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TITLE: Cardiovascular stability during arteriovenous extracorporeal therapy: a randomized controlled study in lambs with acute lung injury ABSTRACT.INTRODUCTION: Clinical application of arteriovenous (AV) extracorporeal membrane oxygenation (ECMO) requires assessment of cardiovascular ability to respond adequately to the presence of an AV shunt in the face of acute lung injury (ALI). This ability may be age dependent and vary with the experimental model. We studied cardiovascular stability in a lamb model of severe ALI, comparing conventional mechanical ventilation (CMV) with AV-ECMO therapy. ABSTRACT.METHODS: Seventeen lambs were anesthetized, tracheotomized, paralyzed, and ventilated to maintain normocapnia. Femoral and jugular veins, and femoral and carotid arteries were instrumented for the AV-ECMO circuit, systemic and pulmonary artery blood pressure monitoring, gas exchange, and cardiac output determination (thermodilution technique). A severe ALI (arterial oxygen tension/inspired fractional oxygen <200) was induced by lung lavage (repeated three times, each with 5 ml/kg saline) followed by tracheal instillation of 2.5 ml/kg of 0.1 N HCl. Lambs were consecutively assigned to CMV treatment (n = 8) or CMV plus AV-ECMO therapy using up to 15% of the cardiac output for the AV shunt flow during a 6-hour study period (n = 9). The outcome measures were the degree of inotropic and ventilator support needed to maintain hemodynamic stability and normocapnia, respectively. ABSTRACT.RESULTS: Five of the nine lambs subjected to AV-ECMO therapy (56%) died before completion of the 6-hour study period, as compared with two out of eight lambs (25%) in the CMV group (P > 0.05; Fisher's exact test). Surviving and nonsurviving lambs in the AV-ECMO group, unlike the CMV group, required continuous volume expansion and inotropic support (P < 0.001; Fisher's exact test). Lambs in the AV-ECMO group were able to maintain normocapnia with a maximum of 30% reduction in the minute ventilation, as compared with the CMV group (P < 0.05). ABSTRACT.CONCLUSION: AV-ECMO therapy in lambs subjected to severe ALI requires continuous hemodynamic support to maintain cardiovascular stability and normocapnia, as compared with lambs receiving CMV support. BODY.INTRODUCTION: Neonatal, pediatric, and adult extracorporeal membrane oxygenation (ECMO), using venoarterial or venovenous modes, have been practised for over 3 decades [1-5]. These modes of ECMO are known to activate the inflammatory cascade [6,7], but the long-term cardiopulmonary outcome (10–15 years follow-up period) and neurodevelopmental outcome (at age 5 years) are relatively comparable to those in control individuals [8-10]. Patients who now receive ECMO therapy may also be different from patients treated in the 1980s and early 1990s because the alternative therapies have improved [11]. A search for safer modes of bypass therapy, including arteriovenous (AV)-ECMO, is warranted because of the cardiovascular and cerebral autoregulatory complications that are common during ECMO operations [12,13]. This new mode of ECMO therapy may have some advantages over conventional venoarterial ECMO or venovenous ECMO techniques because the AV-ECMO technique appears simpler and may involve fewer operational complications [14]. The first investigators to conduct AV-ECMO trials, Kolobow and coworkers [15] studied eight normal and conscious lambs (age 1–8 days) for periods up to 96 hours. They described reductions in hemoglobin concentrations during AV-ECMO therapy, showing some mild postmortem pulmonary pathology in a few cases. In a later study, those investigators [16] also designed a carbon dioxide membrane lung, which was used to reduce ventilation in spontaneously breathing or sedated animals subjected to controlled mechanical ventilation. They suggested that a carbon dioxide membrane lung could ideally be operated in an AV mode without using a pump. The AV shunt of the AV-ECMO circuit requires adequate blood flow from the systemic circulation, which may require an increase in cardiac output (CO). Animal models of AV-ECMO without acute lung injury (ALI) show clinically acceptable cardiorespiratory stability [17-21], whereas models with ALI usually require inotropic and fluid support [13,22-26]. Conrad and coworkers [27], following a series of preclinical studies [14,23-25], evaluated the safety and efficacy of AV-ECMO therapy in a phase I clinical study. They treated eight patients (five males and three females, aged 21–67 years), who had acute respiratory failure and hypercapnia, with AV-ECMO over a 72-hour period. They found no significant changes in hemodynamic variables, whereas arterial carbon dioxide tension (PaCO2) was significantly reduced from 90.8 ± 7.5 mmHg to 51.8 ± 3.1 mmHg after 2 hours of AV-EMCO therapy [23]. At the same time, minute ventilation was reduced from a baseline of 6.92 ± 1.64 l/min to 3.00 ± 0.53 l/min. AV-ECMO technique applied in the presence of ALI requires reasonable hemodynamic stability to permit an extracorporeal AV shunt sufficient for carbon dioxide clearance. Recently, we demonstrated that lambs with normal lungs are able to maintain effective CO and provide efficient ventilator support with a relatively moderate AV shunt of 15% [17]. The aim of the present study was to determine the cardiovascular support needed to maintain hemodynamic stability and the minute ventilation needed to maintain normocapnia in lambs subjected to severe ALI and treated with AV-ECMO (AV shunt flow of up to 15%) or conventional mechanical ventilation (CMV; AV shunt flow of 0%). BODY.METHODS: BODY.SURGICAL PROCEDURES: The experimental protocol for this study was approved by the Institutional Animal Care and Use Committee of the Mount Sinai Hospital Research Institute (Miami Beach, FL, USA). Seventeen lambs (aged 2–6 weeks, weight 3.6–12.7 Kg) and their ewes were transported to the laboratory at least 3 days before the experiments began. On the day of an experiment, an intravenous line was established, and anesthesia was induced (initial dose 50 mg/kg ketamine intravenously) and maintained throughout the experiment (5 mg/kg per hour intravenous ketamine). A 2% xylocaine solution was used to provide local anesthesia at the incision sites. A while after induction of anesthesia (30–45 min), a tracheotomy was performed and the lambs were connected to a ventilator (Adult Star Infrasonics, Inc., San Diego, CA, USA) at a fractional inspired oxygen (FiO2) of 1.0. Animals were then paralyzed with an intravenous bolus of 1.0 mg/kg vecuronium bromide, followed by 0.1 mg/kg per hour. To establish an ECMO circuit, one internal jugular vein and one carotid artery were cannulated using neonatal ECMO catheters (Medtronic Bio-Medicus, Inc., Eden Prairie, MN, USA). A femoral vein was then cannulated using a 5 Fr Swan–Ganz catheter (Baxter Health Care Co., Critical Care Division, Irvine, CA, USA) for periodic measurement of CO employing the thermodilution technique (Oximetrix-3, CO Computer; Abbott Critical Care System, North Chicago, IL, USA) and for continuous recording of the mean pulmonary artery pressure (PAP). A femoral artery was cannulated for continuous monitoring of the mean arterial pressure (MAP; Datascope 2001; Datascope Co., Paramus, NJ, USA) as well as periodic blood sampling for gas analyses. A bolus of 200 U/kg heparin was administered intravenously, followed by a maintenance infusion of 200 U/kg per hour. Normothermia (38 ± 0.5°C) was maintained throughout the experiments. Lactated Ringer's solution (5 ml/kg per hour) was provided for fluid replacement. BODY.PROCEDURES BEFORE INJURY: One hour after the completion of all invasive procedures, pre-ALI baselines were determined for all investigated variables. Arterial blood samples, corrected for body temperature, were measured using a blood gas analyzer (ABL-30; Radiometer, Copenhagen, Denmark). The same samples were used to measure arterial hemoglobin concentration and hemoglobin–oxygen saturation (Hb-O2) using a hemoximeter (OSM-3; Radiometer). CO was determined by the thermodilution technique using the indwelling Swan–Ganz catheter and a CO computer (Oximetrix-3; Abbot Critical Care System). Minute ventilation was measured using a neonatal respiratory monitor (Bicore Neonatal Respiratory System, Model CP-100; Bicore, Irvine, CA, USA). The ventilator tidal volume was set at 7 ml/kg body weight and positive end-expiratory pressure was set at 4 cmH2O. The peak inspiratory pressure was maintained below 30 cmH2O. Because arterial hypercapnia may affect the cardiovascular system [28], maximizing the ability of the heart to drive the AV shunt, we elected to maintain the PaCO2 between 30 and 45 mmHg, rather than allowing permissive hypercapnia to occur. BODY.ACUTE LUNG INJURY MODEL: To establish a model of severe ALI, in a preliminary study we used the above surgical procedures without AV lines in two lambs. This was accomplished with three consecutive saline lavages (5 ml/kg saline for each). The third lung lavage was followed by an intratracheal instillation of a single dose of 2.5 ml/kg 0.1 N HCl. This procedure resulted in substantial increases in the alveolar–arterial oxygen gradient and an average 60% increase in PAP with relatively stable CO over an 8-hour study period (Fig. 1). Saline lavage followed by tracheal instillation of HCl was used in all animals administered CMV and AV-ECMO therapy. This combination may result in surfactant deficiency (caused by the saline lavage), and cellular injury and edema (caused by pulmonary exposure to acid). BODY.POST-ACUTE LUNG INJURY PROCEDURES: In our ALI model significant arterial hypercapnia developed (data not presented), which was adjusted to relative normocapnia by changes in the respiratory frequency. Based on our preliminary results in the ALI model, we allowed a 90-min interval before determination of a postinjury baseline in order to stabilize gas exchange and hemodynamic parameters. During this recovery period, arterial blood gases were determined every 15 min. A postinjury baseline for all variables was then determined (time 0). At this stage, lambs were consecutively assigned either to continued CMV treatment or to AV-ECMO plus CMV therapy. BODY.GROUP I: These lambs received continuous CMV support during a 6-hour study period with a closed AV shunt (n = 8). All hemodynamic, and arterial and venous mixed blood gas exchange variables were recorded every 2 hours. The oxygen content of both arterial and mixed venous blood was determined for calculation of oxygen consumption as a product of oxygen delivery (the difference between arterial oxygen content and mixed venous blood oxygen content) and CO (Fick's equation). Oxygen extraction was calculated using the differences between the measured values of arterial Hb-O2 and venous Hb-O2 saturation. After completion of the study period the lambs were euthanized by lethal dose of pentobarbital (100 mg/kg intravenously). BODY.GROUP II: In this treatment group a set of baseline values were obtained during CMV with a closed AV shunt (n = 9). Subsequently, lambs were subjected to 6 hours of AV-ECMO plus CMV (AV-ECMO therapy) with a maximum AV shunt of 15% (calculated from CO measured during postinjury baseline). The AV-ECMO circuit was established using a hollow fiber oxygenator (Minimax; Medtronic, Inc. Minneapolis, MN, USA) primed with fresh maternal blood (150–200 ml). To test the efficiency of AV-ECMO as compared with that of CMV in terms of carbon dioxide clearance, we attempted to maintain relative normocapnia in both groups. This required changes in minute ventilation that were achieved by modifying the respiratory rate while maintaining peak inspiratory pressure below 30 cmH2O. To control the flow rate through the AV shunt, a clamp was placed on the arterial side of the AV-ECMO circuit and the flow was continuously measured (Medical Volume Flow Meter; Transonic Systems Inc., Ithaca, NY, USA). Carbon dioxide clearance during an AV-ECMO operation is dependent on the gas flow through the oxygenator. The efficacy of carbon dioxide removal and oxygenation of the Minimax hollow fiber oxygenator were previously studied in our laboratory using 15% AV shunt during stepwise decreases in minute ventilation and oxygenation with gas flow of 1 l/min [17]. This gas flow was approximately four times the maximum blood flow through the AV shunt and maintained normocapnia with a 50% reduction in minute ventilation [17]. In the present study, the oxygenator's gas flow was kept constant at 1 l/min of 100% oxygen and was controlled by an in-line gas regulator (Servo pressure limited system; Hudson RCI, Temecula, CA, USA). To ensure proper performance of the oxygenators during AV-ECMO therapy, the post-oxygenator partial oxygen tension and partial carbon dioxide tension were measured at 2 and 6 hours during the study period. BODY.RESUSCITATIVE MEASURES: The outcome measures in our study were the degree of cardiovascular support needed to maintain hemodynamic stability and the minute ventilation needed to maintain normocapnia during both CMV and AV-ECMO therapy. A number of resuscitative measures were used to maintain hemodynamic stability during both CMV and AV-ECMO trials. These included the following: boluses of 10 ml/kg per hour of lactated Ringer's solution, which were provided if MAP fell below 60 mmHg; infusion of dopamine (5 μg/kg per min) and epinephrine (adrenaline; 0.5–2 μg/kg per min) to maintain MAP above 60 mmHg, given if this MAP was not achieved with fluid resuscitation; and 1 mEq/kg sodium bicarbonate, which was given if the base excess was below –5 mmol/l despite institution of other resuscitative measures. The end-point for resuscitation was deemed to have occurred when all of the above measures failed and the MAP fell below 30 mmHg for a period of 15 min. This cutoff point was selected empirically because below this level of MAP the AV-ECMO animals could not maintain an AV shunt of over 5% of baseline CO. BODY.STATISTICAL ANALYSES: All values are expressed as mean ± standard deviation. Differences in specific variables after establishment of postsurgery baseline (60 min after completion of surgery) and post-ALI baseline (90 min after injury), both within the same group at different times and between the CMV and AV-ECMO groups, were evaluated using two-tailed unpaired t-tests. Data from the surviving lambs in the same group over the 6-hour study period were evaluated using analysis of variance (ANOVA), followed by Dunnett multiple comparisons test. For this analysis, we used the postinjury baselines in each variable as controls. Differences in each parameter among the surviving lambs in CMV and AV-ECMO groups and a group of nonsurvivors in the AV-ECMO category were evaluated using ANOVA, followed by Bonferroni multiple comparison test for comparable time periods. The use of resuscitative measures (lactated Ringer's, dopamine, epinephrine and bicarbonate) in all lambs after time zero and in the surviving lambs in the CMV and AV-ECMO groups, as well as mortality (death before completion of the 6-hour study period), were compared using Fisher's exact test. All resuscitative measures before baseline (time zero) were excluded from data analyses. P < 0.05 was considered statistically significant. BODY.RESULTS: BODY.PRE- AND POST-ACUTE LUNG INJURY BASELINES: These data were collected in all animals (survivors and nonsurvivors) before assignment to the CMV or the AV-ECMO groups (Table 1). No significant differences were found between the preinjury values of lambs that were later randomized to CMV and AV-ECMO groups. After ALI, all lambs required significant increases in minute ventilation in order to achieve relative normocapnia (Table 1). Comparison of postinjury PaCO2 and pH between the two treatment groups revealed statistically significant differences in favor of the AV-ECMO group (Table 1). ALI created a arterial oxygen tension (PaO2)/FiO2 ratio of less than 200 (also representing PaO2) in both groups. After ALI, the PAP was significantly increased by approximately 50% in both groups. There were no significant differences in the postinjury baselines of MAP, PAP, and CO between the groups. The average body weight, measured before surgical procedures, was not significantly different between lambs consecutively assigned to CMV and those that were assigned to AV-ECMO (6.3 ± 1.7 kg versus 8.5 ± 2.8 kg, respectively). However, the four surviving lambs in the AV-ECMO group had significantly greater body weight than the five nonsurviving lambs (11.0 ± 2.2 kg versus 6.5 ± 1.3 kg; P < 0.05, by two-tailed unpaired t-test). BODY.CONVENTIONAL MECHANICAL VENTILATORY SUPPORT VERSUS ARTERIOVENOUS EXTRACORPOREAL MEMBRANE OXYGENATION THERAPY: The data presented in Tables 2 and 3, and Figs 1 and 2 are from the surviving lambs only. Six out of eight lambs (75%) in the CMV group and four out of nine lambs (44%) in the AV-ECMO group survived the 6-hour study period after ALI. Three of the five nonsurviving lambs in the AV-ECMO group died within 45–90 min and two others died after 4 hours, despite a combination of resuscitative measures. On average, the surviving lambs in both groups had stable CO and MAP during the 6-hour study period (Tables 2 and 3). The four surviving lambs in the AV-ECMO group were able to maintain CO and MAP with varying degrees of hemodynamic support. This also allowed for a relatively stable AV shunt flow (14.8 ± 0.4% of the CO, measured at 0, 2, 4, and 6 hours) and a significant reduction of 25–30% in minute ventilation, as compared with the CMV group (Fig. 2). There were no significant differences between the PaCO2 in CMV and AV-ECMO treated lambs during the study period, but the alveolar–arterial oxygen gradient was consistently higher in the AV-ECMO group (Fig. 3). The last measurements of MAP, PAP, and PaO2, which were obtained in four out of the five nonsurviving lambs in the AV-ECMO group, were 33.5 ± 9.3, 36.0 ± 6.3, and 53.7 ± 9.2 mmHg, respectively. These values were significantly lower than those recorded in the surviving lambs in either the AV-ECMO or the CMV group (Tables 2 and 3; ANOVA followed by Bonferroni multiple comparison test). Gas exchange of the oxygenators remained stable within the 6 hours of the study period. For example, the postoxygenator partial oxygen tension was 282 ± 8 mmHg and 282 ± 7 mmHg at 2 and 6 hours, respectively, and the postoxygenator partial carbon dioxide tension was 19.7 ± 5.1 mmHg and 21.0 ± 5.0 mmHg at 2 and 6 hours of AV-ECMO therapy. BODY.HEMODYNAMIC STABILITY: Analysis of the use of resuscitative measures as indicators of hemodynamic stability between the CMV and AV-ECMO groups revealed that significantly more lambs in the AV-ECMO group (including survivors and nonsurvivors) were resuscitated than in the CMV group (Table 4; P < 0.001, Fisher's exact test). However, there was no significant difference in 'mortality' between AV-ECMO and CMV groups within the 6-hour period of study (P > 0.05, Fisher's exact test). BODY.DISCUSSION: The cardiovascular effects of AV-ECMO have been studied in adult and neonatal animal models [14-26]. It has been suggested that the resistance of the membrane oxygenator, hemodynamic stability, and the number, size and length of the conducting cannula, as well as the viscosity of the blood, will all affect the exogenous flow rate [22]. In the present study we utilized a low resistance membrane oxygenator, minimized the length of the conducting cannulae, and attempted to maintain MAP above 60 mmHg by using various resuscitative measures (Table 4). These measures in the AV-ECMO group failed to sustain hemodynamic stability in five out of nine lambs (56%), whereas the survivors (44%) were able to maintain normocapnia with a maximum of 30% reduction in minute ventilation over a 6-hour period of study (Fig. 2). The latter implies that AV-ECMO therapy, providing an AV shunt flow of up to 15% of the CO, may be able to reduce ventilator-induced lung injury in hypercapnic respiratory failure. However, in acute respiratory failure or acute respiratory distress syndrome with high intrapulmonary right-to-left shunt, extracorporeal blood flow in the range of 5–15% of CO may not be sufficient to provide adequate arterial oxygenation. The reasons for the relatively poor performance of AV-ECMO therapy in our lamb model, as compared with the findings of studies conducted in adult animals [14,23,25], may be related to a number of factors. These possibilities are considered below. First, differences between our model and other experimental models of ALI could account for differences between our findings and those of other studies. The present model may create a noncardiogenic pulmonary edema, which