8)Severe (0–10)119 (85.6)120 (84.5)115 (81.6)REF ≤ 3b 137 (98.6)138 (97.2)141 (100) nsRP approach (n, %)Open surgery68 (48.9)65 (45.5)56 (39.7)Conventional laparoscopy29 (20.9)31 (21.7)28 (19.9)Robot-assisted laparoscopy31 (22.3)41 (28.7)44 (31.2)Other11 (7.9)6 (4.2)13 (9.2) Total nerve-sparing score post-nsRP (n, %)Perfect (2)117 (84.2)116 (81.1)113 (80.1)Not perfect (>2)22 (15.8)27 (18.9)28 (19.9) BMI body mass index, ED erectile dysfunction, IIEF-EF International Index of Erectile Function-Erectile Function, N total number of patients, n number of patients, nsRP bilateral nerve-sparing prostatectomy, OaD once a day, PRN "pro-re-nata"/on demand, REF residual erectile function, SD standard deviation aBased on intent-to-treat population, excluding one patient from the tadalafil PRN group with no post-baseline data bTwo patients in the tadalafil OaD group and two patients in the tadalafil PRN group had missing values BODY.RESULTS.TIME TO EF-RECOVERY DURING DBT: The proportion of patients achieving IIEF-EF ≥22 at any time point during DBT with OaD, PRN, and placebo was 29.5, 23.9, and 18.4 %, respectively. Based on the Kaplan–Meier analysis, 25 % of patients achieved EF-recovery (IIEF-EF ≥ 22) within 5.8 months for tadalafil OaD, 9.0 months for tadalafil PRN, and 9.3 months for placebo (Fig. 1a). Median time to EF-recovery could not be estimated as <50 % of patients achieved EF-recovery during the 9-month DBT period (Supplementary Figure S3). The Cox proportional hazard model showed a significant overall treatment effect (p = 0.038). Patients in the tadalafil OaD (but not PRN) group had a significantly higher probability for EF-recovery versus placebo (HR [95 % CI]: tadalafil OaD versus placebo: 1.90 [1.16, 3.12], p = 0.011; tadalafil PRN versus placebo: 1.47 [0.88, 2.47], p = 0.140). Age group had no significant effect on time to EF-recovery (p = 0.223; Supplementary Figure S4).Fig. 1EF-recovery (IIEF-EF ≥ 22) and EF category improvement during DBT. CI confidence interval, DBT double-blind treatment, EF erectile function, IIEF-EF International Index of Erectile Function-Erectile Function domain, LSmean least square mean, MCID minimal clinically important difference, MMRM mixed model for repeated measures, n number of events, N number of patients, n.e. not estimable, n.s. not significant, OaD once a day, PLC placebo, PRN "pro-re-nata"/on demand, TAD tadalafil, yrs years. a Event (EF-recovery) was defined as change in IIEF-EF from <22 at screening to ≥22. P values are obtained from Cox proportional hazard model including terms for treatment, country, and age. b p value obtained from an MMRM model, assuming an unstructured covariance structure, including terms for visit, treatment, treatment-by-visit interaction, country, age group, and baseline IIEF-EF score as fixed effects, and patient and error as random effects. Previously published in: Montorsi et al. 2014 [15] BODY.RESULTS.LSMEAN IIEF-EF IMPROVEMENTS: LSmean IIEF-EF improvement during DBT significantly exceeded the MCID (Lower 95 % CI LSmean ΔIIEF-EF ≥ 4) at month 5 in the tadalafil OaD treatment group (LSmean [95 % CI]: 6.9 [5.0, 8.8]) and month 9 in the tadalafil PRN treatment group (6.5 [4.6, 8.5]) (Fig. 2). For placebo, LSmean IIEF-EF did not significantly exceed the MCID before month 10.5 (end of DFW: 6.0 [3.9, 8.0]). The treatment effect versus placebo was statistically significant for tadalafil OaD only (LSmean difference [95 % CI]: 2.8 [0.8, 4.8]; p = 0.007) at month 9.Fig. 2Improvement and maintenance of improvement from baseline in ED severity (based on IIEF-EF categories). DBT double-blind treatment, DFW drug-free washout, ED erectile dysfunction, IIEF-EF International Index of Erectile Function-Erectile function domain, OaD once a day, PLC, placebo, PRN "pro-re-nata"/on demand, N total number of patients, n number of patients. IIEF-EF scores defining ED severity categories: severe, 0–10; moderate, 11–16; mild, 17–25; normal, 26–30. Improvement was defined as reporting an IIEF-EF score of at least 1 category higher than baseline (or maintaining normal EF). Improvement declined was defined as reporting IIEF-EF scores at the end of DFW that were less than the end of the DBT but still at least 1 category higher than baseline. Improvement maintained was defined as reporting IIEF-EF scores at the end of DFW that were at least as high or higher than scores at the end of DBT. Improvement lost was defined as IIEF-EF scores that were less than or equivalent to ED severity at baseline. All percentage are relative to the size of each treatment group ("N," provided below each bar) not relative to the overall population. Missing data: For 33 patients (23.7 %) in the tadalafil OaD group, for 27 (19.0 %) in the tadalafil PRN group, and for 34 (24.1 %) in the placebo group, improvement could not be calculated because the patient either discontinued during DBT or had missing IIEF-EF scores at baseline and/or month 9. For three patients (5.3 %) in the tadalafil OaD group, one (1.8 %) in the tadalafil PRN group, and one (3.1 %) in the placebo group, maintenance of improvement could not be calculated because the patients either discontinued during DFW or had missing IIEF-EF scores at month 10.5 BODY.RESULTS.ED SEVERITY IMPROVEMENT AND MAINTENANCE OF IMPROVEMENT: At baseline (randomization), 83.9 % of patients overall had severe ED (IIEF-EF 0–10; Table 1). During DBT, improvement in ED severity by ≥1 severity grade was achieved by 41.0 % of all tadalafil OaD patients, 38.7 % of all tadalafil PRN patients, and 22.7 % of patients on placebo (Fig. 2). In all groups, the majority of those patients who had improved during DBT maintained an improvement of ≥1 severity grade from baseline through DFW (improvement maintained, Fig. 2). For tadalafil OaD (Fig. 2), 43 of 57 improved patients (75.4 %) were still improved from baseline after DFW, including 16 patients (28.1 %) who maintained the improvement they had reached at the end of DBT and 27 patients (47.4 %) who declined but still