could be associated with loss of intravascular volume. Such conditions may require prolonged fluid and positive inotropic treatments to support a sufficient AV shunt flow. In comparison, Zwischenberger and coworkers [6,25] used an adult sheep model, in which acute respiratory distress syndrome was induced by smoke inhalation and 40% third degree burns. Sheep were then ventilated for 2 days before randomization to CMV and AV-ECMO (AV shunt of 11–14%) groups for a period of 7 days. There were no deaths in the AV-ECMO group (n = 8), as compared with only three survivors in the CMV group (n = 8). That model [6,25] demonstrates that perhaps a longer period of CMV support is needed to achieve relative cardiovascular stability before subjecting animals with severe ALI to the additional stress of an AV shunt. How may a short recovery period after ALI affect hemodynamic stability during an AV-ECMO operation? ALI leads to the release of a variety of bioactive materials, including proinflammatory cytokines and reactive oxygen species [29]. The addition of an ECMO circuit to animals with ALI is known to stimulate the generation of inflammatory mediators, leading to further deterioration in cardiovascular function [6,7,16]. Zwischenberger and coworkers [6] studied the pathophysiology of ovine smoke inhalation lung injury after a relatively short recovery interval of 6 hours during both conventional ECMO therapy and CMV in female sheep. Those investigators demonstrated that animals treated with smoke and ECMO had significantly increased circulating thromboxane B2 levels and oxygen free radical activity, and a significant increase in lung wet:dry weight ratios. They suggested that an ECMO operation could potentiate the pathophysiology of smoke inhalation injury and lead to initial deterioration in native lung function [6]. Therefore, despite the simplicity of AV-ECMO procedures, as compared with conventional ECMO [14,30], it could be still subject to free radical generation because of presence of the membrane oxygenator. Thus, the addition of an AV shunt after ALI may further compromise the cardiovascular system. A second factor that could account for the discrepancy between our findings and those of other investigators is that the AV shunt opening in our study led to a mortality rate in the smaller lambs, resulting in a difference between the body weights of the surviving lambs in two groups. This implies that smaller (and presumably younger) lambs with ALI could be more vulnerable to the presence of an AV shunt than relatively larger or older animals. Thus, studies concerning the safety and efficacy of neonatal AV-ECMO therapy should use animals with a narrow age range (1–7 days in lambs). The third factor is whether the ALI in the CMV and AV-ECMO therapy groups was equal in severity. Whether the severity of ALI was different between the groups may be indirectly evaluated by comparing the indices of pre- and post-injury gas exchange. Our data indicate that pulmonary performance before starting AV-ECMO therapy was comparable with that observed in the CMV group (Table 1). The degree of lung injury was not significantly worsened during the 6-hour study period, as judged by lack of significant changes in alveolar–arterial oxygen gradient in the surviving lambs subjected to CMV or AV-ECMO therapy (Fig. 3). BODY.STUDY LIMITATIONS: The outcome measures in this study were the degree of hemodynamic stability and the minute ventilation required to maintain relative normocapnia, while comparing CMV support with AV-ECMO therapy. Our study was not designed to evaluate mortality as an ultimate clinical outcome. A greater number of lambs would have been required to demonstrate significant differences in mortality between the CMV and AV-ECMO groups. However, the more than 50% mortality rate in the AV-ECMO group may raise questions about the clinical and/or statistical significance of our findings. Technically, we failed to use a narrow range of age and body weight in our lambs. However, the average body weights in lambs consecutively randomized t1). BODY.CONCLUSION: Our study indicates that cardiovascular support is required to maintain hemodynamic stability during application of AV-ECMO therapy in lambs with severe ALI. In this model, AV-ECMO therapy with continuous cardiovascular support and an AV shunt flow of 15% of CO can provide a maximum 30% reduction in minute ventilation. We suggest that AV-ECMO with cardiovascular support [30] could be suitable for use in ALI of mild severity, in which permissive hypercapnia is not an acceptable treatment [28,31]. BODY.KEY MESSAGES: • Continuous hemodynamic support is required during AV-ECMO in lambs subjected to severe ALI. • By using a shunt flow of up to 15% of CO, AV extracorporeal therapy in lambs with severe ALI can reduce minute ventilation by 25–30%. • Neonatal patients with severe ALI and hemodynamic instability may not be suitable candidates for AV-EMCO therapy. BODY.ABBREVIATIONS: ALI = acute lung injury; ANOVA = analysis of variance; AV = arteriovenous; ECMO = extracorporeal membrane oxygenation; CMV = conventional mechanical ventilation; CO = cardiac output; FiO2 = fractional inspired oxygen; Hb-O2 = hemoglobin–oxygen saturation; MAP = mean arterial pressure; PaCO2 = arterial carbon dioxide tension; PaO2 = arterial oxygen tension; PAP = pulmonary artery pressure. BODY.COMPETING INTERESTS: The author(s) declare that they have no competing interests. BODY.AUTHOR'S CONTRIBUTIONS: BRT, JBS and DT completed the proposal writing and experimental design. DT and BRT participated in research coordination, data analysis and presentation. JG, HF, YM, and JLO conducted all experimental aspects of the study. BRT, DT, JBS, and JW prepared the manuscript.

TITLE: Limited β2-adrenoceptor haplotypes display different agonist mediated airway responses in asthmatics ABSTRACT.BACKGROUND: In vitro and some in vivo studies suggested that genetic haplotypes may have an impact on β2-agonist mediated airway responses in asthmatics. Due to strong linkage disequilibrium the single nucleotide polymorphisms (SNPs) in the β2-adrenoceptor gene result in only a limited number of haplotypes. We intended to evaluate the impact of β2-adrenoceptor haplotypes on β2-agonist mediated airway responses and the development of tolerance in mild to moderate asthmatics. ABSTRACT.METHODS: Patients were genotyped for the part of the β2-adrenoceptor gene with a known bearing on receptor function and regulation. Cumulative dose response curves of fenoterol versus PD20 methacholine and FEV1 were constructed after 2 week treatment periods with either terbutaline or placebo in a double blind, randomised and cross-over design. Analysis of the dose response curves was based on a repeated measurement analysis of covariance. ABSTRACT.RESULTS: In our study population comprising 45 asthmatic patients, we found three limited allelic haplotypes, resulting in six different genotypes. Our data support the existence of differences between these six genotypes both in the shape of the dose response relationship of the β2-adrenoceptor agonist fenoterol as well as in the propensity to develop tolerance for these effects by pre-treatment with terbutaline. However, this could only be substantiated for the endpoint PD20 methacholine. ABSTRACT.CONCLUSION: Between β2-adrenoceptor genotypes differences exist both in baseline β2-agonist induced airway responses as well as in the propensity to develop tolerance during maintenance β2-agonist therapy. The net differences after two weeks of therapy are, however, of magnitudes that are unlikely to be of clinical significance. BODY.BACKGROUND: Over the past decade an increasing number of single nucleotide polymorphisms (SNPs) in the β2-adrenoceptor (β2-AR) gene have been identified. Initially the focus of research was on two highly prevalent non-synonymous SNPs in the coding region of the gene that both result in an amino acid substitution in the extra-cellular part of the receptor protein: position 16 Arg→Gly and position 27 Gln→Glu. In vitro these amino acid changes appeared to alter the susceptibility to receptor downregulation by exposure to β2-agonists [1,2]. These observations fuelled a number of clinical and in vitro/ex vivo studies yielding inconsistent and sometimes conflicting results. Arg-16 was found to be associated with a greater acute bronchodilator response to a β2-AR agonist [3,4], but also with loss of asthma control in some studies [5,6], but not in all [7]. In an ex vivo study using human peripheral blood lymphocytes no impact of either polymorphism could be substantiated on baseline receptor expression or responsiveness[8]. It is generally assumed that the in vivo consequence of the downregulation of β2-ARs is tolerance towards the airway smooth muscle mediated effects of β2 agonists. This tolerance has generally been difficult to show for the bronchodilator effects of β2-AR agonists, but is more pronounced and potentially clinically relevant for their bronchoprotective effects [9]. The relationship between the polymorphic amino acids 16 and 27 and the susceptibility to bronchodilator tolerance was the subject of two clinical studies [10,11], but in only one of these such an association could be substantiated [10]. Tolerance development towards the bronchoprotective effects of β2-agonists was the subject of two prospective clinical studies, which did not find differences between amino acid 16 genotypes [12,13]. Results of in vitro studies using either human mast cells or airway smooth muscle cells did not aid in settling the issue [14,15]. More recently additional SNPs in the non-coding regulatory part of the β2-AR gene were described, some of which affect receptor expression and regulation in vitro [16-18]. These SNPs are in strong linkage disequilibrium with those coding for amino acid 27 in the β2-AR protein, which results in only a very limited variation in extended allelic haplotypes [16,18,19]. In vitro studies initially focused on the SNPs in the 5' flanking region of the receptor coding block in isolation. Analysis of the relative promoter activities of serially truncated fragments of the 5' flanking region suggested that the regulatory activity of the β2-AR gene is largely concentrated in the region of 550 base pair 5' to the coding block. In particular, deletion of the region containing the -367 SNP strongly reduced transcription. In a comparative assay, alleles containing the -367 T→ C mutation were shown to result in a lower transcription rate (~17%) [18]. Recently, we were able to confirm this finding and showed that this was associated with the decreased binding of an as yet unidentified transcription factor [20]. The intronless coding region of the β2-AR protein is preceded by a small open reading frame encoding a 19 amino acid peptide, the β2-AR upstream peptide (BUP), which inhibits β2-AR mRNA translation [17]. The -47 C/T SNP leads to a Cys → Arg substitution at position 19 of the BUP. Transfection experiments with constructs containing either variant of this SNP showed that Cys19 resulted in an increase in receptor protein expression through an effect on mRNA translation [16]. However, when the BUP SNP was studied in the context of a validated haplotype, the BUP Cys19 allele was associated with decreased receptor protein and mRNA expression, which appeared to be associated with a decreased bronchodilator response to an inhaled β2-agonist in a cross-sectional study in a cohort of asthmatics [19]. On the basis of this latter observation these authors advocated studying the biological phenotypic consequences of the β2-AR SNPs only within the context of validated haplotypes. In fact, our study extends on this study. For our functional analyses we limited the haplotypes to the SNPs in the 5'region of the gene, of which an influence on transcription and regulation may be expected, combined with the two far most prevalent non-synonymous SNPs in the receptor protein coding block at +46 and +79. Our primary aim was to study the impact of different combinations of allelic haplotypes on tolerance to β2-agonist induced bronchoprotection. To this end, we conducted a double blind cross-over study of two-week treatment periods with either the short-acting β2-agonist terbutaline or a matching placebo. Cumulative dose response curves of the full β2-AR agonist fenoterol versus PD20 methacholine were used as the main physiologic endpoint. We found differences between six distinct β2-AR allelic genotypes in the shape of the dose response relationship and in the propensity to develop tolerance for these effects. These differences are statistically significant and functionally relevant only for bronchoprotection when compared to bronchodilation in terms of recovery from metacholine induced bronchoconstriction. The magnitudes of the net differences are, however, unlikely to be of clinical significance BODY.METHODS: BODY.PATIENTS: Recruitment of patients with mild to moderate asthma and inclusion criteria have been described in detail elsewhere [21]. According to current guidelines all patients used inhaled corticosteroids, of which the dose was kept stable from at least 8 weeks prior to inclusion until the end of the study. If inclusion criteria were met, a blood sample was drawn for isolation of DNA. All subjects gave written informed consent to participate in the study that was approved by the Medical Ethics Committee of the Academic Medical Centre in Amsterdam. BODY.DESIGN: The study had a randomized, placebo-controlled, double-blind, cross-over design. Two treatment periods of two weeks were preceded and separated by wash-out periods of two weeks, during which all β2-agonists were discontinued and only ipratropium bromide pressurized metered dose inhaler (pMDI) was allowed for symptom relieve. During the treatment periods a dry powder inhaler (Turbuhaler®, Astra-Zeneca, Zoetermeer, the Netherlands) containing either 500 μg of terbutaline per inhalation or placebo was used four times daily. The subjects attended to the laboratory 24 hours after the last dose of study medication and after ipratropium bromide had been withheld for at least 8 hours. After baseline FEV1 and PD20 methacholine had been determined, subjects inhaled 200 μg of fenoterol pMDI from an aerochamber as the first of a series of 4 doubling doses, resulting in cumulative doses of 200, 600, 1400, and 3000 μg respectively. One hour after each dose of fenoterol a PD20 methacholine was determined, immediately after which the next dose of fenoterol was inhaled. Lung function measurements and methacholine provocation tests were done as described previously [21]. BODY.ASSESSMENT OF EXTENDED Β:2-adrenoceptor genotypes Genomic DNA was extracted from peripheral blood mononuclear cells. Using allele-specific primers distinguishing between the -367T- and -367C-alleles, DNA was amplified by PCR, applying standard conditions. The fragment between nucleotides -367 and + 377 was amplified using sense primers 7 or 8 (Table 1) and anti-sense primer 2, and the fragment between -367 and -1081 was amplified with anti-sense primers 215 or 216 and sense primer 214. The PCR products were separated by agarose gel electrophoresis and isolated from the gel. Using the same -367 haplotype-specific primer sets, the sequence of the PCR products was determined by automatic sequencing. In case of -367 homozygous patients within the cohort studied, heterozygous polymorphisms downstream or upstream were limited to the +46 SNP, thus still allowing for the assessment of the full haplotypes. Table 1Primers used for allele-specific PCR amplification and sequencing. Primer Fragment direction Sequence 2 +396/+377 Antisense 5'-gtagcgatccactgcgatca-3' 7 -387/-367 Sense 5'-gggccccgcccgggccagcct-3' 8 -387/-367 Sence 5'-gggccccgcccgggccagccc-3' 214 -1081/-1061 Antisense 5'-ctgcaaattcctaaggagggc-3' 215 -349/-367 Antisense 5'-ctcgccctccttctcctga-3' 216 -349/-367 Antisense 5'-ctcgccctccttctcctgg-3' BODY.STATISTICAL ANALYSIS: Patients were divided into subgroups according to their established allelic genotypes, based on combinations of the three found limited allelic haplotypes I, II, and III. FEV1 values are presented as % predicted, methacholine provocation test results as (geometric mean) PD20 (μg). Baseline FEV1 and PD20 are those measured after a two week wash out period followed by a two week placebo treatment period and before administration of the first dose of fenoterol. Because of the markedly skewed distribution of PD20 values, these were logarithmically transformed prior to analysis. Analysis of the dose response curves was based on a repeated measurement analysis of covariance with log(PD20) or FEV1 (% predicted) as dependent variable, fenoterol dose, treatment (terbutaline vs. placebo), combined allelic genotype and period as factors, baseline log(PD20) or FEV1 respectively as a covariate and patient as subject within whom repeated measurements may be correlated. An unstructured covariance matrix was used, implying possible differences in SD's at the 8 different fenoterol doses by treatment combinations, as well as varying within-patient correlations between these 8 measurements (heteroscedastic). In the model, all possible interactions were allowed between the three factors fenoterol dose, treatment (terbutaline vs. placebo) and combined allelic genotype. P-values were calculated from a Wald-based F-test with denominator degrees of freedom from the "within-between" method. The analysis comprised a total of 8 global model and 6 within-genotype comparisons and associated P-values. Standard, P-values were not adjusted for multiple comparisons. For PD20 results (means and means ± SE's) from the analyses were back-transformed to the normal scale. The statistical package SAS 8.2 was used for the calculations. BODY.RESULTS: BODY.PATIENT CHARACTERISTICS: A total of 50 patients were enrolled of whom 45 (11 male/ 34 female), with a mean FEV1 of 84.6 % predicted, and a geometric mean PD20 methacholine of 163 μg completed the study. Two patients discontinued the study because of side effects of the study medication (palpitations and tremor). Two patients were excluded because of not allowed use of β2-AR agonist as was one female patient that turned out to be pregnant during the course of the study. The cohort of the 45 patients that completed the study comprised 5 haplotypes within the part of the β2-AR gene between nucleotides -1023 and + 79 (table 2), two of which occurred only single, one of which (Ic) has not been described previously. As described by others, linkage disequilibrium was found between nucleotides -367 T/C, -47 T/C (coding for Arg/Cys19 of the 5' leader peptide (5'LP)), -20 T/C and + 46 A/G (coding for Glu/Gln27 of the β2AR protein). This resulted in the presence within the cohort of only 3 limited haplotypes with considerably differences in (relative) frequencies (table 3). Baseline patient characteristics of these combined limited allelic haplotypes (or allelic genotypes) (table 3) as well as those of the subgroups based upon the amino acid 16 and 27 polymorphisms displayed no statistically significant differences. Table 2Localization of SNPs and delineation of haplotypes of the β2-AR gene in the cohort. The limited haplotypes correspond to the SNPs in boldface. Haplotypes between brackets correspond to those of Drysdale et al. Nucleotide: -1023 -654 -468 -367 -47 -20 46 79 Alleles G/A G/A C/G T/C T/C T/C G/A C/G Limited Haplotype n Frequency (%) III Arg19Gly16Glu27 (2) A G G C C C G G 39 43,3 Ia Cys19Arg16Gln27 (4) G A C T T T A C 36 40 II Cys19 Gly16Gln27 (6) G A C T T T G C 13 14,4 Ib Cys19Arg16Gln27 (1) A G C T T T A C 1 1,1 Ic Cys19Arg16Gln27 (x) G G G T T T A C 1 1,1 Table 3Frequencies and patient characteristics of the subgroups formed by the combined limited allelic hap1otypes. Baseline FEV1 and PD20 were measured after a two week washout period followed by a two week placebo treatment period, before administration of the first dose of fenoterol. Genotype Frequency Baseline FEV1 (% of predicted), mean (range) Baseline PD20 geometric, mean (range) Inhalation steroid dose, mean (range) I/III 14 (31%) 85 (59 – 107) 141 (24 – 761) 470 (200 – 1200) III/III 10 (22%) 86 (54 – 104) 313 (92 – 1240) 440 (200 – 1000) II/III 5 (11%) 81 (72 – 99) 110 (20 – 371) 380 (200 – 500) I/I 10 (22%) 85 (60 – 101) 176 (29 – 672) 530 (200 – 1000) I/II 4 (9%) 82 (70 – 94) 74 (30 – 157) 500 (400 – 800) II/II 2 (4%) 88 (82 – 93) 148 (33 – 668) 500 (200 – 800) BODY.INFLUENCE OF THE GENETIC POLYMORPHISMS AND OF TERBUTALINE PRE-TREATMENT ON THE Β:2-agonist mediated effects on asthmatic airways BODY.A. BRONCHOPROTECTION: Baseline PD20 methacholine values after placebo pre-treatment as compared to terbutaline pre-treatment were of a similar magnitude and not statistically different. Figure 1 shows the back-transformed means and SE's for PD20 methacholine, estimated from the model described above and corrected for baseline. There was no evidence that the shape differences on the log-scale between the dose-response curves for the two treatments vary between the allelic genotypes (interaction: pre-treatment * fenoterol dose * genotype, P = 0.23). However, there was evidence that the difference in PD20 between the two treatments, averaged over fenoterol dose, is related to allelic genotype (interaction: pre-treatment * genotype, P = 0.0029) and also that the shape of the dose-response curve, averaged over both treatments, varies between the allelic genotypes (interaction: fenoterol dose * genotype, P = 0.0011). No firm evidence was found that the relative difference in PD20 between terbutaline pre-treatment and placebo pre-treatment varies over the fenoterol dose (interaction: pre-treatment * fenoterol dose, P = 0.071), giving additional support for the averaging over fenoterol dose. Averaged over fenoterol dose and genotype, terbutaline pre-treatment reduced PD20 compared to placebo pre-treatment (P = 0.0026). Terbutaline pre-treatment reduced PD20 by 53% (95% CI: 31–68%, P = 0.0004) on average for I/III patients and by 66% (95% CI: 35–82%, P = 0.0019) for II/III patients (table 4). I/II patients showed a comparable, but non-significant reduction. For the other genetic groups, reductions are lower, if existing at all (Table 4). Adjustment for multiple comparisons – by for instance the Bonferroni correction – would not change these conclusions qualitatively. After application of a most conservative approach, i.e. by multiplying the uncorrected P-values by 14, the highest significant P-value of 0.0029 for the pre-treatment * genotype interaction would still remain below the level of 0.05, namely 0.041. For the within-genotype significant P-values for genotypes I/III and II/III the values would become 0.0056 and 0.0154 respectively. Figure 1PD20 methacholine (mean ± SE) before and after cumulative doses of fenoterol in patients with different genotypes, pre-treated with placebo or terbutaline for two weeks. Averaged over treatment, the shape of the dose response curves varies between the genotypes (p = 0.0011). Averaged over fenoterol dose and genotype, terbutaline pre-treatment reduced PD20 compared to placebo pre-treatment (P = 0.0026). For the reductions in PD20 per genotype and associated p-values: see table 4. Drawn line: placebo pre-treatment, dashed line: terbutaline pre-treatment Table 4The reduction in bronchoprotection by fenoterol after terbutaline pre treatment as compared to placebo pre treatment. The response was averaged over fenoterol dose 200–3000 μg. A 50% reduction corresponds to one double dose reduction of PD20 methacholine. Negative numbers indicate an increase in response. Reduction in PD20 Allelic genotype Median 95% CI P-value I/III 53% 31% 68% 0.0004 III/III 22% -24% 50% 0.30 II/III 66% 35% 82% 0.0019 I/I -51% -139% 4% 0.078 I/II 50% -2% 76% 0.059 II/II 25% -106% 73% 0.58 BODY.B. RECOVERY BY FENOTEROL OF METHACHOLINE INDUCED BRONCHOCONSTRICTION: Figure 2 shows means and SE's for FEV1 measured one hour after fenoterol inhaled directly after the previous PD20 1 between terbutaline pre-treatment and placebo pre-treatment varied over the fenoterol dose (interaction: pre-treatment * fenoterol dose, P = 0.46), nor that the difference in FEV1 between the two treatments, averaged over fenoterol dose, is related to allelic genotype (interaction: pre-treatment * genotype, P = 0.29, see table 5). There was weak evidence that the shape of the dose-response curve, averaged over both treatments, varies between the allelic genotypes (interaction: fenoterol dose * genotype, P = 0.060). There was some evidence that averaged over fenoterol dose and genotype terbutaline decreases FEV1 compared to placebo (P = 0.027) by an estimated 1.58 (SE 0.68) percent points. Only for genotype I/I this decrease reached statistical significance (see table 5). Adjustment for multiple comparisons, by for instance the Bonferroni correction, reduced all these findings to non-significance. Figure 2FEV1 (mean ± SE) before and after cumulative doses of fenoterol in patients with different genotypes, pre-treated with placebo or terbutaline for two weeks. FEV1 was measured one hour after fenoterol inhalation, directly after the previous PD20 measurement. For the reductions in FEV1 per genotype and associated p-values: see table 5. Drawn line: placebo pre-treatment, dashed line: terbutaline pre-treatment. Table 5Decrease in FEV1 response to fenoterol after terbutaline pre-treatment as compared to placebo pre-treatment. Response was averaged over fenoterol dose 200–3000 μg. Negative numbers indicate an increase in response. Decrease in FEV1 (percent points) Allelic genotype Mean 95% CI P-value I/III 0.71 -1.79 3.21 0.57 III/III -0.28 -3.19 2.63 0.85 II/III 1.84 -2.16 5.84 0.36 I/I 3.02 0.09 5.95 0.044 I/II -0.80 -5.28 3.68 0.72 II/II 6.04 -0.18 12.26 0.057 BODY.DISCUSSION: This is the first study finding differences between β2-AR genotypes in the shape of the dose response relationship of β2-AR mediated airway effects in asthmatics in vivo. Our data analysis, however, does not allow a further distinction in differences with respect to specific characteristics of the dose response curves, such as the maximum effect at infinite drug dose (Emax) or the dose at which 50% of this maximum effect is obtained (ED50). We found no evidence for an interaction between treatment and genotype influencing the shape of the dose response curve of PD20 methacholine. Neither was there evidence that the relative differences in PD20 after terbutaline pre-treatment and placebo pre-treatment vary over the fenoterol dose, as illustrated in figure 5. These latter two findings suggest that, at least with respect to protection against methacholine induced bronchoconstriction, there is a genotype-specific way by which binding of a β2-AR agonist to its receptor translates into a clinical response as well as a genotype specific but β2-agonist-dose-independent impact of tolerance development on this response. The latter is illustrated by the parallel course of the two dose responses curves within the different genotypes on semilogscale (figure 5). The functional differences we observed between the allelic genotypes cannot be explained by the known functional consequences of individual SNPs or of haplotypes as delineated in vitro. Moreover, the functional phenotype of heterozygotic genotypes does not appear to fit in with that of the homozygotic variants. For instance, in our study genotypes I/I and III/III appear to be resistant to downregulation, while genotype I/III showed a significant degree of downregulation for bronchoprotection by fenoterol. Patients heterozygous on position 19 of the BUP and position 27 of the β2-AR, genotypes I/III and II/III, appeared to be most affected by desensitization, with reductions in PD20 of 53 and 66%, respectively. This is in line with a study that found more desensitization in human airway smooth muscle cells derived from individuals who were heterozygous on position 27 [15]. Our findings illustrate why previous studies focusing on single SNPs in the receptor protein coding block may have yielded negative or even contradictory results. For example, the sub-group of Gly-16 homozygotes consists of three genotypes, III/III, II/III and II/II, with apparently different baseline β2-AR agonist mediated responses and propensities to develop downregulation. This implicates that the results of functional studies based solely on variation in amino acid 16 will depend upon the distribution of genotypes within the subgroup of Gly-16 homozygotes. The absence of a clear-cut relationship between genotypes and functional phenotypes suggests the influence of other yet unidentified co-factors. The identification of one such factor may come from recent findings in mouse models suggesting the existence of "cross-talk" in airway smooth muscle between the β2-AR system and Gq-phospholipase C coupled receptors responding to contractile agonists such as methacholine [22]. The findings in this animal model of an increase in cholinergic sensitivity in the absence of chronic β2-AR stimulation and vice versa, fit in with our observation of a combination of apparent resistance to downregulation with respect to bronchoprotection (figure 1 and table 4) combined with the numerative (and borderline significant) largest degree of loss of bronchodilation (figure 2 and table 5) within genotype I/I. Since subjects of this genotype are homozygous Arg16/Arg16, our data in this genotype agree with those of Israel et al[6] who found an increased response to anti-cholinergic therapy in patients of this genotype when they were off β2-agonist therapy, in combination with no improvement in lung function when they were on β2-agonist therapy. Some potential limitations of our study need to be discussed. Our active treatment arm consisted of the short acting β2-agonist terbutaline, where long-acting β2-agonists are nowadays the standard for maintenance bronchodilator therapy in asthma. What matters, however, is whether the degree of "receptor stimulation" we obtained is representative for the usual situation in maintenance therapy. In this respect it is relevant that the dose of terbutaline we employed is generally considered to be about therapeutically equivalent to the standard doses of the two long-acting β2-agonists formoterol and salmeterol. Furthermore, in a direct comparison 500 μg of terbutaline induced a degree of bronchoprotective subsensitivity of a same order of magnitude as the usually employed doses of formoterol [23]. Using either formoterol or salmeterol would also have limited the extent to which findings with either of this drugs can be generalized in view of their differences in intrinsic efficacy. Relevant in this respect may be that in vitro [24] the intrinsic efficacy of terbutaline appears to be in between those of salmeterol and formoterol. Next, the "test drug" we employed for the functional studies was fenoterol which is a full β2-AR agonist, like formoterol, but unlike salbutamol, salmeterol and also terbutaline that are partial agonists in vitro as well as in vivo [21,25,26]. It cannot be ruled out that the responses induced by fenoterol are stronger than those that would have been induced by a partial agonist, but it is unlikely that this would have changed the main conclusions of this study. Furthermore, the cohort we studied was of a relatively limited extent, especially in relation to the low numbers of individuals in some subgroups, particularly genotype II/II, and to the uneven presence of the different genotypes in asthmatic cohorts, as also noticed previously [19]. This implicates that the functional implications of especially genotype II/II need further study either in larger groups of patients or after pre-selection of specific genotypes. Our genotype analyses contained all the SNPs with a known bearing on gene regulation or receptor expression, thus ignoring the three SNPs downstream from basepair +79. In our opinion this is only of limited impact. The SNPs at +252 and +523 are synonymous and so do not result in amino acid substitution, while the one at + 491 is very uncommon. However it cannot be ruled out completely that these three SNPs have an impact on receptor expression e.g. via an effect on mRNA stability. Our study confirms that downregulation of β2-agonist induced airway responses is more easily substantiated for protection towards a bronchoconstrictive stimulus than for bronchodilation form "baseline", in this case: recovery form methacholine-induced bronchoconstriction one hour earlier. This is in line with a recent study showing that susceptibility to bronchodilator tolerance increases when the degree of induced bronchoconstriction increases [27]. The degree of tolerance development was much less for FEV1 and at the most borderline statistically significant for the cohort as a whole. With respect to the potential clinical implications of our findings it must be realised that for all genotypes some degree of protection against bronchoconstriction remained after two weeks of β2-agonist use. After terbutaline pre-treatment, the differences in the dose response curves between genotypes were attenuated and the maximum difference in improvement in PD20 by the highest dose of fenoterol between the genotypes (II/III versus I/I, figure 1) was 1.5 doubling dose, where differences larger than about one doubling dose are generally considered to be clinically significant, in view of the confidence intervals for repeated determinations of methacholine bronchoprovocation thresholds [28]. It is evident that at lower doses of β2-agonist, such as normally used by asthmatics in a clinically stable state, the differences are even smaller. Altogether, this implicates that the functional phenotypical differences between the genotypes are probably only of limited clinical significance, at least in stable mild to moderate asthmatics as in our cohort. As we previously argued, such differences are likely to be more relevant in situations with a high state of functional antagonism, such as in asthma exacerbations with severe bronchoconstriction and functionally antagonized receptors by inflammatory mediators, when high doses of short acting β2-agonists are used [21]. BODY.CONCLUSION: Our data and analyses in a cohort of asthmatic patients indicate differences between six distinct β2-AR allelic genotypes in the shape of the dose response relationship of a β2-AR agonist and in the propensity to develop tolerance for these effects. The genotypes are based upon combinations of three limited allelic haplotypes containing the functionally relevant parts of the β2-AR gene. The differences we found are statistically significant and functionally relevant only for bronchoprotection when compared to bronchodilation in terms of recovery from methacholine induced bronchoconstriction, and of a magnitude unlikely to be of clinical significance. BODY.COMPETING INTERESTS: The department of Pulmonology of the AMC (authors: AV, FRW, HMJ, REJ) received an unrestricted research grant for the conduction of this study and one additional study. There are no competing interests for the other authors. BODY.AUTHORS' CONTRIBUTIONS: AV conducted the study, was involved in the analysis of the data and was involved in drafting the manuscript. EAW aided in the carry out the molecular genetic assays and was involved in drafting the manuscript. RW carried out the molecular genetic assays FRW participated in the design of the study GAMH performed the statistical analysis HMJ participated in the design of the study and interpretation of the data REJ participated in the design of the study and was involved in the analysis of the data and the drafting of the manuscript

TITLE: Harmonic Scalpel-Assisted Laparoscopic Cholecystectomy vs. Conventional Laparoscopic Cholecystectomy - A Non-randomized Control Trial Introduction Laparoscopic cholecystectomy (LC) is the most commonly done, minimally invasive surgical procedure. Routinely used electrocautery produces more smoke, which masks the operating field, thereby prolongs the surgery and posing an increased risk of gallbladder (GB) perforation. The titanium clips used for clipping the cystic artery and cystic duct have a risk of slippage, which may lead to bleeding, and an increased risk for bile leakage. In addition, it may act as a nidus for stone formation. Advanced energy sources, such as the harmonic scalpel, though expensive, may provide the advantage of shorter operating time by reducing smoke, bloodless dissection in the GB bed, lower risk of bleeding from the cystic artery due to secure vessel sealing, and avoiding the use of a larger number of titanium clips. However, evidence to substantiate this advantage is limited. Aim To compare the operating time and perioperative complications between conventional laparoscopic cholecystectomy (CLC) and harmonic scalpel assisted laparoscopic cholecystectomy (HLC). Methodology All consecutive patients who underwent elective LC were included. Patients with acute infection, impaired liver function tests, concomitant common bile duct calculi, chronic liver disease/cirrhosis, suspected GB carcinoma, and pregnant women were excluded from the study. Patients were allocated into two groups. In the CLC group, both the cystic duct and the cystic artery were divided after conventional titanium clip application and electrocautery was used for thermal energy. In the HLC group, the cystic duct was clipped with a titanium clip and the rest of the procedure was carried out using Harmonic Ace (Ethicon, New Jersey, United States) and Harmonic Hook (Ethicon, New Jersey, United States). Outcome parameters analyzed were operating time in minutes, post-operative pain using visual analogue scale (VAS) scoring, frequency and route of analgesic requirement after 24 hours, and intraoperative complications, including bleeding, bile duct injury, GB perforation, and surgical site infection (SSI) in the postoperative period, per the Centers for Disease Control (CDC) criteria. Results Both the groups were comparable with respect to age, gender, body mass index (BMI), and the presence of comorbidity and an indication of cholecystectomy. The duration of surgery did not significantly differ between the groups (67.3 vs. 64.3 mins; p = 0.30). Other parameters, such as analgesic required on postoperative Day 1 (3.2 vs. 3; p = 0.67), VAS scores on Day 0 (4.55 vs. 4.65; p = 0.59), VAS scores on Day 1 (2.3 vs. 2.2; p = 0.84), superficial SSI (15% vs. 10%; p = 0.63), intraoperative GB perforation (30% vs. 20%; p = 0.71), and intraperitoneal drain (30% vs. 20%; p = 0.71) did not significantly differ between the groups. Conclusion HLC has no significant advantage over CLC with respect to operating time, postoperative pain, and perioperative complications. BODY.INTRODUCTION: Laparoscopic cholecystectomy (LC) is the gold standard for the treatment of gallstone disease. Conventionally, during LC, titanium clips are used for sealing the cystic duct and the cystic artery before dividing them. There have been reports of clip dislodgement, which poses an increased risk of bile leakage [1-3]. While using electrocautery, there is excess smoke production and an increased risk of lateral tissue damage. Due to the transmission of energy through the titanium clips, there is also an increased risk of gallbladder (GB) perforation. Slipped titanium clips also act as a nidus for stone formation [1,4]. The harmonic scalpel can seal vessels of up to 5 mm thickness without an increased risk of leakage [5-6]. The use of the harmonic scalpel in LC has been accepted by many surgeons. It is mainly used for the dissection of Calot’s triangle and the lifting of the GB from the liver bed. However, the cystic duct and cystic artery are divided after the application of conventional clips due to a fear of cystic artery and cystic duct leakage. The harmonic scalpel, which seals up to 5 mm thickness of luminal structures (vessels), can be used to divide both cystic duct and artery and has been shown as a safe method in a few studies [7-10]. The cystic duct diameter and thickness may vary between patients due to existing pathology, such as acute/chronic cholecystitis, fibrotic GB, etc. Hence, the ‘clipless’ LC has not been accepted widely due to the routine use of clips for the fear of cystic duct leak. Using the harmonic scalpel for the entire operative procedure of LC except cystic duct division may provide the advantage of a shorter operating time with a reduced risk of cystic duct leakage [11]. Hence, this study was carried out to assess the role of harmonic in LC (harmonic scalpel-assisted laparoscopic cholecystectomy - HLC), wherein the entire operating procedure of laparoscopic cholecystectomy was done using the harmonic scalpel, except for cystic duct division, which was done using titanium clip application, as compared to conventional LC (CLC). BODY.MATERIALS AND METHODS: This prospective, parallel arm, non-randomized controlled trial was carried out in a tertiary care center in south India over a period of two years. Institute Human Ethics Committee (IEC) approval was obtained for the study. The nature, methodology, and risks involved in the study were explained to the patient and informed consent was obtained. All the information collected was kept confidential and the patients were given full freedom to withdraw at any point during the study. All provisions of the Declaration of Helsinki were followed in this study. The study included all