maintained improvement from baseline after DFW. BODY.DISCUSSION: This trial was the first RCT in patients with established ED post-nsRP which investigated the effect of early treatment with tadalafil OaD and PRN on EF-recovery. As previously reported, tadalafil OaD significantly improved drug-assisted EF-recovery, as measured by the proportion of patients achieving IIEF-EF ≥22 at the end of DBT (25.2 % of OaD patients versus 14.2 % in placebo group; p = 0.016; Supplementary Figure S3). Unassisted EF-recovery after DFW was not improved by tadalafil OaD or PRN [15]. Here, we show that tadalafil OaD (but not PRN) significantly shortened the time to EF-recovery during DBT when compared with placebo: with placebo, it took 9.3 months until 25 % of patients had reached EF-recovery; this period was shortened by 3.5 months (i.e., to 5.8 months) in the tadalafil OaD treatment group. An early start of penile rehabilitation does seem to be important; Mulhall et al. [20] showed that patients who started PDE5-inhibitor treatment early post-nsRP reached significantly higher mean IIEF-EF scores than patients who started later at ≥6 months post-nsRP (p < 0.0001). EF-recovery rates during 9-month DBT were <50 % in all treatment groups, which is in line with the published data on natural EF-recovery post-nsRP. Without treatment, time to EF-recovery averages 18 months [4] and can extend well beyond 2 years [5, 6]. In a study by Gallina et al. [21], only 35.8 % of untreated patients reached EF-recovery (IIEF-EF ≥ 22) after an average of 26.8 months post-nsRP. However, LSmean IIEF-EF improvement in the tadalafil OaD group significantly exceeded the MCID (ΔIIEF-EF ≥ 4 [19]) already at month 5 of DBT, as compared to month 9 for tadalafil PRN; improvement with placebo did not significantly exceed the MCID before month 10.5 (end of DFW). At the end of DBT, the treatment effect versus placebo was statistically significant for tadalafil OaD only (p = 0.007). Chronic (daily) dosing of tadalafil, but not PRN treatment, will lead to steady state PDE5-inhibition [22] which may be associated with prolonged (continuous) periods of increased tissue oxygenation during the post-operative regenerative process. Preclinical data suggest that chronic low-dose administration may protect from structural changes of penile cavernous corpora and is associated with EF enhancement [23–25]. None of the other RCTs on PDE5-inhibitors post-nsRP has reported time to EF-recovery data based on Kaplan–Meier analysis. However, data from 2 non-RCTs indicated that PDE5-inhibitor treatment may shorten time to EF-recovery [26, 27]. Bannowsky et al. [26] reported a significant difference in time to EF-recovery between patients receiving nightly low-dose sildenafil for up to 12 months when compared with patients receiving no treatment (p < 0.001). In agreement with these results, Briganti et al. showed that patients receiving any PDE5-inhibitor (OaD or PRN) achieved significantly higher 3-year EF-recovery rates (IIEF-EF ≥ 22) than patients receiving placebo (72 vs. 38 %, p ≤ 0.001, Kaplan–Meier analysis). For the overall population studied, no significant difference was observed between OaD and PRN treatment. However, patients with an intermediate risk of ED (66–69 years or IIEF-EF 11–25, and Charlson Comorbidity Index ≤1), who shared key criteria with our patient population of low/intermediate ED risk (average patient ≤61 years; IIEF-EF ≥ 22 at baseline), achieved significantly higher 3-year EF-recovery rates with OaD compared to PRN treatment (74 vs. 52 %; p = 0.02) [27]. As suggested by Castiglione et al. [28], the effect of PDE5-inhibitor treatment post-nsRP may be maximal in patients with intermediate ED risk. To date, trials have focused on populations with low ED risk [11, 13, 15]. In our trial, age group had no significant effect on the time to EF-recovery during DBT or on the proportion of patients achieving EF-recovery during DBT [15]. However, after DFW (Month 10.5), younger patients (<61 years) were significantly more probable to achieve EF-recovery than older patients (p = 0.020; [15]). These results are in line with literature. A 2010 study by Briganti et al. [29] showed that younger patients (≤65 years) were more likely to recover EF (IIEF-EF ≥ 22) than older patients. A meta-analysis by Kilminster et al. and studies by Nelson et al. and Gallina et al. also showed that younger patients were significantly more likely to recover EF post-nsRP than older patients [5, 7, 21]. A clear limitation of the current trial was that the 9-month DBT phase was too short for full assessment of EF-recovery. We cannot exclude that the treatment effect of tadalafil OaD may be lost by the end of 2 years due to spontaneous EF-recovery in the placebo arm. Valid statistical analysis of time to EF-recovery could not be performed on IIEF-EF data collected after DBT (i.e., after DFW at month 10.5 or OLT at month 13.5) due to the break in ED treatment for 6 weeks. The significant treatment effect on EF-recovery was lost during the DFW. However, after 3-month OLT with tadalafil OaD (month 13.5), the proportion of patients with EF-recovery increased in all treatment groups (32.4 % in the tadalafil OaD group; 33.1 % in the tadalafil PRN group; and 27.0 % in the placebo group) [15]. Further, the results of the Briganti study indicate that the significant effect of OaD treatment on time to recovery may persist after longer follow-up periods (up to 3 years) [27]. In this context, the demonstrated maintenance of tadalafil's treatment effect could play an important role in future studies that allow for longer treatment or follow-up periods. Even after DFW, over 75 % of patients treated with tadalafil OaD maintained an improvement in ED severity from baseline. In conclusion, patients taking tadalafil OaD (but not those taking PRN) significantly shortened the time to EF-recovery during DBT when compared with placebo. No statistically significant difference in time to EF-recovery was observed between younger and older patients. These data suggest that tadalafil OaD, if started early, may accelerate EF-recovery post-nsRP. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material 1 (PDF 333 kb)