patients between 18 and 70 years of age scheduled for elective LC. Patients with acute infection, impaired liver function tests, concomitant common bile duct calculi, chronic liver disease/cirrhosis, suspected GB carcinoma, and pregnant women were excluded from the study. The patients were randomly allocated to either CLC or HLC using a computer-generated random number table. Allocation concealment was carried out with the sequentially numbered, opaque, sealed envelopes (SNOSE) technique. The envelope was opened after the patients were induced under general anesthesia. The primary outcome parameter studied was operating time in minutes, which was measured starting from skin incision for port placement to the last port closure. Other outcome parameters, such as postoperative pain, analgesic requirement, rate of GB perforation, intraperitoneal drain placement, conversion to open cholecystectomy, and surgical site infection (SSI) were also compared between HLC and CLC. Considering the reduction of 15 minutes in operating time as clinically significant, assuming a 99% confidence interval (alpha=0.01), 90% power, the sample size was calculated as 20 in each group (OpenEpi, version 3). Patients were categorized into two groups, A and B. For group A patients, the conventional four-port cholecystectomy was performed. The cystic artery and duct were clipped using titanium clips and divided. The GB was dissected from the GB fossa with monopolar diathermy. For group B patients, the harmonic scalpel was used in the 10 mm epigastric working port for securing the cystic artery. This was done at a power setting of two and the energy source was applied continuously till the end gave way. The cystic duct was divided after securing with titanium clips. The harmonic scalpel was used for dissection throughout the procedure wherever the energy device was required. The GB was separated from the liver bed using the harmonic shear and harmonic hook. All patients received prophylactic antibiotics unless contraindicated due to hypersensitivity, renal failure etc. The duration of surgery, from skin incision to skin closure, was noted in both the groups. Postoperative pain was assessed using the visual analogue scale (VAS) score at 12 and 24 hours after surgery. At the time of discharge, during the first postoperative visit and at the 30th postoperative day, patients were examined for wound infection. SSI was diagnosed according to the Centers for Disease Control (CDC) criteria. Ultrasonography (USG) of the abdomen was done during the first follow-up visit to look for any intraabdominal collection. The outcome parameters were recorded during the preoperative, intraoperative, and postoperative periods using a specified proforma. A statistical analysis was done using the SPSS 19.0 software (IBM, Armonk, New York, United States) for Windows. All categorical data between both groups were compared using the chi-square test. Percentage of wound infections and proportions were analyzed using the chi-square test. Data related to continuous variables, such as operative time, were compared using the Independent Student t-test. Continuous variables were assessed using the unpaired t-test when they follow the normal distribution. In case of non-continuous variables as well as continuous variables not following normal distribution, appropriate statistical tests were used. BODY.RESULTS: A total of 40 patients were enrolled in the study, 20 in the CLC group and 20 in the HLC group. There was no mortality in patients included in the study. The demographic profiles of the two groups were comparable. The mean age for the group undergoing conventional LC was 46.6 years ± 11.39 years, ranging from 29 years to 65 years. The mean age for the HLC group was 40.25 years ± 14.85 years, ranging from 21 years to 73 years. This suggests that cholelithiasis is most common in the age group of 40-50 years. Out of the total 40 patients enrolled in the study, 25 (62.5%) were female and 15 (37.5%) were males and the gender ratio in both the groups was comparable. Both the groups had a comparable body mass index (BMI) and distribution of various comorbidities in the study population. The American Society of Anaesthesiologist (ASA) fitness category did not significantly differ between the two groups (Table 1). Table 1BODY.COMPARISON OF BASELINE PARAMETERS BETWEEN THE GROUPS: BMI: body mass index; SD: standard deviation; ASA: American Society of Anaesthesiologist Parameters Group A (n=20) Group B (n= 20) p- value Age (mean±SD) 46.6±11.39      40.25±14.85 0.259 BMI (mean±SD) 23.62±4.22 23.55±4.75 0.612 Sex (n)  Male  9(45%)            6(30%)            -  Female  11(55%) 14(70%) - Comorbities (n) Diabetes 9(45%)            3(15%)            - Hypertension 6(30%)            4(20%)              ASA category (n) ASA l 11(55%) 12(60%) - ASA ll 9(45%)            8(40%)            - The mean duration of surgery in the CLC group was 67.3 ± 9.65 minutes, ranging from 54 minutes to 90 minutes (CI: 62.79-71.81). The mean duration of surgery in the HLC group was 64.3 ± 8.5 minutes, ranging from 49 minutes to 78 minutes (CI: 64.3-72.2). The difference in the duration of surgery between the two groups was not significant (p = 0.30) (Table 2). Table 2BODY.COMPARISON OF DURATION OF SURGERY (IN MINUTES) BETWEEN THE GROUPS: CI: confidence interval Surgery duration Group A   (n=20) Group B (n=20) p-value   Minimum 54 49     0.30 Maximum 90 78 Mean 67.3±9.65 64.3±8.5 Median 65 64.5 95% CI 62.79-71.81 60.32-68.28 The intraoperative complications between the two groups were compared (Table 3). Table 3BODY.COMPARISON OF INTRAOPERATIVE COMPLICATIONS BETWEEN THE GROUPS: GB: gallbladder Variables Group A (n=20) Group B (n=20) p-value No. of patients requiring drain 6(30%)            4(20%)            0.71 GB perforation 6(30%) 4(20%)            0.71 Out of 40 patients enrolled in the study, five patients (12.5%) had intra-operative GB perforation. Three patients (15%) in the CLC group had GB perforation while two (10%) patients in the HLC group had GB perforation. The rate of GB perforation between the two groups was not statistically significant (p = 0.714). Out of 40 patients, an intraperitoneal drain was kept in seven patients (17.5%). Six patients (30%) in the CLC group and four patients (20%) in the HLC group required an intraperitoneal drain. The difference was not significant (p = 0.716). Postoperative pain and analgesic requirement between both the groups was comparable (Table 4). Table 4BODY.COMPARISON OF POSTOPERATIVE PAIN AND ANALGESIC REQUIREMENT BETWEEN THE GROUPS: VAS: visual analogue scale Variables Group A (n=20) Group B (n=20) p-value VAS day 0 4.55±0.51 4.65±0.67        0.59    VAS Day 1     2.3±0.8 2.25±0.78 0.84 Analgesic requirement (No. of diclofenac tablets) 3.1±0.8 3±0.7 0.67 All patients were given injectable analgesics in the immediate postoperative period and were given oral diclofenac tablet 50 mg from postoperative Day 1 if the patient complained of pain. An average of three diclofenac tablets was required in the HLC group while an average of 3.2 diclofenac tablets was required in the CLC group. The number of diclofenac tablets required on the first postoperative day was not statistically significant in both the groups. Visual analogue scale (VAS) on Day 0 and Day 1 of surgery for pain assessment also did not show a significant difference. The mean VAS score on Day 0 for the CLC group was 4.55 ± 0.51, ranging from 4 to 5 (CI: 4.31-4.79). The mean VAS score on Day 0 for the HLC group was 4.65 ± 0.6, ranging from 3 to 6 (CI: 4.34-4.96). The mean VAS score on Day 1 of surgery was 2.3 ± 0.8, ranging from 1 to 4 in the CLC group. The mean score of VAS on Day 1 for the HLC group was 2.25 ± 0.78, ranging from 1 to 4. A total of five (12.5%) patients had superficial SSI. None of the patients had deep SSI. Three patients (15%) in the CLC group had superficial SSI and two patients (10%) in the HLC group had superficial SSI. The SSI rates between the two groups were not significant (p = 1.000) (Table 5). Table 5BODY.COMPARISON OF POSTOPERATIVE COMPLICATIONS BETWEEN THE GROUPS: SSI: surgical site infection Variables Group A (n=20) Group B (n=20) p-value SSI 3(15%) 2(10%)            0.63 Intra-abdominal Collection 1(5%) 0          1.0 Only one patient in the CLC group had an intraabdominal collection, which was detected on USG carried out for persistent abdominal pain on postoperative Day 5. Therapeutic aspiration showed 100 ml of serous fluid. The patient was managed conservatively and the follow-up USG did not show any residual collection. None of the patients in the HLC group developed a postoperative intra-abdominal collection. There was no incidence of major bleeding intraoperatively in both the groups and no patients in the study group required a blood transfusion. None of the patients in this study had any major postoperative complications, such as bile leakage or biliary peritonitis. None of the patients required readmission or a second surgery. BODY.DISCUSSION: The safety of the harmonic scalpel as a vessel-sealing device in a high-pressure system, such as the superior, inferior mesenteric arteries and veins, has already been established. Considering the fact that the biliary tract is a low-pressure system and the thickness of the cystic duct and artery is usually less than 5 mm except in cases having previous cholecystitis/cholangitis episodes that lead to inflammatory changes, the harmonic scalpel can be safely used. The harmonic scalpel also produces less smoke and minimizes blood loss. There is also a decreased risk of GB perforation [1].   The terminology “clipless cholecystectomy” has been used in a few studies to indicate that the total operating procedure is carried out by using the harmonic scalpel including the division of the cystic artery and cystic duct. In the present study, the harmonic scalpel was used for the dissection of Calot’s Triangle, sealing the cystic artery and the dissection of the GB from the GB fossa. The cystic duct was divided after clipping with conventional titanium clips. This was carried out to reduce the risk of bile leakage from the ligated cystic duct using the harmonic scalpel, as sealing the harmonic scalpel is limited to lumens with 5 mm thickness. Predicting the cystic duct diameter by radiological means with the help of contrast-enhanced computed tomography (CECT) also required the harmonic division of the cystic duct, which is, at times, expensive and time-consuming. In various studies done previously, there is a significant difference in the operating time between the harmonic and electrocautery groups. This can be explained by the fact that the harmonic scalpel is a multifunctional instrument [1-2,7]. It replaces four instruments routinely used in conventional LC, namely, the dissector, clip applier, scissors, and electrosurgical hook/spatula. Hence, there is no need to change instruments repeatedly, and this saves time. Also, there is no smoke production while using the harmonic scalpel. This also saves time, as the camera lens need not be cleaned repeatedly and provides a clear operative field for the surgeon to work. Operating time was significantly less in the HLC group in the study conducted by Jain et al. (64.7 ± 13.74 vs. 50 ± 9.36; p = 0.001) and Kadil et al. (61.88 ± 16.17 vs. 52.14 ± 9.8; p < 0.0001) [1,7]. In our study, we did not find a significant difference in operating times between the two groups (67.3 ± 9.65 vs. 64.3 ± 8.5; p = 0.30). This could be partly attributed to the use of clips for cystic duct ligation, which leads to increased operating time due to multiple instruments needed for clip application and the cutting of the cystic duct with scissors. Among the previous studies, the difference observed in most other studies, ranging from five minutes to seven minutes, has a questionable advantage when compared to the cost of harmonic scalpel [1,7,12]; only two of the previous studies had shown a reduction in operating time of 15 minutes in the harmonic group [2,13]. Bleeding during LC either from a slippage of clips from the cystic artery or bleeding from the GB fossa may make the operating field blurred, which may pose an advertent injury to the biliary system and prolongs the operating time. The safety of the harmonic scalpel for vessel ligation has been shown by Vu et al. in laparoscopic surgery [14]. As the cystic artery system is not a high-pressure zone, ligation and division of the cystic artery with a harmonic scalpel may be advantageous, as ligation and division can be done using a single instrument. Bleeding from the liver bed is also a commonly encountered problem that prolongs the operating time, as it takes time to control diffuse bleeding from the liver bed. Electrocautery with the ball tip is conventionally used to stop bleeding from the GB fossa; however, the crust formation and stickiness of electrocautery make  In their studies, Jain et al. and Kandil et al. have observed a significant reduction in blood loss, which was measured indirectly by means of a fall in hemoglobin and hematocrit [1,7]. No intraoperative bleeding or injury to adjacent structures was encountered in the present study. None of the patients in the present study had any fall in hemoglobin and no patients required a blood transfusion. However, a reduction in such indirect parameters for measuring intraoperative blood loss has been questioned by a few studies [13]. The clinical significance of a reduction in these parameters, which measures the few mL of additional blood loss as a statistically significant difference is also criticized by some studies [1,7]. Comparing the additional cost incurred due to the use of the harmonic scalpel, the clinically insignificant blood loss carries a questionable advantage. GB perforation is one of the most common intraoperative complications while doing LC. The perforation also makes the operating procedure difficult and long due to the continuous leakage of bile from the perforated GB during the dissection of GB from the liver bed. Causes for GB perforation are laceration due to grasper traction and lateral tissue damage due to electrocautery dissection. The harmonic scalpel reduces the lateral thermal spread and decreases the risk of GB perforation. Kandil et al., in their study, showed that the risk of GB perforation was significantly higher in the traditional group than in the harmonic group (18.6% vs. 7.1%, respectively; p = 0.04) [7]. Conversely, the risk of GB perforation was not found significant in the study conducted by Mukesh et al. [13]. Intraoperative complications also increase the risk of GB perforation, as evident in the study of Mahabaleshwar et al., which revealed a 14.23 times greater risk of GB perforation in the presence of complications [2]. In our study, six patients (30%) in the CLC group had GB perforation while four patients (20%) in the HLC group had GB perforation; the difference was not significant with a p-value of 0.78. None of the patients in the present study had any intraoperative complications in terms of bleeding, bile duct injury, etc. The experience of surgeons and experience in handling conventional electrocautery and harmonic scalpel instruments also play a vital role in GB perforation. Careful use of electrocautery in experienced hands may lead to less incidence of GB perforation, as in the present study, where the HLC group had no significant reduction in GB perforation. Pain in the immediate post-operative period is mostly due to visceral irritation. In the CLC group, the effect spreads laterally up to 0.5 cm as compared to 1.5 mm in ultrasonic shears. This leads to increased thermal damage and charring in surrounding tissues and nerve structures, leading to increased postoperative pain in the CLC group. Jain et al. noted that post-operative pain was significantly less in the harmonic shear group [1]. This is due to less release of inflammatory mediators, as there is less lateral tissue and nerve damage. Also, the duration of peritoneal distension is less due to the shorter surgery duration, thereby directly affecting the duration and degree of traction to vessels and nerve. Since there is less risk of GB perforation while using the harmonic scalpel, the degree of pain also reduced because of less inflammation due to bile leakage. Mahabaleshwar et al. also concluded that the postoperative pain is less in the harmonic scalpel group [2]. Post-operative pain scores after 24 hours were found to be significantly better in HCL by Kandil et al. as well (4.48 ± 1.89 vs. 3.12 ± 1.84; p = 0.000) [7]. Postoperative pain depends on the duration of surgery, personality and sensitivity of the patient, and intraoperative complications, such as biliary spillage. Since in this study, duration of surgery and GB perforation are not statistically significant, this could have contributed to the statistical insignificance of the postoperative pain score in this study. Only a few studies compared the analgesic requirement in the CLC and HLC groups [1]. As previous studies have shown, pain scores are less in patients undergoing harmonic LC, so less analgesic is needed. But, the study conducted by Jain et al. found a significantly lower analgesic requirement in the ultrasonically activated scalpel group (2.66 ± 0.66 vs. 1.89 ± 0.59; p = 0.001) [1]. However, in the present study, no difference in the analgesic requirement was found between the two groups which could be explained by the fact that in both the groups, the VAS pain scores were similar, indicating that the postoperative pain and analgesic consumption expected to be comparable. If bile leakage or oozing/bleeding from the vessel is suspected, intraperitoneal drainage can effectively decrease the morbidity by effectively draining out the intraabdominal contents and reducing the risk of stasis and infection. The placement of the drain increases operative time and increases morbidity. In this study, drains were kept for patients in whom significant bile spillage occurred due to GB perforation. Intraperitoneal drains were kept for six patients in the CLC group and four patients in the HLC group. The test was not significant (p-value: 0.74). This could be explained by the fact that the rate of GB perforation was not statistically significant. Also, in the study conducted by Kaul et al., no statistical difference was found in intraperitoneal drain placement in the two groups [12]. The drains in each group were removed within 48 hours. The risk of SSI is less in laparoscopic procedures as compared to open surgeries, as the size of the incision is small. The risk of SSI depends on various factors, such as duration of surgery, spillage of bile, intra-abdominal collection due to postoperative bile leak, retrieval of GB through the port, presence of drain, and comorbidities such as diabetes. In our present study, three patients (15%) in the CLC group had developed superficial SSI. Out of three, two patients were diabetic and one patient had an intraperitoneal drain. Two patients (10%) in the harmonic group had developed superficial SSI and both patients were diabetic. The overall SSI in this study was 12.5%. In this study, the incidence of perforation of GB was comparable between the two groups and nor had we encountered complications such as bleeding or bile leakage in this study. Hence, the SSI rate of both the groups did not vary significantly with a p-value of 1.000. In all patients, infection subsided with oral cloxacillin for five days. Similar results have been shown in many studies [2,12,15]. The length of hospital stay couldn’t be assessed in the present study. Being a tertiary care and teaching hospital, the cancellation of cases were inevitable, hence, we could not assess the hospital stay of our patients. In this study, only an indirect assessment, such as a drop in postoperative hemoglobin, was carried out and an accurate quantification of intra-operative blood loss could not be assessed, which could be an important parameter. We could not randomize patients due to logistics. Further randomized trials with a bigger number of patients may provide added evidence that is not addressed in the present study. BODY.CONCLUSIONS: There was no significant difference in operating time, incidence of GB perforation, and intraperitoneal drain placement between the HLC and CLC groups. Further, there was no significant reduction in postoperative pain and analgesic requirements between the groups. In addition, there was no significant reduction in the postoperative SSI or intra-abdominal collection. In our study, HLC has no significant advantage over CLC. Further randomized trials are required to substantiate a clear advantage of the harmonic scalpel over conventional electrocautery for LC.

TITLE: The effect of sensor-based exercise at home on functional performance associated with fall risk in older people – a comparison of two exergame interventions ABSTRACT.BACKGROUND: There is good evidence that balance challenging exercises can reduce falls in older people. However, older people often find it difficult to incorporate such programs in their daily life. Videogame technology has been proposed to promote enjoyable, balance-challenging exercise. As part of a larger analysis, we compared feasibility and efficacy of two exergame interventions: step-mat-training (SMT) and Microsoft-Kinect® (KIN) exergames. ABSTRACT.METHODS: 148 community-dwelling people, aged 65+ years participated in two exergame studies in Sydney, Australia (KIN: n = 57, SMT: n = 91). Both interventions were delivered as unsupervised exercise programs in participants’ homes for 16 weeks. Assessment measures included overall physiological fall risk, muscle strength, finger-press reaction time, proprioception, vision, balance and executive functioning. ABSTRACT.RESULTS: For participants allocated to the intervention arms, the median time played each week was 17 min (IQR 32) for KIN and 48 min (IQR 94) for SMT. Compared to the control group, SMT participants improved their fall risk score (p = 0.036), proprioception (p = 0.015), reaction time (p = 0.003), sit-to-stand performance (p = 0.011) and executive functioning (p = 0.001), while KIN participants improved their muscle strength (p = 0.032) and vision (p = 0.010), and showed a trend towards improved fall risk scores (p = 0.057). ABSTRACT.CONCLUSIONS: The findings suggest that it is feasible for older people to conduct an unsupervised exercise program at home using exergames. Both interventions reduced fall risk and SMT additionally improved specific cognitive functions. However, further refinement of the systems is required to improve adherence and maximise the benefits of exergames to deliver fall prevention programs in older people’s homes. ABSTRACT.TRIAL REGISTRATIONS: ACTRN12613000671763 (Step Mat Training RCT) ACTRN12614000096651 (MS Kinect RCT) BODY.BACKGROUND: Unstable balance, lower extremity muscle weakness and impaired cognitive functions (executive functions, processing speed) are important risk factors for falls in older adults [1]. There is strong systematic review evidence that exercise interventions including high intensity balance training (i.e., exercises that reduce the base of support, minimize upper limb support and include weight shifting) are most effective in preventing falls in older people [2]. To our knowledge, there is no study investigating the impact of cognitive training on falls, but robust evidence suggests that physical exercise may improve cognition [3]. One study found that an exercise program known to reduce falls led to improved response inhibition in addition to physical improvements of balance and muscle strength [4]. It has been suggested that in order to reduce falls, older people should engage in at least 2 h of strength and balance training per week over an extended period of time [5]. In order to reach this exercise training dose, without overwhelming specialist services, older people need to perform at least part of the training at home. However, incorporating a new habitual exercise regimen is challenging for many older people due to poor exercise tolerance and enjoyment [6]. Furthermore, additional challenges arise when conducting unsupervised exercise programs in home settings where factors such as adherence, progression, safety, quality and dosage are harder to control compared to exercise under supervision of an exercise professional. Recently, videogame technology has been increasingly used to deliver exercise programs (often referred to as “exergames”) to address this problem. Exergames offer engaging video games instead of repetitive conventional exercises, provide instant performance feedback and unlock levels of difficulty according to the individual’s performance. Accordingly, exergames might enhance motivation and replace or complement more resource-demanding traditional approaches of exercise delivery [7, 8]. However, while a plethora of such exergames is available, evidence-based programs in a home setting remain sparse. A recent systematic review of 37 studies concluded that there is preliminary evidence that exergame interventions can improve physical and cognitive fall risk factors in older people [9]. It further suggested that these exergame interventions are of equivalent efficacy in reducing fall risk as traditional training programs. However, the review also found that the methodological quality of the included studies was often poor, sample sizes were mostly small, and only three [10–12] of the 37 studies were carried out unsupervised by older people at home. Clearly, more research is required before exergames could be recommended as a fall prevention strategy. A wide variety of exergames has been explored which can provide challenging balance exercises. Two promising exergame interventions that require further investigation are step mat training (SMT) and Microsoft Kinect® (KIN) exergames. As part of the iStoppFalls research program [13], we compared the feasibility and efficacy of SMT (trial registration ACTRN12613000671763) and KIN strength and balance exercises (trial registration ACTRN12614000096651). Specifically, we compared the two home-based interventions that took place in Sydney, Australia, with respect to adherence and efficacy in relation to functional performance outcomes in older people. SMT was played by using a step mat as input device on which participants had to repetitively step in multiple directions under varying cognitive load that required attention, executive control and fast processing. KIN player’s movements were mirrored by an avatar in a virtual environment on a television (TV). KIN games were specifically designed to improve balance and lower extremity strength. Based on the exergame components and principle of task specificity in exercise training, we hypothesised that the SMT would have a greater effect on improving cognitive functioning whereas KIN would have greater effects on muscle strength and balance. BODY.METHODS: BODY.PARTICIPANTS: The sample comprised 148 participants from the SureStep study and the Australian arm of the iStoppFalls study: (KIN: n = 57, SMT: n = 91). Participants were recruited from retirement villages and the community in Sydney, Australia. Individuals were eligible if they were: 1) aged 65 years or older, 2) living independently, 3) able to walk with or without a walking aid. For the SMT randomised controlled trial (RCT), potential participants were 70 years or older, and were excluded if they were not able to step unassisted on a step pad (step size 25–30 cm), or had severe lower extremity pain that prevented them from step training. For the KIN RCT, participants had to be able to watch TV with or without their glasses from 3 m distance. General exclusion criteria were: 1) major cognitive impairments (Mini-Cog score <3) [14], 2) medical conditions preventing regular exercise (i.e., neurodegenerative disease, cardiovascular disease, psychiatric disorder), 3) colour blindness and 4) insufficient language skills to understand the study procedures. All participants gave written informed consent prior to inclusion. Ethical approval was given by the Human Research Ethics Committee of the University of New South Wales. BODY.RANDOMIZATION: In both studies, eligible participants were randomised (ratio 1:1) by permuted block-randomisation using computer-generated random numbers. Block size was random and ranged from four to eight. Participants living in the same household were treated as one unit and randomised into the same block. BODY.INTERVENTION DESIGN: The interventions were delivered as unsupervised exercise programs in participants’ homes. Both, the KIN and SMT systems are described in detail elsewhere [13, 15]. Both contained an input device to record exercise data, a computer to deliver the intervention tasks and store the data, and universal serial bus (USB) modems to provide remote access to the computers by research staff. Additionally, the participants’ TVs were used as projection screens for the exergames. During a 90 min introductory session the systems were installed in an appropriate location within participants’ homes. Participants were provided with teaching manuals and instructed on how to use the system and play the games safely. Exercise data (i.e., frequency, duration, game level) for both 16-week interventions were stored on the systems’ computers. Participants had continuous access to their performance scores on the physical tests (KIN) and games (SMT, KIN). To discuss any issues related to system use and exercises, participants were contacted by monthly phone calls. In case of technical issues, participants could call the research team for phone support, or to request additional home visits. The KIN exergames consisted of three specifically developed balance exergames (i.e., walking, stepping, weight shifting) based on the Weight-bearing Exercise for Better Balance (WEBB) program (www.webb.org.au), and five strength exercises (i.e., knee extension/flexion, hip abduction, calf/toe raises) based on the Otago Exercise Program [16, 17] targeting fall risk factors such as unstable balance and muscle weakness of the lower limbs. Participants’ movements were recorded by the Kinect (3D depth sensor) and displayed as an avatar on screen. Participants were encouraged to perform 120 min of balance exergames per week and 60 min of strength exercises per week. Progression of training intensity was possible by increasing the level of difficulty (i.e., by including secondary cognitive memory tasks (dual-tasking) for balance exergames) and by increasing the number of repetitions and sets or ankle cuff weights (1 kg, 2 kg or 3 kg) for strength exercises. The SMT comprised of exergames each targeting specific cognitive functions associated with fall risk and stepping: a modified StepMania game (divided attention, inhibition and processing speed; timed and coordinated stepping according to arrows on screen that differed in their direction and drift speed), Stepper (processing speed, selective attention; rapid stepping in four directions), Trail-stepping (visual attention, set-shifting; stepping to connect numbers and letters) and Tetris-stepping (visuo-spatial skills, problem-solving; stepping used to rotate and control blocks of different shapes). Participants were able to play the exergames by stepping onto six arrows (pointing to the front, side and back) on a pressure-sensitive electronic mat. Participants were instructed to perform a minimum of three 20 min sessions per week each including all four exergames. Progression was possible by the selection of more difficult exergame levels with higher cognitive load, stepping speed and complexity. The control group was provided with an educational booklet about evidence-based health and fall prevention advice [18], and asked to remain their usual activities for the duration of the study period. BODY.OUTCOMES: Participants were assessed at baseline and at the end of the intervention period (16 weeks) by blinded assessors. Socio-demographic and medical information was collected by self-report questionnaires. The 12-item World Health Organization Disability Assessment Schedule (WHODAS) 2.0 was used as a generic assessment instrument of health and disability. Participants reported their level of impairment for several instrumental activities of daily living on a five-point Likert scale (www.who.int/classifications/icf/whodasii/en/). The Mini-Cog was used as screening test to detect major cognitive impairments. It consists of two tasks, delayed recall of three items and the clock drawing test as a measure of executive functioning and visuo-spatial ability [2]. Anthropometric measures were obtained during the assessments. The following assessment measures were included in the comparison analyses. Fall risk was assessed by the Physiological Profile Assessment (PPA) which includes five sensorimotor tests [19]: (i) the Melbourne Edge Test of visual contrast sensitivity; (ii) a lower limb-matching task to assess proprioception, with errors in degrees recorded using a protractor marked on a vertical clear acrylic sheet placed between the legs; (iii) quadriceps strength measured isometrically in the dominant leg with participants seated with the hip and knee flexed 90°; (iv) finger-press reaction time assessed using a light as stimulus and a finger-press as the response; (v) postural sway measured using a sway meter recording displacements of the body at the level of the pelvis with participants standing on a foam rubber mat with eyes open. Weighted contributions from these measures have been shown to discriminate between older fallers and non-fallers with an accuracy of up to 75 % [20]. Functional mobility was assessed using the timed up and go test (TUG) [21], and the five times sit-to stand test (STS) [22]. For the TUG, participants were asked to stand up from a chair, walk to a three meter mark at their self-selected usual pace, return, and sit down in the chair again. For the STS, participants were instructed to cross their arms over the abdomen, and rise from a chair for five times as quickly as possible. For both tasks, the time to complete was recorded in seconds. Cognitive performance was assessed using the Attention Network Test (ANT) [23], the Victoria Stroop Test (VST) [24], and the Digit span backward (DSB) Test [25]. The ANT requires participants to determine whether a central arrow points to the left or right, and was used to quantify processing efficiency within three attentional networks (alerting, orienting, executive attention) by measuring how response times are influenced by alerting cues, spatial cues, and flankers. The VST requires participants to state a colour under three conditions, while supressing habitual responses related to the conditions. The VST was used to assess executive control by response inhibition. For this secondary analysis we only used the following VST outcomes: time to complete and number of errors made during the colour-word interference task (condition 3) and the efficiency score of inhibition calculated as the ratio of colour-word interference and colour only tasks (condition 3/condition 1). The DSB requires participants to repeat sequences of numbers with increasing length (two to nine digits) in reverse order as stated by the investigator, and was used as a measure of working memory. BODY.STATISTICAL ANALYSES: Variables used in this comparison analysis were normally distributed with or without log-transformation. Differences between groups at baseline were analysed by analysis of variance (ANOVA) with Tukey’s post-hoc tests for pairwise comparisons for continuous variables and chi square tests for categorical data. Generalized linear modelling (GLM) was used to determine the intervention effects for both training modes and comparing it with the combined control group. Age and baseline performance of the variable under investigation were entered as covariates in all models. The alpha level was set at 5 %. Analyses were performed with SPSS version 22 for Windows (SPSS, Inc., Chicago, IL). BODY.RESULTS: Of the 148 people recruited (KIN = 57, SMT = 91); 76 were allocated to the intervention groups (KIN = 29, SMT = 47) and 72 to the control groups (KIN = 28, SMT = 44). Participant characteristics are summarised in Table 1. Participants across the two studies did not differ in demographic characteristics (Table 1). However, there were differences in some of the outcome measures at baseline (Table 2). At baseline, the KIN intervention group performed better than the SMT intervention group and combined control group in a number of measures: PPA overall score (F = 7.69, df = 2, p = 0.001), proprioception (F = 5.14, df = 2, p = 0.007), postural sway (F = 6.16, df = 2, p = 0.003), and DSB (F = 7.449, df = 2, p = .001). One-hundred and twenty-four participants were re-assessed after the completion of the training period (KIN = 43, IG = 24, CG = 19; SMT = 81, IG = 39, CG = 42) and included in the analyses using intention-to-treat principles. Figure 1 shows the flow of participants through the studies (Fig. 1).Table 1Participant characteristics (mean and standard deviation) KINSMTControl p-value(n = 24)(n = 39)(n = 61)Age (years) 80.1 (6.3) 82.5 (7.0) 80.2 (6.5) 0.195 Height (cm) 161.8 (7.7) 163.2 (9.7) 162.5 (10.1) 0.863 Weight (kg) 69.8 (12.3) 73.4 (18.3) 70.1 (13.9) 0.526 Medication use (n) 4.6 (3.0) 4.2 (3.0) 4.4 (3.0) 0.872 Comorbidities (n) 3.2 (1.5) 3.0 (1.6) 3.1 (1.8) 0.934 WHODAS (score) 16.8 (3.7) 16.4 (5.0) 16.2 (4.0) 0.822 Mini-Cog (score) 4.6 (0.7) 4.4 (0.8) 4.5 (0.7) 0.571 Gender (female) 62.5 % 69.2 % 65.6 % 0.853 Fallen past year (yes) 37.5 % 35.9 % 24.6 % 0.352 Use of walking aids (yes) 16.7 % 23.1 % 27.9 % 0.544 ®KIN Microsoft-Kinect®, SMT step-mat-training, WHODAS World Health Organization Disability Assessment Schedule Table 2Group comparison of physical and cognitive outcomes at baseline (mean and standard deviation, SD) KINSMTControl p-valuePPA score 1.1 (0.8) 2.0 (0.9) 1.8 (1.1) 0.001 *,** Contrast sensitivity scoreb 21.6 (1.9) 20.8 (2.2) 21.0 (2.6) 0.412 Proprioception (degree)a 1.8 (1.1) 2.9 (1.7) 2.6 (1.6) 0.007 *,** Knee extension strength (kg)b 20.8 (9.4) 24.2 (10.3) 21.9 (8.6) 0.318 Standing balance (mm)a 261 (124) 377 (152) 379 (188) 0.003 *,** Finger-press reaction time (ms) 240 (45) 259 (45) 247 (45) 0.225 Timed up & go (s)a 11.5 (3.5) 11.5 (3.1) 12.4 (3.7) 0.317 Sit-to-stand transfer (s)a 13.6 (4.8) 13.1 (3.8) 14.4 (4.9) 0.340 ANT alert (ms)b 34 (32) 34 (35) 39 (33) 0.789 ANT orient (ms)b 40 (43) 60 (43) 44 (45) 0.128 ANT conflict (ms)a 118 (67) 154 (120) 137 (74) 0.533 Stroop CW_incongruency - time (s) 68.3 (32.3) 65.0 (28.4) 75.5 (38.0) 0.314 Stroop CW_incongruency - errorsa 4.5 (3.4) 6.1 (4.0) 6.2 (5.4) 0.270 Stroop efficiency (CW_incongruent/C) 2.0 (0.5) 2.2 (0.8) 2.4 (1.0) 0.147 Digit span backwards (length of sequence)ab 6.8 (2.6) 5.1 (1.0) 5.3 (1.4) 0.001 *,** aLog transformed bHigher values indicate better performance KIN Microsoft-Kinect®, SMT step-mat-training, PPA Physiological Profile Assessment, ANT Attentional Network Test, CW colour-word, C colour *Post-hoc tests (Tukey) results indicate difference between KIN and Control **Post-hoc tests (Tukey) results indicate difference between KIN and SMT Table 3Between-group intervention effects KIN (IG 1)SMT (IG 2)ControlPrePostPrePostPrePost p-value p-value p-valueMean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)Mean (SD)IG1-IG2IG1-CGIG2-CGPPA score 1.1 (0.8) 0.9 (1.1) 2.0 (0.9) 1.5 (0.8) 1.8 (1.1) 1.6 (0.9) .866 .057 .036 Contrast sensitivity scoreb 21.6 (1.9) 23.0 (1.1) 20.8 (2.2) 21.4 (1.6) 21.0 (2.6) 21.9 (2.0) .003 .010 .462 Proprioception (degree)a 1.8 (1.1) 1.8 (1.3) 2.9 (1.7) 2.0 (1.3) 2.6 (1.6) 2.5 (1.6) .486 .192 .015 Knee extension strength (kg)b 20.8 (9.4) 26.2 (10.3) 24.2 (10.3) 25.8 (9.2) 21.9 (8.6) 23.8 (9.1) .131 .032 .578 Standing balance (mm)a 261 (124) 263 (159) 377 (152) 332 (131) 379 (188) 337 (189) .481 .431 .967 Finger-press reaction time (ms) 240 (45) 239 (49) 259 (45) 238 (40) 247 (45) 247 (41) .057 .611 .003 Timed up & go (s)a 11.5 (3.5) 11.1 (3.3) 11.5 (3.1) 11.5 (2.6) 12.4 (3.7) 12.6 (4.4) .298 .190 .848 Sit-to-stand transfer (s)a 13.6 (4.8) 12.1 (3.7) 13.1 (3.8) 11.4 (2.7) 14.4 (4.9) 13.3 (3.8) .370 .197 .011 ANT alert (ms)b 34 (32) 45 (36) 34 (35) 35 (33) 39 (33) 37 (38) .285 .243 .990 ANT orient (ms)b 40 (43) 47 (47) 60 (43) 57 (38) 44 (45) 57 (40) .886 .375 .418 ANT conflict (ms)a 118 (67) 111 (46) 154 (120) 103 (41) 137 (74) 111 (46) .196 .267 .720 Stroop time (s) 68.3 (32.3) 62.5 (28.3) 65.0 (28.4) 26.8 (21.0) 47.1 (33.1) <.001 <.001 .001 Stroop errorsa 4.5 (3.4) 3.9 (3.8) 6.1 (4.0) 4.7 (4.2) 6.2 (5.4) 5.6 (5.0) .516 .437 .085 Stroop efficiency 2.0 (0.5) 1.9 (0.7) 2.2 (0.8) 1.9 (0.7) 2.4 (1.0) 2.2 (0.8) .298 .589 .054 Digit span backwardsab 6.8 (2.6) 6.5 (2.6) 5.1 (1.0) 5.2 (1.0) 5.3 (1.4) 5.3 (1.4) .634 .263 .473 aLog transformed bHigher values indicate better performance IG inervention group, CG, control group, KIN Microsoft-Kinect®, SMT step-mat-training, PPA Physiological Profile Assessment, ANT Attentional Network Test, CW colour-word, C colour Note: Significant p-values in bold, p-values between .1 and .05 in italics Fig. 1Study flow chart For participants allocated to the intervention arms, the median exercise time for the KIN system was 4.5 (interquartile range, IQR 8.4) hours and 12.7 (IQR 25) hours for the SMT system during the 16 week intervention periods. The median time played each week was 17 min (IQR 32) for KIN and 48 min (IQR 94) for SMT. No adverse events associated with the intervention were reported for either study. However, technical difficulties impacted on training time. Between-group differences (controlling for age and baseline performance) for the two interventions are presented in Table 3, compared to the combined control group and compared to each other. Compared to the control group, the KIN intervention group improved significantly in tests of contrast sensitivity and isometric knee extension strength and showed a trend towards improved PPA fall risk scores. A significant between-group difference in Stroop time was due to an improved performance in the control group compared to little change in the KIN intervention group. The SMT intervention group had significantly improved PPA fall risk scores at reassessment compared with the control group and showed significant improvements in the proprioception, finger-press reaction time, sit-to-stand and Stroop time as well as trends for improvements in the number of errors made and the efficiency score in the Stroop test. When comparing the two intervention groups, the following outcome measures were different at re-assessment: KIN participants had significantly better contrast sensitivity than SMT participants and SMT participants had a faster Stroop time than KIN training participants. There was also a trend indicating SMT participants had faster finger-press reaction times than KIN participants. BODY.DISCUSSION: Our findings are partly in line with the hypotheses that SMT would have a greater effect on cognitive functioning and KIN training would have greater effects on strength and balance. SMT, with its strong motor-cognitive component, did improve cognitive processing measures (processing speed, inhibition) as well as proprioception, sit-to-stand times and overall physiological fall risk. The KIN training, with its specific strength and balance exercises, improved knee extension strength as well as visual contrast sensitivity. Both intervention groups reduced their overall physiological fall risk (as measured by the PPA) compared to the control group suggesting the interventions have potential efficacy for fall prevention. However, when comparing the two intervention groups directly, KIN participants improved more than SMT participants in the contrast sensitivity test and SMT participants improved more than KIN participants in the Stroop and finger-press reaction time tests. Some effects of the two interventions require comment. The obtained findings reflect task-specific changes. First, the SMT included games that required fast central processing, dual-tasking and inhibiting irrelevant stimuli. Therefore, the improvement in finger-press simple reaction time in the SMT group suggests SMT-induced improvements in central processing and is consistent with a previous step training study [26]. Improvements in time and efficiency of the Stroop task may be underpinned by this faster processing. However, the reduced number of errors suggests additional improvements in selective attention and inhibitory processes. Second, improvements in PPA scores in both intervention groups are due to changes in different functions. SMT showed significant improvements in finger-press reaction time and proprioception, the KIN training group improved significantly in muscle strength and contrast sensitivity. The improvement in vision following KIN training is consistent with previous research that has found associations between computer game play and visual improvements. For example, Hale et al. [27] have reported that simple computer skills training improved contrast sensitivity in older people and Li et al. [28] found that young expert action video game players had superior contrast sensitivity compared to age- and gender-matched non-action game players. Therefore, navigation through the system menu from a 3 m distance and playing exergames that required central and peripheral vision under changing contrast conditions and accurate, visually guided actions could have resulted in improved vision. Finally, both exergames emphasised balance training but did not improve postural sway suggesting that participants did not challenge their postural stability at the appropriate intensity and/or duration to improve balance skills. While we acknowledge that other more task-specific measures of dynamic balance might have been more suitable, it should be noted that both trials did not find beneficial effects for other balance measures [papers under revision]. A pilot study of the SMT found significant improvements of postural sway after eight weeks of Stepmania training in well-functioning older people [7]. The different finding may be partially explained by a higher proportion of frail people in the current SMT study, suggesting that this type of training might be more suitable for vigorous older people. On the other hand, the current SMT study added games with an emphasis on cognitive tasks resulting in reduced training time of dynamic postural control challenge [29] compared to the Stepmania game training only. As the KIN exergames were adapted from successful training programs, the lack of improvement was most likely due to insufficient exercise time. Therefore, while some differences between the interventions were due to different training content and task-specificity, adherence to the intervention was another major factor impacting on the results. Adherence in both studies was below the recommended doses (SMT 60 min and KIN 120 min per week), and this may have resulted in an insufficient exercise dose to induce benefits. However, despite low adherence rates several improvements were achieved, suggesting a favourable dose-response relationship. The two exergames used in this study both have advantages and disadvantages. Both systems require significant space in participants’ homes. A major difference between the two systems is the physical mat necessary for SMT. A stepping mat placed on the floor could be viewed as an advantage, because it provides real (tangible) targets for participants during step games. On the other hand, such fixed targets could be seen as a disadvantage as participants have to keep touch with the stance position in order to perform a correct step. Step mats could also present trip hazards and restrict games to those that require stepping. By providing greater flexibility in game designs, step directions and distances, KIN addresses some of these limitations. However, current camera capture abilities are sometimes limited (e.g., bright sun light may disturb sensor capturing). Finally, as the KIN system requires participants to interact with the game through gestures, a greater level of tech-savviness is required. Both exergame interventions are in initial research and development stages. Therefore, the low exercise adherence can to a large extent be explained by technical difficulties related to the prototype versions of the systems. While individuals frequently reported enjoying playing the games, they often could not manage to start them due to technological issues (bugs), system complexity (multiple components) and usability problems (navigating through the menu). The majority of participants in both interventions required additional visits by research staff to fix technical problems and provide additional training. As part of the studies, participants were offered extensive support which would not be feasible in a real-life setting. These findings therefore suggest the need for substantial system refinement (i.e., reliability and usability) in order to be suitable for older individuals with limited technical experience and skills. Particularly, the development of one-touch solutions is promising to maximise participant acceptability and subsequent adherence and efficacy. This study had a number of strengths and limitations. A major strength was the comparison of two exergame interventions that were carried out in an unsupervised home setting. It thus provides results of “real world” training and older people’s ability to use exergame technology. The findings that both exergames were safe and improved some aspects of fall risk are encouraging. We also acknowledge certain limitations related to the statistical analysis design. Although considerable efforts were made to use common methodologies across the two studies, logistical issues resulted in study samples that differed with respect to a number of baseline measures. For example, the different minimum age-limit resulted in one participant aged below 70 years in the KIN study. However, the adjustments for baseline test scores and age in the statistical analyses will have controlled for such differences. Sample size differences may have resulted in a greater likelihood of detecting significant differences for SMT, and this appears to be the case for PPA scores where the effect sizes for improvements in both interventions were similar (and significant improvements were evident in the iStoppFalls sample as a whole in preparation). Finally, as indicated above, technological problems contributed to relatively high dropout rates and reductions in training time. Future studies investigating the effectiveness of technology-based intervention programs should include a process evaluation to explore how and to what extent technological problems affect the intervention [30]. However, in this emerging field, improvements to equipment (cameras and step mats), software, internet connectivity and game interfaces are highly feasible. With increasing information technology (IT) awareness among older people, such refinements would present significant scope for acceptable and efficacious training systems in the future. BODY.CONCLUSION: The study findings indicate the safe use of exergame interventions in the home setting. Both interventions reduced physiological fall risk while SMT additionally improved central processing speed and specific executive functions. Neither intervention improved balance control. Adherence to both interventions was impacted by technical problems with the exergame systems. Further refinement is required to maximise the benefits and potential that exergames may have for the delivery of home-based fall prevention programs in older people. BODY.INFORMED CONSENT: All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.

TITLE: Evaluation of the safety and efficacy of an intravenous nanocrystal formulation of meloxicam in the management of moderate-to-severe pain after bunionectomy ABSTRACT.OBJECTIVE: This randomized, double-blind, placebo-controlled study evaluated the safety and efficacy of an intravenous (IV) nanocrystal formulation of meloxicam in subjects with moderate-to-severe pain following a standardized unilateral bunionectomy. ABSTRACT.METHODS: Fifty-nine subjects aged 18–72 years were randomized to receive doses of either 30 mg (n=20) or 60 mg (n=20) meloxicam IV or placebo (n=19), administered once daily as bolus IV injections over 15–30 seconds (two or three doses). Safety, the primary objective, was assessed by physical examination, clinical laboratory tests, and the incidence of adverse events (AEs). Efficacy was evaluated by examining summed pain intensity differences over the first 48 hours (SPID48) using analysis of covariance models. Use of opioid rescue analgesic agents was evaluated. ABSTRACT.RESULTS: Generally, AEs were mild-to-moderate in intensity, and their incidence was similar across the three treatment groups. No serious AEs were reported; there were no withdrawals due to AEs, including injection-related AEs. The estimated effect size for SPID48 versus placebo was 1.15 and 1.01 for meloxicam IV doses 30 mg and 60 mg, respectively (P≤0.01). Both doses produced significantly greater pain reductions versus placebo (P≤0.05) at all evaluated times/ intervals during the 48-hour period. The proportions of subjects with ≥30% and ≥50% overall reduction in pain from baseline after 6 and 24 hours were significantly higher with meloxicam IV 30 mg doses versus placebo, but not with meloxicam IV 60 mg doses. The time to first use of rescue medication was significantly longer versus placebo with meloxicam IV 60 mg (P<0.05), but not with meloxicam IV 30 mg doses. ABSTRACT.CONCLUSION: Meloxicam IV was generally safe and well tolerated in subjects with moderate-to-severe post-bunionectomy pain. Once-daily administration of meloxicam IV 30 mg and 60 mg exhibited rapid onset of analgesia (as early as 15 minutes) with maintenance of analgesic effect for two consecutive 24-hour periods. ABSTRACT.VIDEO ABSTRACT: ABSTRACT.INTRODUCTION: Intense pain is common after bunionectomy,1 particularly in the first few days after surgery. Post-bunionectomy pain is generally classified as a hard-tissue pain model,2 and its evaluation is useful for assessing the effectiveness of analgesic agents that have a rapid onset of action.3 As with other postoperative pain settings, effective management of pain is an important component of patient care and affects the patient’s ability to resume normal activities after surgery.4 Although opioids traditionally have been the mainstay of peri- and postoperative pain management, opioid-related adverse events (AEs) such as respiratory depression, sedation, nausea/vomiting, and constipation can potentially limit the benefit of these medications. Thus, there is a need for non-opioid analgesics that provide pain control while reducing the risk of AEs.5,6 Selection of pain control strategies for bunion surgery – which include local infiltration of anesthetics and/or peripheral nerve block procedures,1,7,8 opioids,9–11 nonsteroidal anti-inflammatory drugs (NSAIDs),12 and various other analgesics (eg, acetaminophen) – is generally guided by the intensity of the post-bunionectomy pain, the duration of the analgesia provided, and the tolerability and patient acceptance of the chosen strategy. Patient acceptance of opioid analgesics may be influenced by AEs such as respiratory depression, nausea/vomiting, urinary retention, dysphoria, and possible long-term dependence. Potential AEs of nonselective (ie, COX-1 and COX-2 inhibitors) NSAIDs include impaired platelet and renal function, and gastrointestinal intolerance, which could limit patient acceptance of these agents. Patient tolerability of the chosen medication is crucial for attaining rapid and adequate pain relief. Meloxicam, a preferential COX-2 inhibitor with analgesic, antipyretic, and anti-inflammatory properties, with better gastrointestinal tolerability compared with nonselective NSAIDs, has been proven effective when administered orally for ameliorating the signs and symptoms of rheumatoid arthritis and osteoarthritis.13–16 However, largely due to its poor solubility, orally administered meloxicam is not rapidly absorbed; peak plasma concentrations after a dose of 30 mg are not reached until 9–11 hours after administration.14,17,18 Consequently, this slow onset of action is a reason why oral meloxicam is not currently approved for the management of acute pain. A novel nanocrystal colloidal dispersion formulation of meloxicam (N1539; Recro Pharma, Inc., Malvern, PA, USA) has recently been developed for bolus intravenous (IV) administration, providing faster onset of analgesia than can be achieved with oral administration.19 In a randomized, double-blind, placebo-controlled study in females who underwent abdominal hysterectomy, the IV nanocrystal formulation of meloxicam was effective in relieving moderate-to-severe postoperative pain.20 All single doses of meloxicam IV evaluated (5 mg to 60 mg) resulted in significantly lower pain intensity (PI) scores and better global pain-control scores than placebo; doses >5 mg also achieved significantly better pain-relief scores than morphine (10 mg to 15 mg), and the use of rescue medication was lower in all meloxicam IV dose groups than in the morphine and placebo groups.20 The present Phase 2 study (NCT02675907) was designed to evaluate the safety and efficacy of the IV nanocrystal formulation of meloxicam administered in doses of 30 mg and 60 mg compared to placebo in subjects with moderate-to-severe pain following a standardized bunionectomy procedure. The primary objective was to evaluate the safety of meloxicam IV administered as a bolus injection over 15–30 seconds (rather than 1–2 minutes, as in earlier studies) by assessing vital signs, clinical laboratory findings, electrocardiography (ECG) changes, wound healing, and the occurrence of AEs. The principal efficacy objective was the estimated effect size of the two doses of meloxicam IV, determined via time-weighted summed PI differences (PID) over the first 48 hours (SPID48), relative to placebo. ABSTRACT.METHODS: ABSTRACT.STUDY DESIGN AND SUBJECTS: This Phase 2, randomized, double-blind, placebo-controlled trial was performed at a single center in the United States (Chesapeake Research Group, Pasadena, MD) in males and females aged 18–75 years in good health (American Society of Anesthesiology class 1 or 2) who were scheduled to undergo a primary, unilateral, first metatarsal osteotomy and internal fixation (without collateral procedures) during the period August 10 to November 20, 2015. On the first postoperative day, subjects eligible for study participation were required to have moderate-to-severe pain on a 4-point Likert scale and a score of ≥4 on the 11-point numeric pain rating scale (NPRS) after cessation of a popliteal sciatic nerve block and discontinuation of other pain management measures. Ineligible participants included those with known hypersensitivity to aspirin, NSAIDs, or any of the peri- or postoperative medications used in the study; active gastrointestinal bleeding or a history of peptic ulcer disease; known bleeding disorders affecting coagulation; evidence of respiratory insufficiency, hypotension, or bradycardia; a history of migraine, frequent headaches, seizures, or significant renal, hepatic, cardiovascular, metabolic, neurologic, or psychiatric disease; or a history of alcohol or drug abuse, hepatitis B or C, or human immunodeficiency virus infection. Further ineligibility criteria included subjects with other painful physical conditions that could interfere with the assessment of postoperative pain; those with a body mass index >35 kg/m2; and pregnant or lactating females. Subjects receiving various other medications were also excluded, including those taking opioids long term (ie, for >30 consecutive days in the past year) and those taking antiepileptic and antidepressant drugs, neuroleptics, systemic or intra-articular corticosteroids (within 6 weeks prior to the study), sedative/hypnotic drugs, warfarin, lithium, and/or combinations of furosemide with either an angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Also prohibited were other analgesics (except prespecified drugs for postoperative rescue use), prophylactic antiemetics, epidural or spinal anesthetics, and pre-, intra-, or postoperative corticosteroids. The study was approved by an independent institutional review board (Sterling IRB, Atlanta, GA, USA). All subjects enrolled provided written