TITLE: The effect of perhexiline on myocardial protection during coronary artery surgery: a two-centre, randomized, double-blind, placebo-controlled trial ABSTRACT.OBJECTIVES: Perhexiline is thought to modulate metabolism by inhibiting mitochondrial carnitine palmitoyltransferase-1, reducing fatty acid uptake and increasing carbohydrate utilization. This study assessed whether preoperative perhexiline improves markers of myocardial protection in patients undergoing coronary artery bypass graft surgery and analysed its effect on the myocardial metabolome. ABSTRACT.METHODS: In a prospective, randomized, double-blind, placebo-controlled trial, patients at two centres were randomized to receive either oral perhexiline or placebo for at least 5 days prior to surgery. The primary outcome was a low cardiac output episode in the first 6 h. All pre-specified analyses were conducted according to the intention-to-treat principle with a statistical power of 90% to detect a relative risk of 0.5 and a conventional one-sided α-value of 0.025. A subset of pre-ischaemic left ventricular biopsies was analysed using mass spectrometry-based metabolomics. ABSTRACT.RESULTS: Over a 3-year period, 286 patients were randomized, received the intervention and were included in the analysis. The incidence rate of a low cardiac output episode in the perhexiline arm was 36.7% (51/139) vs 34.7% (51/147) in the control arm [odds ratio (OR) 0.92, 95% confidence interval (CI) 0.56–1.50, P = 0.74]. Perhexiline was associated with a reduction in the cardiac index at 6 h [difference in means 0.19, 95% CI 0.07–0.31, P = 0.001] and an increase in inotropic support in the first 12 h (OR 0.55, 95% CI 0.34–0.89, P = 0.015). There were no significant differences in myocardial injury with troponin-T or electrocardiogram, reoperation, renal dysfunction or length of stay. No difference in the preischaemic left ventricular metabolism was identified between groups on metabolomics analysis. ABSTRACT.CONCLUSIONS: Preoperative perhexiline does not improve myocardial protection in patients undergoing coronary surgery and in fact reduced perioperative cardiac output, increasing the need for inotropic support. Perhexiline has no significant effect on the mass spectrometry-visible polar myocardial metabolome in vivo in humans, supporting the suggestion that it acts via a pathway that is independent of myocardial carnitine palmitoyltransferase inhibition and may explain the lack of clinical benefit observed following surgery. ABSTRACT.CLINICALTRIALS.GOV ID: NCT00845364. BODY.INTRODUCTION: The population of patients undergoing cardiac surgery is becoming older, with more comorbidities and complex patterns of disease, requiring more urgent surgery [1]. Such factors are often associated with abnormal myocardial metabolism and may contribute towards increasing the length of ischaemic arrest, which remains an independent predictor of mortality in contemporary practice [2]. With current techniques for myocardial protection using blood cardioplegia, up to 30% of patients have a period of clinically detectable transient dysfunction due to myocardial stunning. An episode of non-fatal low cardiac output in the early postoperative period is associated with a significant reduction in late survival following coronary artery bypass graft (CABG) surgery [3] and inadequate protection may lead to permanent injury due to diffuse necrosis, fibrosis, remodelling and long-term impairment of ventricular function. The need for longer periods of ischaemia in more metabolically vulnerable patients necessitates further research to improve protection strategies [4]. Pharmacological manipulation of myocardial metabolism aims to induce a shift in substrate utilization from fatty acids to glucose, thereby increasing energy efficiency and decreasing the potentially harmful effects of β-oxidation. Suppression of fatty acid utilization improves coupling of glycolysis to glucose oxidation, reduces lactate production, diminishes mitochondrial proton leakage and increases the efficiency of adenosine triphosphate (ATP) production per mole of oxygen [5]. Metabolic drugs may have a role in myocardial protection as adjuncts to cardioplegia and hypothermia by increasing metabolic efficiency and reducing the impact of ischaemia–reperfusion injury, thereby translating into enhanced recovery of cardiac function during reperfusion, and improved clinical outcomes. We have previously shown that glucose–insulin–potassium (GIK) therapy improves myocardial protection in patients with multivessel disease undergoing CABG [6] and in those with left ventricular hypertrophy undergoing aortic valve replacement [7]. However, GIK therapy has not been widely adopted due to the lack of large multicentre randomized, controlled trials or a standardized protocol, increased monitoring requirements with high-dose insulin, the potential negative impact of perioperative hyperglycaemia and uncertainty over its mechanisms of action. An agent with a similar metabolic efficacy but without the complexity of administration is desirable although the evidence of benefit for such an agent in myocardial protection is lacking. Perhexiline is a modulator of myocardial metabolism that is effective in patients with refractory angina unsuitable for revascularization [8] and chronic cardiac failure [9, 10]. However, marked interindividual variation in its metabolism via cytochrome p450 2D6 (CYP2D6) previously resulted in severe adverse events due to chronic toxicity in a small number of poor metabolizers; toxicity can be prevented by therapeutic drug monitoring and dose adjustment to achieve plasma concentrations within the therapeutic range (0.15–0.6 mg/l) [11]. In the rat heart, it has been shown to inhibit carnitine palmitoyltransferase-1 (CPT-1), the key