informed consent prior to their participation. All clinical work was conducted in compliance with Good Clinical Practices (as referenced in the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guideline E6), local regulatory requirements, and the principles of the Declaration of Helsinki. ABSTRACT.STUDY PROCEDURES: All subjects underwent the Austin bunion procedure21 involving osteotomy of the first metatarsal with internal fixation, which was required to be completed within 120 minutes and no later than approximately 6 pm on Day –1 of the study. The surgery was performed while the subject was under a standardized anesthesia regimen consisting of a continuous popliteal sciatic nerve block and local anesthesia, with IV midazolam and propofol for sedation. On postoperative Day 1, the continuous popliteal sciatic nerve block was maintained until 3 am, and other analgesic measures (eg, topical ice and ketorolac and/or morphine) were discontinued prior to this time. After cessation of the popliteal sciatic nerve block and discontinuation of all other analgesic measures, subjects with an NPRS score of ≥4 and a rating of moderate or severe pain on a 4-point categorical pain rating scale (ie, none, mild, moderate, severe) prior to 12 pm on postoperative Day 1 were deemed eligible for study participation (n=59) and were randomized (1:1:1 via a computer-generated method) within 15 minutes to one of three treatment groups: meloxicam IV 30 mg, meloxicam IV 60 mg, or placebo IV (5% dextrose in water). The study medication, meloxicam IV or placebo, was administered as an IV bolus over 15–30 seconds under double-blind conditions, such that the subjects, investigators, and site staff involved with collecting safety and efficacy data were unaware of the assigned treatment. A second IV dose of study medication was administered 24 hours after the first dose, with the option of a third dose prior to discharge from the study center (after the initial 48 hours), at the discretion of the investigator and the subject. Subjects were assessed for AEs and PI at various time points after administration of the first dose of study medication (Hour 0). PI was evaluated using the 11-point NPRS (0= no pain, 10= worst pain imaginable) prior to and at 0.25, 0.5, 0.75, 1, and 2 hours after the first dose. Thereafter, pain assessments were made every 2 hours until Hour 48. PI also was assessed within 5 minutes before administration of each dose of rescue medication (oral oxycodone 5 mg administered up to every 2 hours as needed). Subjects were discharged following the 48-hour evaluations. Follow-up assessments were performed on Day 7 (at a clinic visit) and Day 30 (via a telephone call). ABSTRACT.OUTCOME MEASURES: ABSTRACT.SAFETY ENDPOINTS: These included the occurrence of AEs and serious AEs; wound-healing status; changes in vital signs from baseline and the incidence of clinically significant changes in vital signs; changes from baseline in clinical laboratory test parameters and the incidence of abnormal values; and the incidence of clinically significant abnormal ECG findings. Vital signs including resting blood pressure, resting pulse, respiratory rate, and peripheral oxygen saturation were measured and recorded 15 minutes before dosing and 0.5, 1, 2, and 6 hours after dosing (±15 minutes). Vital signs were also recorded at Hour 48 and at Day 7. A 12-lead ECG was completed at screening, at check-in on Day −1 (if screening ECG was done >7 days prior to Day −1), at Hour 48 prior to discharge, at the Day 7 visit, and at time of early discontinuation. Urine and blood samples were collected for routine clinical laboratory testing during the screening visit, on Day −1 during check-in, at the Day 7 visit, and in the event of subject early discontinuation. ABSTRACT.EFFICACY ENDPOINTS: The PID at each time point was calculated, and SPID values were determined by multiplying a weight factor to each score prior to summation; the weight factor at each time point was the time (in minutes) elapsed since the previous observation. The primary efficacy endpoint, the effect size (point estimates with 95% CIs), was determined based on the difference in SPID48 values for each dose of meloxicam IV as compared to placebo. Secondary efficacy endpoints were as follows: SPID values at other time points, specifically 6, 12, and 24 hours (SPID6, SPID12, and SPID24, respectively) and for the intervals 12–24, 12–48, and 24–48 hours (SPID12–24, SPID12–48, and SPID24–48, respectively), all of which were determined by the same method used for SPID48 values. The proportions of subjects with ≥30% and ≥50% overall reductions in pain from baseline within the first 6 and 24 hours after administration of the first dose of study medication. Patients’ global assessment (PGA) of their pain-control method at 24 and 48 hours after the first dose, scored on a 5-point scale (0= poor, 1= fair, 2= good, 3= very good, 4= excellent). The time to administration of the first dose of rescue analgesia (oral oxycodone 5 mg administered up to every 2 hours, if necessary), and the number of times rescue analgesia was required during each of the following periods: 0–24, 24–48, and 0–48 hours. ABSTRACT.STATISTICAL ANALYSIS: All randomized subjects (ie, the intention-to-treat population) were included in the safety and efficacy analyses. For the safety analysis, AEs were summarized by treatment group. The Medical Dictionary for Regulatory Activities (version 18.1) was used to classify all AEs by system organ class and preferred term. Changes in vital signs at each assessment point were summarized by treatment group using descriptive statistics. The numbers and proportions of subjects with abnormal ECG findings at each assessment point were summarized by treatment group. For the efficacy analysis, differences in SPID values between the study groups were assessed by analysis of covariance, with the baseline pain score as a covariate. Least-squares (LS) mean and standard error (SE) SPID values for each treatment group were determined, and the effect size was calculated from differences in LS mean values versus placebo and the pooled SD derived from the analysis of covariance model. Between-group differences in LS mean SPID values were also tested using paired-sample Student’s t-tests. Differences in the proportions of subjects achieving ≥30% and ≥50% overall reductions in pain from baseline were tested using Fisher’s exact test. Exact CIs of the differences and common odds ratios for the proportions of subjects meeting the criteria were calculated. Differences in PGA scores between treatment groups were evaluated by the exact Mantel-Haenszel chi-square test. Time to first use of rescue analgesia was analyzed by Kaplan-Meier methodology, and the differences in these times were evaluated by a log-rank test. All tests were two-sided, and P-values of ≤0.05 were deemed significant. Nominal P-values were reported without adjustment for multiple comparisons. To address the impact of rescue analgesia on the response to study medication, two methods were used: a last observation carried forward (LOCF) method and a 2-hour windowed LOCF (W2LOCF) method. For the LOCF analysis, the PI score obtained before the first dose of rescue medication was carried forward to replace all PI scores obtained after the rescue dose. For the W2LOCF analysis, the PI score obtained before each dose of rescue medication was carried forward to replace the PI scores collected in the following 2-hour window. Although LOCF initially was considered the primary analysis method for this study, it was later determined, from consultations with the regulatory agency, that W2LOCF was a more appropriate method for imputation in this setting. Consequently, the W2LOCF method was used for all results, and the LOCF method became a supportive analysis tool. All analyses were performed using SAS versions 9.1 and 9.3 (SAS Institute Inc., Cary, NC, USA). ABSTRACT.RESULTS: Of the 93 subjects screened for suitability for participation, 59 who satisfactorily completed the surgery and met all study criteria (including the required level of postoperative pain) were randomized to one of the three study groups. All randomized subjects received study medication and were included in the safety and efficacy analyses. The demographic and clinical characteristics of the study groups were comparable (Table 1). The age range of the study population was 18–72 years. Most subjects (81.4%) were female; 50.8% were black or African American, and 47.5% were white. The study site did not target recruitment to any demographic subgroup and the predominance of females is consistent with the epidemiology of this condition. During the study, treatment was discontinued in two subjects. One subject in the meloxicam IV 60 mg group was withdrawn at her request. The other subject, a placebo recipient, was withdrawn by the investigator. Therefore, 57 subjects completed the study. ABSTRACT.SAFETY FINDINGS: All randomized subjects received at least one dose of study medication; 57 subjects (96.6%) received two doses, and 30 (50.8%) received a third dose after the 48-hour assessment. The findings indicated that both doses of meloxicam IV (30 and 60 mg) were generally well tolerated. Although most subjects experienced at least one treatment-emergent AE (Table 2), the majority of AEs were rated as mild and there were no meaningful differences between the study groups. No deaths or other serious AEs were reported, and no subject was discontinued from the study due to an AE. Moreover, there were no injection-related events and no apparent trends in clinically meaningful abnormal laboratory results between the study groups. The results of liver function tests were normal in subjects who received meloxicam IV, as were all findings of renal function tests and urinalyses. The only clinically meaningful laboratory abnormality among recipients of meloxicam IV was a decreased white blood cell count in one subject. Because the subject had a history of this blood disorder, the abnormality was not considered related to the study treatment. Three subjects in the placebo group had clinically meaningful laboratory abnormalities (including two with increased gamma-glutamyl transferase levels), all of which resolved subsequently. There were no clinically meaningful changes in blood pressure or heart rate in subjects who received meloxicam IV or placebo. Two subjects, one who received meloxicam IV 60 mg and one who received placebo, had abnormal wound healing. The subject who received meloxicam IV had local cellulitis (reported as an AE) associated with erythema and edema, none of which were considered treatment related. The placebo subject’s wound was described as slightly macerated; this finding was not considered clinically significant or an AE. No clinically significant abnormal ECG findings were detected during the study, and there were no injection-related AEs reported. ABSTRACT.EFFICACY FINDINGS: ABSTRACT.SPID:48 and other post-dose SPID values The effect sizes for doses of 30 mg and 60 mg meloxicam IV during the first 48 hours after administration, using the W2LOCF and LOCF analysis methods, are shown in Figure 1 and Table 3. Subjects treated with either dose of meloxicam IV had significantly greater reductions in SPID48 than subjects who received placebo (P≤0.01) according to both methods of analysis (Table 3). Both doses of meloxicam IV also produced significantly greater pain reductions than placebo at other post-dose intervals (ie, in SPID6, SPID12, SPID24, SPID12–24, SPID12–48, and SPID24–48 values) according to W2LOCF and LOCF analysis methods (P≤0.05; Table 4). ABSTRACT.PID AT EACH TIME POINT: Mean PID values versus baseline for the three treatment groups over 48 hours, using the W2LOCF assessment method, are shown in Figure 2A. Statistically significant decreases in pain from baseline (ie, negative PID values) were detected as early as 15 minutes after the first dose of both meloxicam IV 30 mg and meloxicam IV 60 mg (Figure 2B). At this time, mean PID values versus baseline with the two doses were −1.5 ± 2.44 (P<0.05) and −0.7 ± 1.22 (P<0.05), respectively. The mean PI changes from baseline at all assessment points were less than zero with both doses of meloxicam IV (ie, the pain level was lower than at baseline); at the 48-hour assessment, the changes were −4.6 ± 3.61 and −4.3 ± 3.06 with meloxicam IV doses of 30 mg and 60 mg, respectively (both P<0.001 versus baseline). With placebo, the mean PID at 0.25 hours was −0.1. Thereafter, PI changes from baseline were positive until the 20-hour assessment, and a significant difference versus baseline was not detected until Hour 28. ABSTRACT.RESPONSE ANALYSIS: The proportions of subjects with pain reduction of ≥30% and ≥50% from baseline during the first 6 and 24 hours were determined by the W2LOCF and LOCF analysis methods. According to W2LOCF analysis, significantly more subjects who received meloxicam IV 30 mg doses had pain reductions of ≥30% and ≥50% over 6 hours and 24 hours after receiving the first dose in comparison with those who received placebo (P≤0.05; Figure 3). However, the LOCF analysis method did not show a statistically significant difference versus placebo with doses of 30 mg, and neither analysis method demonstrated a significant difference with meloxicam IV doses of 60 mg versus placebo. ABSTRACT.PGA OF THE PAIN-CONTROL METHOD: No statistically significant differences in PGA scores between the three study groups were observed at 24 or 48 hours after administration of the first dose of study medication. However, compared with placebo recipients, more subjects in both meloxicam IV groups reported “good” or better pain control (ie, a rating of ≥2 on the 5-point scale) at these times (Figure 4). ABSTRACT.RESCUE ANALGESIA: Oral oxycodone 5 mg, the rescue medication used in this study, was required by ≥90% of subjects in all three study groups within the first 24 hours of receiving the initial dose of study medication. However, the percentage of subjects who required rescue analgesia during the second 24 hours (Hours 24–48) was lower with both doses of meloxicam IV than with placebo (55.0%, 52.6%, and 77.8%, respectively). Although the time to first use of rescue medication was significantly longer with meloxicam IV doses of 60 mg than with placebo (median, 3.10 hours versus 1.57 hours; P<0.05), there was no significant difference between the meloxicam IV 30 mg dose and placebo. Subjects treated with either dose of meloxicam IV required fewer doses of rescue analgesia than those who received placebo (average number of rescue doses: 8.2, 6.9, and 11.1 with meloxicam IV 30 mg and 60 mg doses and placebo, respectively), but the differences were not statistically significant. ABSTRACT.DISCUSSION: This randomized, double-blind, placebo-controlled study was conducted in subjects who experienced moderate-to-severe pain after bunionectomy, a postoperative pain model that has proven useful for assessing the analgesic efficacy of NSAIDs, COX-2 inhibitors, and other drugs.3 The study’s design was consistent with current Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) recommendations for short-duration, acute-pain trials.2 This included using standardized surgical, anesthetic, and postoperative care regimens. The primary objective of the study was to evaluate the safety of the IV nanocrystal formulation of meloxicam administered as a bolus injection over 15– Although the study originally was not statistically powered to demonstrate efficacy, results numerically favored both doses of meloxicam IV push (30 mg and 60 mg, given once daily) over placebo in relieving moderate-to-severe post-bunionectomy pain at all assessment times during the 48-hour study period, regardless of the analysis method (LOCF or W2LOCF). A statistically significant difference versus placebo was observed with both doses of meloxicam IV for SPID48 values, and also for SPID6, SPID12, SPID24, SPID12–24, SPID12–48, and SPID24–48 values. The significant differences in SPID12–24 values versus placebo with the two doses indicate that the analgesic effect of meloxicam IV remains significant throughout the second half of the dosing interval. Meloxicam IV had a rapid onset of action; significant decreases in PID values from baseline were detected with both doses as early as 15 minutes after administration. Moreover, compared with placebo, meloxicam IV 30 mg resulted in significantly more subjects with PI reductions of ≥30% and ≥50% from baseline within the first 6 and 24 hours after the initiation of treatment, according to W2LOCF analysis. The time to first use of rescue medication was significantly longer with the meloxicam IV 60 mg doses than with placebo and, overall, fewer doses of rescue medication were used by subjects who received meloxicam IV. Although differences in rescue medication usage between the meloxicam IV and placebo groups were not statistically significant (likely due to the small sample size), the rescue analgesia findings from this study indicate that, as with other NSAIDs administered intravenously in postoperative pain settings,22–26 meloxicam IV may have an opioid-sparing effect. This may be beneficial in reducing the occurrence of opioid-induced AEs that, in some patients, may slow postoperative recovery. Determining whether the opioid-sparing effect of meloxicam IV is clinically significant will require investigation in larger studies. To address the impact of rescue medication on the treatment response in this study, two methods of analysis were used: LOCF and W2LOCF. Although LOCF was initially stated to be the primary method of analysis, the W2LOCF method was ultimately used for the primary analysis, following its identification as a more appropriate method for imputation. The LOCF method served as a supportive analysis. For most efficacy evaluations, PI results determined by the two methods were consistent. The main difference pertained to the proportions of subjects with pain reductions of ≥30% and ≥50% from baseline in the first 6 hours and 24 hours after the initial dose of meloxicam IV 30 mg. The proportions of subjects with these overall pain reductions were significantly greater with the 30 mg dose than with placebo according to the W2LOCF analysis method but not the LOCF method. Limitations of this study include the relatively small sample size and the possibility of some intersubject variability. Although the surgical procedure, anesthesia protocol, and initial postoperative management regimens were standardized as much as possible to minimize intersubject variability in this study, it is recognized that hemodynamic fluctuations and other intraoperative events may have necessitated some deviation from standard regimens, which may have resulted in intersubject variability. Further, while the extended duration of hospital stay (48 hours) was required for clinical assessment, this may not reflect the current standard of care. ABSTRACT.CONCLUSION: Meloxicam IV was generally safe and well tolerated in subjects with moderate-to-severe post-bunionectomy pain, yielding a low incidence of AEs and no injection-related events. Most AEs were rated mild in intensity, and their incidence was similar to that in the placebo group and did not increase over time. In terms of efficacy, doses of 30 mg and 60 mg meloxicam IV administered once daily by bolus injection over 15–30 seconds produced meaningful effect sizes according to W2LOCF and LOCF analysis methods, and achieved significantly better LS mean SPID48 values than placebo. Moreover, once-daily dosing of meloxicam IV produced a rapid onset of analgesic activity (within 15 minutes after administration) and maintained analgesia for the 48-hour study period. Consequently, findings from this study support further (Phase 3) investigations of the efficacy and safety of once-daily administration of meloxicam IV to subjects with moderate-to-severe postoperative pain.