uptake enzyme for long-chain fatty acids into mitochondria, and this is thought to be its primary mechanism of action [12]. We report a prospective, double-blind, randomized, placebo-controlled trial to investigate whether preoperative administration of oral perhexiline improves clinical markers of myocardial protection in patients undergoing CABG at two centres in the UK. To examine the impact of perhexiline on myocardial metabolism in humans, we analysed the polar metabolic profile of left ventricular biopsies obtained prior to ischaemia from patients within the therapeutic range for perhexiline compared with controls using high-resolution mass spectrometry (MS) [13]. The polar metabolome was chosen to provide the greatest insight into energy metabolism, including glycolysis and the citric acid cycle. BODY.MATERIALS AND METHODS.STUDY DESIGN: A prospective, two-centre, double-blind, randomized, placebo-controlled trial of preoperative perhexiline (PEXSIG, Aspen Australia, Croydon, Victoria, Australia) was conducted in patients undergoing elective or urgent (during the same hospital admission) isolated, first-time CABG for multivessel coronary artery disease. The study was approved by the Cambridgeshire 1 Research Ethics Committee (06/Q0104/41) and the Medicines and Healthcare products Regulatory Agency (2006-003164-62). The trial was registered with ClinicalTrials.gov (NCT00845364), and patients were enrolled between February 2007 and April 2010 at the Queen Elizabeth Hospital Birmingham and the Royal Sussex County Hospital, Brighton, UK. Informed consent was obtained from each participant and all research was performed in accordance with the Declaration of Helsinki and the UK Human Tissue Act 2004 within a research governance framework. The exclusion criteria were diabetes mellitus, significant renal or hepatic impairment, peripheral neuropathy, porphyria, atrial fibrillation, recent amiodarone therapy, emergency surgery (before the next scheduled operating list), known hypersensitivity to perhexiline or prospectively identified as requiring a significant deviation from the protocol on clinical grounds. Patients were randomized to either perhexiline or placebo in a 1 : 1 ratio using a computer-generated random allocation sequence with minimization for surgeon, priority of surgery and left ventricular function. All tablets were identical in appearance to conceal allocation. Trial medication was commenced a minimum of 5 days prior to the planned date of surgery and continued for up to 31 days. A standardized loading and maintenance regime was used: 200 mg b.i.d. for 3 days followed by 100 mg b.i.d. until the morning of surgery. Blood was drawn prior to anaesthesia and stored; serum perhexiline concentrations were determined by high-performance liquid chromatography at the end of the trial. BODY.MATERIALS AND METHODS.SURGERY, ANAESTHESIA, CARDIOPULMONARY BYPASS AND MYOCARDIAL PROTECTION: Anaesthesia, cardiopulmonary bypass (CPB) and myocardial protection with intermittent anterograde cardioplegia using St Thomas' solution buffered in cold blood were all standardized as previously described [6], except that phenylepherine was used as the first-line vasoconstrictor. Distal anastomoses were performed during cardioplegic arrest and proximal anastomoses during partial aortic occlusion. A pulmonary artery catheter was used to assess haemodynamic variables, and the thermodilution technique was used to measure cardiac function. Baseline haemodynamic studies were performed prior to sternotomy. Following weaning from CPB, further measurements were taken before and ∼10 min after the administration of protamine, and then at 2, 4, 6, 9 and 12 h after release of the aortic cross-clamp. Standardized protocols were used to guide perioperative management including heart rate, volume expansion, inotrope use, temperature and glycaemic control. Serial blood samples were drawn at baseline at 6, 12 and 24 h to determine troponin-T by ECLIA (Roche Diagnostics, Burgess Hill, UK) and plasma non-esterified free fatty acids by enzymatic colorimetric assay (Wako Chemicals, Neuss, Germany). BODY.MATERIALS AND METHODS.END-POINTS: The primary end-point was the incidence of a low cardiac output episode (LCOE), defined as a cardiac index of <2.2 l/min/m2 refractory to appropriate intravascular volume expansion after correction of dysrhythmias in the first 6 h after cross-clamp release. A blinded end-points committee assessed all episodes of LCOE and a consensus opinion was reached. A planned exploratory analysis compared patients in the perhexiline arm who reached the lower threshold of the therapeutic range (0.15 mg/l) with propensity score-matched controls. Secondary end-points were a comparison of cardiac index and inotropic support in the first six and 12 h, peak and area under the concentration–time curve (AUC) for troponin-T in the first 24 h and perioperative myocardial injury on 12-lead electrocardiogram (ECG). ECG changes were assessed by an independent blinded cardiologist and defined as new Q waves ≥2 mm in two or more contiguous leads, new bundle branch block or loss of R wave progression by the 4th postoperative day. BODY.MATERIALS AND METHODS.METABOLOMIC ANALYSIS: Transmural biopsies of the left ventricular free wall between the left anterior descending artery and the first diagonal branch were obtained prior to application of the aortic cross-clamp and immediately snap-frozen. Metabolites were extracted using a methanol : water : chloroform solvent system [14], dried using a centrifugal concentrator and stored at −80°C. Subsequently, each dried polar extract was re-dissolved in 80 : 20 methanol:water containing 20 mM ammonium acetate, vortexed and centrifuged prior to MS. Quality control (QC) samples were prepared by pooling an aliquot of each sample. MS analyses were conducted using a hybrid 7-Tesla linear ion trap Fourier transform ion cyclotron resonance (FT-ICR) MS (LTQ FT Ultra, Thermo Fisher Scientific, Germany) equipped with a Triversa chip-based nano-electrospray ion source (Advion Biosciences, NY, USA) using conditions as described previously [15]. Three mass spectra for each sample were collected using a selected-ion-monitoring stitching method from m/z (mass-to-charge ratio) 70–740 in negative ion mode [15, 16], processed, normalized and generalized log-transformed as reported previously [17, 18]. This produced a peak intensity matrix representing the metabolic profile of each extracted biopsy. Using the MI-Pack software [19], m/z measurements were putatively annotated (see Supplementary material). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS.CLINICAL TRIAL: All analyses were prespecified and conducted according to the intention-to-treat principle. The trial had a statistical power of 90% to identify a relative risk of 0.50, which was statistically significant, assuming an incidence of LCOE in the control group of 0.37 and a conventional one-sided α of 0.025. Analyses were conducted with SAS software version 9.1 or above (SAS Institute, Inc., Cary, NC, USA). Continuous data are presented as mean (standard deviation, SD) or median (interquartile range, IQR). P-values other than for the primary end-point were nominal. All analyses were stratified for left ventricular function and urgency of surgery as patient-level covariates, and surgeon as a random effect. Dichotomous outcomes were analysed with the use of nonlinear mixed models and continuous data with mixed models. Propensity scoring approaches were used to identify matched controls, based on age, race, weight, days of trial therapy, left ventricular function and priority of surgery. Supportive analyses were conducted using generalized linear models and repeated measures. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS.METABOLOMICS: Student's t-tests were conducted on the non-generalized log-transformed peak intensity matrix, using a false discovery rate (FDR) of 5%, to determine if individual peaks changed significantly between groups [20]. In addition, principal components analyses (PCA) and partial least squares-discriminant analyses (PLS-DA) were conducted to discover metabolic differences between groups. Internal cross-validation was applied to assess for over-fitting of the optimal PLS-DA model. Additional methods appears in the Supplementary material. BODY.RESULTS.STUDY POPULATION: Three hundred and twenty-seven patients were randomized of whom 286 were included in the analysis (Fig. 1), 139 randomized to perhexiline and 147 to placebo (see Supplementary material). Baseline preoperative characteristics were similar between the groups (Table 1) including median logistic EuroSCORE: 1.82 (1.23–3.06) in the treatment arm and 1.82 (1.07–2.94) in the control arm. Participants took the trial medication for a median of 10 days (6–12 days) prior to surgery. Eighteen (6.3%) patients reported potential side effects of perhexiline, principally nausea or dizziness, of whom 15 were in the treatment group and three in the placebo group; 6 (4.3%) patients in the perhexiline group reported non-compliance with the dosing protocol due to side effects. Serum perhexiline at the time of surgery was measured in 280 patients including 135/139 (97.1%) in the treatment group in whom the median serum concentration was 0.24 mg/l (0.33 mg/l) with a range of 0.04–1.97 mg/l. In the perhexiline arm, 37 (27.4%) patients were below the lower threshold of the therapeutic range (0.15 mg/l); there was no correlation between serum perhexiline level and length of therapy. In the placebo group, all patients measured had a serum perhexiline level of zero, confirming the difference between the groups. Table 1:Baseline patient characteristicsPerhexiline (n = 139)Control (n = 147)Age, median (IQR) (years)66.1 (59.4–73.2)65.7 (60.2–73.6)Male gender, n (%)128 (92.1)134 (91.2)Race, n (%) Caucasian133 (95.7)136 (92.5) South Asian5 (3.6)10 (6.8) Black1 (0.7)1 (0.7)CCS class, n (%) 012 (8.6)7 (4.8) I9 (6.5)14 (9.5) II70 (50.4)71 (48.3) III39 (28.1)43 (29.3) IV9 (6.5)12 (8.2)NYHA class, n (%) I58 (41.7)59 (40.1) II68 (48.9)79 (53.7) III13 (9.4)9 (6.1) IV0 (0)0 (0)Previous MI, n (%)49 (35.3)48 (32.7)Previous coronary stent, n (%)13 (9.4)10 (6.8)Left main stem disease ≥50%, n (%)54 (38.9)47 (32.0)Left ventricle function, n (%) Good118 (84.9)122 (83.0) Moderate20 (14.4)24 (16.3) Poor1 (0.7)1 (0.7)Priority, n (%) Elective120 (86.3)124 (84.4) Urgent19 (13.7)23 (15.7)Smoker, n (%) Non38 (27.3)55 (37.4) Current14 (10.1)16 (10.9) Ex-smoker87 (62.6)76 (51.7)Pulmonary disease, n (%)17 (12.2)12 (8.2)Peripheral vascular disease, n (%)14 (10.1)10 (6.8)Recent MI (last 90 days), n (%)19 (13.7)17 (11.6)Number of antianginal agents, n (%) 03 (2.2)2 (1.4) 168 (48.9)65 (44.2) 248 (34.5)55 (37.4) 315 (10.8)19 (12.9) 45 (3.6)6 (4.1)Haemoglobin, mean (SD) (g/dl)14.32 (1.3)14.11 (1.2)Creatinine, mean (SD) (mg/dl)1.20 (0.2)1.12 (0.2)EuroSCORE, median (IQR)3 (1 to 4)3 (1 to 4)Logistic EuroSCORE, median (IQR)1.82 (1.23 to 3.06)1.82 (1.07 to 2.94)CCS: Canadian Cardiovascular Society; NYHA: New York Heart Association; MI: myocardial infarction; SD: standard deviation; IQR: interquartile range. Figure 1:Participant flow diagram. All patients underwent multivessel CABG. One patient in the perhexiline and 3 in the control group had an additional mitral valve procedure and 1 further patient in the control group underwent aortic valve replacement. Operative variables including CPB and aortic cross-clamp times were similar between groups (Table 2). Table 2:Operative variablesPerhexiline (n = 139)Control (n = 147)Number of grafts, mean (SD)3.30 (0.73)3.29 (0.82)Internal mammary artery graft used, n (%)132 (95.0)136 (92.5)Additional procedure, n (%)1 (0.7)4 (2.7)Operation performed by trainee, n (%)38 (27.3)a50 (34.0)CPB time, median (IQR) (min)109 (93–131)b110 (90–134)Reinstitution of CPB, n (%)3 (2.2)6 (4.1)Aortic cross-clamp time, median (IQR) (min)56 (45–70)b57 (45–72)Total cardioplegia dose, median (IQR) (l)1.95 (1.70–2.30)1.90 (1.62–2.24)Reperfusion VF/VT, n (%)3 (2.2)3 (2.0)Antifibrinolytic used, n (%)97 (69.8)100 (68.0)Cell salvaged blood, median (IQR) (ml)470 (260–700)485 (300–700)Intra-aortic balloon pump used, n (%)10 (7.2)a21 (14.3) Preop for unstable angina1 (0.7)0 (0) Preop elective in theatre3 (2.2)6 (4.1) Pre-CPB for intraoperative instability2 (1.4)0 (0) During CPB for anticipated instability1 (0.7)4 (2.7) Post-CPB for instability3 (2.2)a11 (7.6)Reinstitution of CPB relates to episodes of unsuccessful weaning from CPB requiring a second period of CPB to prevent cardiovascular collapse.CPB: cardiopulmonary bypass; VF: ventricular fibrillation; VT: ventricular tachycardia; SD: standard deviation; IQR: interquartile range.aNot significant with Fisher's exact test.bNot significant with Student's t-test. BODY.RESULTS.PRIMARY OUTCOME: A LCOE was diagnosed in 102 of 286 (35.7%) patients analysed; however, there was no significant difference in incidence between the two groups: 51/139 (36.7%) in the perhexiline arm and 51/147 (34.7%) in the control arm (OR 0.92, 95% CI 0.56–1.50, P = 0.74) (Table 3). In the exploratory analysis, patients in the perhexiline arm who were above the lower threshold of the therapeutic range (≥0.15 mg/l) at the time of surgery were compared with propensity score-matched controls (n = 97 in each group, 1 patient excluded due to missing data). There remained no significant difference in the incidence of LCOE between groups: 38/97 (39.2%) for perhexiline and 31/97 (32.0%) for controls (OR 0.73, 95% CI 0.40–1.33, P = 0.30). Table 3:Study outcomes, the perhexiline group compared with the control groupOutcomesPerhexilineControlOdds ratio (95% CI)P-valuePrimary outcome, n (%)139147 Low cardiac output episode51 (36.69%)51 (34.69%)0.92 (0.56 to 1.50)0.74Secondary outcomes, categorical, n (%)139147 Inotrope use in first 6 ha39 (28.06%)36 (24.49%)0.84 (0.49 to 1.44)0.52 Inotrope use in first 12 h67 (48.20%)50 (34.01%)0.55 (0.34 to 0.89)0.015 New myocardial injury on ECG23 (16.55%)25 (17.01%)1.02 (0.55 to 1.92)0.94Secondary outcomes, continuous, mean (SD)135144 Cardiac index at 6 h (l/min/m2)2.51 (0.43)2.70 (0.54)0.19 (0.07 to 0.31)b0.001 Peak troponin-T (ng/ml)0.78 (0.71)0.89 (0.92)0.11 (−0.09 to 0.30)b0.28 AUC troponin-T (ng h/ml)3.98 (3.79)4.71 (5.32)0.73 (−0.40 to 1.86)b0.12cAccounting for baseline left ventricular function and priority of surgery, with surgeon as a random effect.SD: standard deviation; AUC: area under the concentration-time curve.aBefore cardiopulmonary bypass, during cardiopulmonary bypass and 0–6 h.bDifference in means (95% confidence interval).cOne-tailed. BODY.RESULTS.SECONDARY OUTCOMES.HAEMODYNAMIC DATA: At 6 h following reperfusion, the mean cardiac index was significantly lower in the perhexiline group (2.51 l/min/m2, SD 0.43) than in the control group (2.70 l/min/m2, SD 0.54) (difference in means 0.19, 95% CI 0.07–0.31, P = 0.001) (Fig. 2). By 12 h after reperfusion, there was no difference between the groups: 2.73 l/min/m2 (SD 0.54) for perhexiline vs 2.79 l/min/m2 (SD 0.48) for controls (difference in means 0.058, 95% CI −0.06–0.18, P = 0.34). Throughout the study, heart rate, mean arterial pressure, central venous pressure and pulmonary artery wedge pressure were similar between groups. Figure 2:Mean cardiac index (l/min/m2) between treatment groups. Error bars represent 95% confidence intervals (CI) for the mean (P = 0.018 at 6 h). Cardiac index measured prior to ischaemia was found to be significantly lower in the perhexiline group (2.09 l/min/m2, SD 0.57) than in the control group (2.31 l/min/m2, SD 0.53) (difference in means 0.22, 95% CI 0.09–0.35, P = 0.001). Using repeated measures, the cardiac index was found to be significantly affected by perhexiline therapy (difference in means 0.13, 95% CI 0.08–0.17, P < .0001). BODY.RESULTS.SECONDARY OUTCOMES.INOTROPE AND VASOCONSTRICTOR USE: There was no difference in the prevalence of inotropic support in the first 6 h after reperfusion: perhexiline 39/139 (28.1%) vs control 36/147 (24.5%) (OR 0.84, 95% CI 0.49–1.44, P = 0.52). However, by 12 h, inotrope use was significantly more frequent in the perhexiline group (67/139, 48.2%) versus the control group (50/147, 34.0%) (OR 0.55, 95% CI 0.34–0.89, P = 0.015). There were no differences between groups in the dose requirements for phenylepherine (P = 0.26), norepinephrine (P = 0.12) or insulin (P = 0.43) in the first 12 h. BODY.RESULTS.SECONDARY OUTCOMES.MYOCARDIAL INJURY: There was no difference in the mean peak serum troponin-T concentration during the first 24 h after surgery: 0.78 ng/ml (SD 0.71) in perhexiline patients vs 0.89 ng/ml (SD 0.92) in control patients (difference in means 0.11, 95% CI −0.09–0.30, P = 0.28). The AUC in the first 24 h was also not significantly different between groups: perhexiline 3.98 ng h/ml (SD 3.79) vs control 4.71 ng h/ml (SD 5.32) (P = 0.12) (Fig. 3). There was no difference in the frequency of ECG changes consistent with new myocardial injury: 23/139 (16.6%) in the perhexiline group vs 25/147 (17.0%) in the control group (OR 1.02, 95% CI 0.55–1.92, P = 0.94). Figure 3:Area under the concentration–time curve for serum troponin-T (ng h/ml) (P = 0.12). Plasma non-esterified free fatty acids were measured in 35 patients (perhexiline n = 16, control n = 19) and found to be significantly higher prior to ischaemia in the perhexiline group (0.75 mmol/l, SD 0.08) than in the control group (0.50 mmol/l, SD 0.13) (P < 0.001). This difference was lost by 6 h into reperfusion: 0.73 mmol/l (SD 0.15) in the perhexiline group vs 0.70 mmol/l (SD 0.21) in the control group (P = 0.64). No significant relationship between plasma free fatty acid concentration and pre-ischaemic cardiac index was observed on regression (R2 = 0.09, P = 0.08). There were no significant differences in the prespecified safety end-points of death, stroke, the need for renal replacement therapy, reoperation or length of ICU or hospital stay between groups (Table 4). Table 4:Other clinical outcomes and complicationsPerhexiline (n = 139)Control (n = 147)Postoperative death, n (%)2 (1.4)2 (1.4)Stroke, n (%)3 (2.2)1 (0.7)Neurological, n (%)18 (12.9)12 (8.2) Type I (stroke, TIA)3 (2.2)2 (1.4) Type II (confusion, disorientation)15 (10.8)10 (6.8)Chest tube drainage at 12 h, mean (SD)701 (498)704 (448)Reoperation, n (%)9 (6.5)a13 (8.8) Bleeding6 (4.3)11 (7.5) LCOE/tamponade1 (0.7)1 (0.7) Arrest2 (1.4)1 (0.7) Need for CPB at reoperation1 (0.7)1 (0.7)Arrhythmia, n (%)55 (39.6)a66 (44.9) Atrial fibrillation53 (38.1)64 (43.5) Atrial flutter2 (1.4)2 (1.4)Pneumonia, n (%)15 (10.8)14 (9.5)Tracheostomy, n (%)4 (2.9)4 (2.7)Any pulmonary complication, n (%)33 (23.7)34 (23.1)Creatinine day 4, mean (SD) (mg/dl)1.08 (0.35)1.10 (0.45)Creatinine peak, mean (SD) (mg/dl)1.34 (0.51)1.30 (0.54)AKIN score, n (%) 0121 (87.1)126 (85.7) 113 (9.4)16 (10.9) 22 (1.4)0 (0%) 33 (2.2)5 (3.4)RRT requirement, n (%)3 (2.2)2 (1.4)Abdominal complication, n (%)5 (3.6)4 (2.7) Gastrointestinal bleed2 (1.4)2 (1.4) Prolonged paralytic ileus0 (0)2 (1.4) Diarrhoea3 (2.2)0 (0)Sternal infection, n (%)5 (3.6)5 (3.4) Superficial infection/dehiscence5 (3.6)4 (2.7) Deep infection requiring surgery0 (0)1 (0.7)Any treated infective episode, n (%)27 (19.4)a25 (17.0)Transfusion, mean (SD) (units) Blood2.26 (2.62)2.02 (2.58) Platelets0.82 (1.30)0.85 (1.18) Fresh frozen plasma1.89 (2.55)1.85 (2.64)Fluid volume in 12 h, mean (SD) (ml/kg)67.4 (27.5)62.7 (31.3)Discharge, n (%)137 (98.6)145 (98.6) Home130 (93.5)138 (93.9) Convalescence5 (3.6)6 (4.1) Another hospital/department2 (1.4)1 (0.7)TIA: transient ischaemic attack; LCOE: low cardiac output episode; CPB: cardiopulmonary bypass; AKIN: Acute Kidney Injury Network; RRT: renal replacement therapy; SD: standard deviation.aNot significant with Fisher's exact test. BODY.RESULTS.METABOLOMIC ANALYSIS: Polar extracts from preischaemic left ventricular biopsies were analysed from 43 patients (perhexiline n = 22, control n = 21). All biopsies from the perhexiline group were from patients above the lower end of the therapeutic range (median 0.36 mg/l, IQR 0.24–0.58). The median spectral relative standard deviation, a benchmark to assess the reproducibility in metabolomics [21], for each sample analysed in triplicate by MS was relatively small and consistent across all samples (mean 11.6%, SD 1.6%). The final intensity matrix after data processing consisted of 4039 peak intensity measurements for each sample. All peaks were examined using univariate statistics to determine if any intensity changed significantly in response to perhexiline treatment. No significant peak intensity changes were found (FDR 5%). Multivariate PCA was used to reduce the dimensionality of the data and visualize the metabolic similarities and differences between the two groups; the plot of PCA scores did not show any metabolic effects due to perhexiline treatment (Fig. 4). The clustering of the QC samples demonstrates the consistency in instrument performance over time. Additionally, PLS-DA was conducted to maximize the separation of the metabolic profiles of the two groups (see Supplementary material). The control and treatment samples were minimally separated, and the associated mean classification error rates of the model for predicting class membership were high at 43 and 47%, respectively, suggesting no metabolic differences between groups. More than 200 m/z measurements in the FT-ICR MS dataset were assigned to at least one putative named metabolite including ATP, creatine, phosphocreatine, glycolytic and citric acid cycle intermediates (see Supplementary material). Figure 4:Principal components analysis scores plot from negative ion Fourier transform ion cyclotron resonance mass spectra of left ventricular extracts: 21 control samples (blue) and 22 perhexiline samples (red), shows no metabolic response to perhexiline along the PC1 and PC2 axes. All eight quality control samples (X) are in a narrow cluster, confirming the consistency of mass spectrometry instrument performance over time. BODY.DISCUSSION: In this double-blind, randomized, placebo-controlled trial of preoperative oral perhexiline as an adjunct to cold blood cardioplegia for myocardial protection during CABG, there was no effect on the primary end-point, the incidence of a LCOE. This was confirmed in a propensity score-matched analysis in which patients in the treatment group who were below the therapeutic range were excluded. In addition, there was no difference in the use of inotropes in the first 6 h or the incidence of myocardial injury determined by troponin release or significant ECG changes between groups. Indeed, cardiac index was found to be lower in the treatment arm compared with controls both prior to ischaemia and at 6 h after release of the aortic cross-clamp. On studying the polar metabolome of the left ventricle prior to ischaemia using MS-based metabolomics, we found that the metabolic profiles of patients on perhexiline, with confirmed therapeutic plasma levels, were indistinguishable from those in the placebo group. Metabolic therapy provides an opportunity to improve myocardial protection by inducing a shift in substrate utilization from fatty acids to glucose, thereby increasing the efficiency of energy production and reducing the harmful effects of β-oxidation during reperfusion [4]. The most extensively studied metabolic strategy is altering the availability and use of substrates using intravenous GIK solution. Insulin has multiple metabolic and non-metabolic effects on the myocardium but their relative importance is unclear. These include suppression of lipolysis with reduced uptake and β-oxidation of fatty acids; increased glucose flux, myocardial oxygen efficiency and glycolytic ATP production; replenishment of citric acid cycle intermediates via anaplerosis; and activation of prosurvival reperfusion injury salvage kinases (RISK) including Akt and AMPK [7]. GIK has been shown to improve postoperative haemodynamics, decrease inotrope requirements and reduce myocardial injury in patients undergoing cardiac surgery [6, 7, 22], but has not been widely adopted principally due to practical difficulties in its administration. While perhexiline has not previously been studied as an adjunct to myocardial protection in cardiac surgery, it has been shown to be clinically effective in the treatment of refractory angina [8], chronic cardiac failure [9] and symptomatic hypertrophic cardiomyopathy [10]. Its mechanism of action has been widely accepted as inhibition of mitochondrial CPT-1, leading to reduced uptake and β-oxidation of long-chain fatty acids [12]. A recent proteomic and metabolomic study in a murine model suggested that perhexiline activates the pyruvate dehydrogenase complex and may cause a complex rebalancing of carbon and nucleotide phosphate fluxes to increase metabolic flexibility [23]. However, our metabolomic analysis demonstrates that, in patients with ischaemic heart disease undergoing cardiac surgery, perhexiline has no significant effect on the polar myocardial metabolome with no change in the intermediates of energy transfer, glycolysis or the citric acid cycle. This lack of up-regulation in glucose metabolism may account for the lack of clinical benefit in this trial and suggests that the mechanism of action of perhexiline in vivo in humans may not be primarily metabolic. The possibility that perhexiline acts via a CPT-independent pathway has previously been raised following the observation that there is a temporal dissociation between its effects on cardiac efficiency and metabolism in a rat heart model [24]. Recent work has shown that it suppresses expression of thioredoxin-interacting protein (TXNIP), the key regulator of the antioxidant thioredoxin system, and increases expression of the energy sensor AMPK and its downstream effector PGC-1α [25], although the relevance of these observations remains to be proven. While its predominant mechanism of action in non-surgical patients remains unconfirmed, we found neither an improvement in contractility at any time point nor a reduction in myocardial injury following ischaemia–reperfusion. Patients treated with perhexiline had a lower cardiac index prior to ischaemia although the mechanisms underlying this finding are unknown. Despite a weak calcium channel inhibitory effect [11], perhexiline has not previously been found to be negatively inotropic although none of the previous studies involved anaesthesia or surgery. Plasma free fatty acids prior to ischaemia were significantly higher in the perhexiline group than the control group; high levels of fatty acids are known to reduce cardiac efficiency although there was no correlation between plasma free fatty acids and preischaemic cardiac index. In the design and conduct of the trial, randomization was stratified for left ventricular function, although it was not precisely quantified on echocardiogram prior to commencing the trial therapy. All patients in whom the primary end-point was available were analysed on an intention-to-treat basis and no differences between groups were observed for the primary end-point, other secondary outcomes apart from the cardiac index, safety end-points or any other postoperative variable. One of the limitations of perhexiline is the marked interindividual variation in its metabolism mainly due to genetic polymorphisms of CYP2D6 [11]. The dosing regimen used in this trial attempted to achieve prompt but effective loading with perhexiline in the community with a low incidence of toxicity, especially in poor metabolizers of the drug. We found that ∼27% of the perhexiline group were below the lower threshold of the therapeutic range (0.15 mg/l) at the time of surgery but there was no correlation with length of therapy, suggesting that the minimum period of loading was sufficient. These sub-therapeutic patients comprised all of those found to be ultrarapid metabolizers of perhexiline plus some of the patients who had reported side effects and were either known or presumed to have stopped taking the drug. While this failure to attain a therapeutic concentration at the time of surgery is a limitation of perhexiline use, the comparison of therapeutic patients with propensity score-matched controls confirmed that perhexiline had no benefit in reducing the incidence of LCOE in this trial. In conclusion, the addition of oral perhexiline as an adjunct to cardioplegia in patients undergoing CABG did not improve clinical or biochemical end-points of myocardial protection or any other outcome measures, and was associated with a reduction in the perioperative cardiac index. Therefore, preoperative perhexiline does not improve the outcomes of patients undergoing cardiac surgery and may in fact impair cardiac function in the perioperative period, increasing the need for inotropic support. We also present novel human data suggesting that perhexiline has no significant effect on the MS-visible polar myocardial metabolome in vivo at therapeutic serum concentrations; this supports the suggestion that perhexiline acts via a pathway that is largely or entirely independent of myocardial CPT-1 inhibition [24] and may explain the lack of clinical benefit observed following surgery. BODY.SUPPLEMENTARY MATERIAL: Supplementary material is available at EJCTS online. BODY.FUNDING: This work was supported by the British Heart Foundation (grant numbers PG/06/044/20703, PG/10/036/28341) and the Sussex Heart Charity. The clinical trial was sponsored by University Hospitals Birmingham NHS Foundation Trust. Funding to pay the Open Access publication charges for this article was provided by the British Heart Foundation. Conflict of interest: Michael P. Frenneaux is inventor of method of use patents for perhexiline in heart muscle diseases. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree with the manuscript as written. Neither the funders nor the sponsor had any intellectual input into the design, analysis, report or submission for publication. BODY.SUPPLEMENTARY MATERIAL: Supplementary Data