TITLE: Assessing the external validity of a randomized controlled trial of anthelminthics in mothers and their children in Entebbe, Uganda ABSTRACT.BACKGROUND: The ‘external validity’ of randomized controlled trials is an important measure of quality, but is often not formally assessed. Trials concerning mass drug administration for helminth control are likely to guide public health policy and careful interpretation of their context is needed. We aimed to determine how representative participants in one such trial were of their community. We explore implications for trial interpretation and resulting public health recommendations. ABSTRACT.METHODS: The trial assessed was the Entebbe Mother and Baby Study (EMaBS), a trial of anthelminthic treatment during pregnancy and early childhood. In a novel approach for assessing external validity, we conducted a two-stage cluster sample community survey within the trial catchment area and compared characteristics of potentially-eligible community children with characteristics of children participating in the trial. ABSTRACT.RESULTS: A total of 173 children aged three to five-years-old were surveyed from 480 households. Of children surveyed, we estimated that mothers of 60% would have been eligible for recruitment, and of these, 31% had actually been enrolled. Children surveyed were compared to 199 trial children in the same age group reviewed at annual trial visits during the same time period. There were significant differences in ethnicity between the trial participants and the community children, and in socioeconomic status, with those in the trial having, on average, more educated parents and higher maternal employment. Trial children were less likely to have barefoot exposure and more likely to use insecticide-treated bed nets. There were no significant differences in numbers of reported illness events over the last year. ABSTRACT.CONCLUSIONS: The trial had not enrolled all eligible participants, and those enrolled were of higher socioeconomic status, and had lower risk of exposure to the parasitic infections targeted by the trial interventions. It is possible the trial may have underestimated the absolute effects of anthelminthic treatment during pregnancy and early childhood, although the fact that there were no differences in reported incidence of common infectious diseases (one of the primary outcomes of EMaBS) between the two groups provides reassurance. Concurrent community surveys may be an effective way to test the external validity of trials. ABSTRACT.EMABS TRIAL REGISTRATION: ISRCTN32849447, registered 22 July 2005 ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1745-6215-15-310) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: The rise of evidence-based medicine has seen much emphasis placed on the internal validity of clinical trials but less attention has been given to external validity. Measures adopted for ensuring internal validity include the design of a trial which is appropriately powered to detect a clinically significant effect, the use of a control group selected by randomization and the ‘blinding’ of both participants and investigators to the intervention. Statistical analysis of the trial results then allows the classification of data on the basis of generally accepted levels of ‘significance’ [1, 2]. However, there have been increasing calls to take into account other factors when assessing the quality of evidence generated by trials [3–7]. These include biological plausibility, reproducibility and external validity [8]. External validity can be considered as the extent to which the results can be generalized to other circumstances. Whilst important, these factors may not have received the attention they deserve because their quality is not always easy to assess. However, failure to take these factors into account may limit a study’s usefulness. The implementation of findings that are not clearly applicable to the population in question has been called ‘evidence-biased medicine’ [9]. Increased awareness of external validity as a measure of study quality has led to its incorporation into several high-profile frameworks for the reporting and assessment of clinical trials [10–12]. The assessment of external validity is particularly important for trials in resource-poor settings, as these may be used to guide wide-ranging public health policy decisions, often in several settings or countries [13–15]. We aimed to assess the ‘external validity’ of the Entebbe Mother and Baby Study (ISRCTN32849447), a trial designed to investigate the effects of anthelminthic treatment in pregnancy and in childhood [16]. The primary outcomes included immunological responses to immunization and incidence of infectious and allergic disease in early childhood. This trial has now been reported and demonstrated a possible benefit of anthelminthic treatment during pregnancy for maternal anaemia, restricted to women with moderate to heavy hookworm infection, and a reduction in malaria incidence among children receiving quarterly anthelminthic treatment. However, there were none of the expected benefits for anaemia, birth weight, perinatal mortality, infant mortality or infant responses to immunizations. By contrast, there was an apparent adverse effect on infantile eczema [17–20]. There are relatively few studies which aim to assess the external validity of clinical trials. Most published studies focus on assessing the number of people included in the trial, as a proportion of those who would have been eligible for participation in the trial given the trial’s inclusion and exclusion criteria [21–27]. Other published studies rate trials on a scoring system devised for the purpose [28, 29], assess the adequacy of reporting of exclusion criteria [30, 31] or other generalizability measures [14], compare inferences derived from randomized controlled trial data with inferences derived from population-based studies addressing similar outcomes [32], compare outcomes between persons included and excluded from a trial [33, 34] or assess the representation of certain groups [35, 36]. Here, we report a novel approach to assess the external validity of a trial. Specifically, we conducted a community survey to assess whether participants in the Entebbe Mother and Baby Study were representative of the trial’s target population. By conducting a community-wide survey in which any appropriately aged child in the trial catchment area could potentially be enrolled, we hope to offer a more comprehensive assessment of external validity than studies to date. Our findings have implications for the generalizability of this trial, but also demonstrate an approach that may be of use in assessing the external validity of other trials. BODY.METHODS: BODY.SETTING: The catchment area for the Entebbe Mother and Baby Study (EMaBS) was comprised of the Entebbe Municipality and Katabi sub-country, a peninsula on the northern shore of Lake Victoria, Uganda. Entebbe town is located approximately 40 km southwest of the capital, Kampala, has a population of approximately 90,500 and is the site of Uganda’s main international airport. Katabi sub-county borders Entebbe Municipality, has a population of approximately 59,000 and consists of semi-urban, rural and fishing communities. The EMaBS trial recruited pregnant women between April 2003 and November 2005. At the time of this investigation (between July and August 2008), EMaBS cohort children were aged three, four and five years. We therefore conducted a survey within the same catchment area, consisting of three, four and five-year-old children, both male and female. BODY.STUDY DESIGN: The community survey used a sampling strategy designed to reduce bias within a setting with limited prior demographic data. The study area comprised 47 administrative units known as wards. Census data detailing the number of households in each ward was available. A sample of 15 wards within the survey area was selected by random number generation, with probability of selection being proportional to the number of households. It was possible for one ward to be selected twice. Each ward was then mapped onto satellite imagery of the area with the help of locally available maps. Uninhabitable areas were excluded from mapping. The wards were divided into segments of equal geographical size (the same size across all wards) based on lines of latitude and longitude (degrees, minutes, seconds position format). These segments were then numbered and four segments from each ward were randomly selected using random number generation. The midpoint of each segment was identified by its coordinates and this was used as the starting point for sampling. The starting point was identified using a geographic information system (GIS) device (eTrex®, Garmin ™ Ltd, Kansas, United States) and the nearest house selected for sampling. Eight houses were then surveyed sequentially from this point, the next house to be sampled being the nearest to the previous house. A household was defined as a habitable roofed structure whose primary function was residence or, if used for dual purposes, had at least one active resident using the structure as their primary residence. In selected households that included a three, four or five-year old child, the parent or guardian was counselled and provided with written information in English and the vernacular of the area prior to obtaining written consent. If two or more eligible children lived in the same house, they were all surveyed if possible. A questionnaire was then administered for each child. This was designed to match with data collected at the yearly trial visits undertaken by children enrolled in the Entebbe Mother and Baby Study, in order to obtain comparable information from both sources. In addition, during the period of the community survey supplementary information sheets were completed by trial participants during these yearly visits. These covered questions asked in the community survey but not routinely asked in the trial, or which had been asked in screening at enrolment into the trial, but were felt likely to have changed since that time. BODY.RECRUITMENT TO EMABS AND COMMUNITY PARTICIPATION: EMaBS trial participants were recruited at the antenatal clinic at Entebbe Hospital over a two and a half year period. At the same time, the community was sensitized to the study. The mayor of Entebbe and sub-county chief of Katabi were informed and the research team visited all villages in the catchment area and held meetings with the local council (LC) leaders. LC leaders were asked to select community field workers, who were trained in simple data collection and subsequently followed up on participating children every two weeks until they were five-years-old. They met monthly and provided the main link between the research team and the community throughout the study period. Inclusion criteria for the EMaBS trial required women to be resident in the study area, attending the Entebbe Hospital antenatal clinic and intending to give birth at the hospital, with no age limits. The exclusion criteria for the trial included not wishing to participate, not being willing to receive an HIV result, bloody diarrhoea, previous adverse reaction to anthelminthics or sulfadoxine-pyrimethamine (Fansidar™), already having a child in the trial, antenatal abnormalities, failure to complete screening or re-attend for enrolment, not being pregnant and anaemia (hemoglobin <8 g/dL). BODY.ELIGIBILITY AND UPTAKE ASSESSMENT: In the survey, we first aimed to assess what proportion of community children would have been potentially eligible for EMaBS trial participation. Children were deemed to have been potentially eligible if, at the time of the child’s birth, the mother was resident within the study area and attended the Entebbe Hospital antenatal clinic. We then aimed to estimate what proportion of potentially eligible children had actually been enrolled in the trial. We were unable to directly assess the impact of the other EMaBS inclusion and exclusion criteria. BODY.OUTCOMES: Socio-demographic characteristics: The outcomes assessed were primary carer/s, maternal and paternal age and health status, level of maternal and paternal education, maternal employment and wage, maternal tribe and other socioeconomic parameters (including housing materials, crowding, water and electricity provision). These factors are unlikely to have been affected by the trial intervention and therefore reflect inherent characteristics of the study population. Disease risk factors and comorbidities: Outcomes assessed were barefoot exposure, lake exposure (both risk factors for helminth infection), mosquito net usage and insecticide-treatment of household nets (risk factors for malaria). Diseases and anthropometry outcomes were as follows: we recorded self-reported disease episodes of malaria, diarrhoea, pneumonia, measles and tuberculosis. We did not ask directly about HIV because of the limitations placed on confidentiality in the field setting and concerns over the reliability of any results obtained. We also measured height, weight, mid to upper arm circumference and head circumference. These outcomes may have been influenced by the trial interventions. BODY.STATISTICAL ANALYSIS: Data were analyzed using Stata version 12 (StataCorp, Texas, United States). Data from the youngest of each pair or group of siblings in the community survey were excluded from the analysis, in order to make the inclusion criteria comparable with EMaBS (where one of the exclusion criteria was having a child already in the trial). Clustering at the ward and segment level was taken into account in the analysis. Clustering at the household level was not allowed for in the analysis due to the small number of households remaining with multiple children after younger siblings were excluded from the analysis. The svy commands in Stata were used to allow for the clustering. Distributions of parental and child characteristics in the two groups were first compared using simple tabulations, with design-based Pearson’s F statistics calculated to test for differences in characteristics between the community survey and EMaBS children. Logistic regression was used to calculate crude and adjusted odds ratios (ORs) and associated 95% confidence intervals (CIs), allowing for the sampling design. Multivariable logistic regression analysis was conducted to control for potential confounding. A hierarchical approach was used to decide which variables should be treated as potential confounders in the multivariable analysis. Maternal and paternal sociodemographic parameters were considered as potential confounders for each other, for household characteristics, for disease risk factors and comorbidities, and for diseases and anthropometry. Household characteristics were considered as potential confounders for each other, for disease risk factors and comorbidities and for diseases and anthropometry. Finally, disease risk factors and comorbidities were considered as potential confounders for diseases and anthropometry. BODY.ETHICAL APPROVAL: Both EMaBS and the community survey received ethical approval from the Science and Ethics Committee of the Uganda Virus Research Institute (GC/127), Uganda National Council for Science and Technology (MV 625) and the London School of Hygiene and Tropical Medicine ethics committee (07/303). BODY.RESULTS: In total, 480 households were surveyed; eight households from each of four locations in 15 wards. The breakdown of these is provided in Figure 1. One hundred and seventy three children were eligible for inclusion in the analysis. During the survey period 199 trial children visited the trial clinic, of whom 128 completed supplementary information questionnaires. There were no differences in characteristics of those who completed supplementary questionnaires compared to those who did not.Figure 1 Breakdown of households approached during the community survey and number of children in each. Of the 173 children seen in the community, 104 (60%) had mothers who would have been potentially eligible for recruitment into the trial. Of the remaining children, 38 (55%) had mothers who were not resident in Entebbe or Katabi at the time of delivery, an additional 28 (41%) had mothers who did not receive antenatal care at Entebbe Hospital and were therefore not available for recruitment, and there was no information available on where mothers had received antenatal care for 3 (4%). Of the 104 children who were potentially eligible for inclusion, 32 (31%) were in the trial. There were significant differences between the ethnic makeup of the two populations, based on the mother’s tribe. Of particular note only half the mothers in the trial were Buganda compared to 65% in the community, and being a member of the Banyankole, Batoro or Banyarwanda tribes was twice as common in the trial mothers (Table 1).Table 1 Comparison of characteristics of EMaBS trial annual visit children and community survey children Trial annual visit (n = 199)Community survey (n = 173)OR (95% CI)1 P 1(P trend) Sociodemographic variables 2 Maternal tribe Baganda 99 (50%) 110 (65%) 1 0.05 Banyankole/Batoro/Banyarwanda 37 (19%) 17 (10%) 0.41 (0.20, 0.87) Basoga 11 (6%) 6 (4%) 0.49 (0.16, 1.50) Other 52 (27%) 37 (22%) 0.64 (0.34, 1.21) Maternal education (mv: 0, 13) None 8 (4%) 22 (14%) 4.43 (1.85, 10.57) <0.001 Primary 103 (52%) 64 (40%) 1 (0.08) Secondary 67 (34%) 68 (43%) 1.63 (0.97, 2.76) Tertiary 21 (11%) 6 (4%) 0.46 (0.17, 1.21) Paternal education (mv: 37, 38) None/primary 31 (19%) 46 (34%) 1 <0.001 Secondary 79 (49%) 75 (56%) 0.64 (0.36, 1.14) (<0.001) Tertiary 52 (32%) 14 (10%) 0.18 (0.09, 0.37) Maternal age (mv: 0, 12) 24.2 (4.7) 23.1 (5.6) 0.96 (0.92, 1.00) 0.04 Paternal age (mv: 121, 54) 34.8 (7.1) 33.8 (7.4) 0.98 (0.95, 1.02) 0.33 Maternal employment (mv: 73, 8) Unemployed 56 (44%) 123 (75%) 1 <0.001 Employed 70 (56%) 42 (25%) 0.27 (0.16, 0.46) Annual maternal income (Ugandan Shillings) (mv: 77, 1) Zero 67 (55%) 126 (73%) 1 0.002 0-50000 26 (21%) 15 (9%) 0.31 (0.15, 0.64) (0.08) 50001-100000 23 (19%) 18 (10%) 0.42 (0.21, 0.83) 100001- 6 (5%) 13 (8%) 1.15 (0.40, 3.29) Household circumstances Crowding (mv: 74, 1) <=3 people/room 77 (62%) 126 (73%) 1 >3 people/room 48 (38%) 46 (27%) 0.59 (0.37, 0.93) 0.02 Roofing (mv: 74, 1) Iron/tiles 118 (94%) 169 (98%) 1 Banana leaves/grass 7 (6%) 3 (2%) 0.30 (0.06, 1.56) 0.13 Walls (mv: 74, 1) Bricks 108 (86%) 164 (95%) 1 Mud/metal 17 (14%) 8 (5%) 0.31 (0.10, 0.92) 0.03 Fuel source (mv: 73, 1) Firewood 25 (20%) 27 (16%) 1 0.04 Charcoal 89 (71%) 141 (82%) 1.47 (0.67, 3.22) Paraffin/gas/elec 12 (10%) 4 (2%) 0.31 (0.08, 1.16) Electricity supply (mv: 71, 1) Yes 60 (48%) 67 (39%) 1 No 66 (52%) 105 (61%) 1.42 (0.81, 2.50) 0.21 Water source (mv: 71, 1) Lake/well/borehole 15 (12%) 14 (8%) 1 Standpipe/domestic tap 111 (88%) 158 (92%) 1.53 (0.52, 4.51) 0.44 Disease risk factors Barefoot exposure (mv: 71, 6) Never/rarely 47 (37%) 7 (4%) 1 <0.001 Often 79 (63%) 160 (96%) 13.60 (5.55, 33.30) Lake exposure (mv: 71, 1) Never 108 (86%) 145 (84%) 1 Ever 18 (14%) 27 (16%) 1.12 (0.59, 2.13) 0.74 Child sleeps under net (mv: 55, 1) Always 113 (78%) 88 (51%) 1 <0.001 Sometimes 13 (9%) 28 (16%) 2.77 (1.32, 5.79) (<0.001) Never 18 (13%) 56 (33%) 3.99 (2.11, 7.56) Household nets treated? None 56 (44%) 109 (94%) 1 <0.001 Some/all 71 (56%) 7 (6%) 0.05 (0.02, 0.12) Reported disease episodes Malaria (mv: 14, 1) No 88 (48%) 74 (43%) 1 Yes 97 (52%) 98 (57%) 1.20 (0.78, 1.86) 0.41 Malaria (slide proven; mv: 44, 2) No 103 (66%) 111 (65%) 1 Yes 52 (34%) 60 (35%) 1.07 (0.69, 1.65) 0.76 Diarrhoea (mv: 14, 2) No 120 (65%) 130 (76%) 1 Yes 65 (35%) 41 (24%) 0.58 (0.35, 0.97) 0.04 Pneumonia (mv: 7, 4) No 188 (98%) 164 (97%) 1 Yes 4 (2%) 5 (3%) 1.43 (0.38, 5.35) 0.59 Measles (mv: 7, 2) No 181 (94%) 147 (86%) 1 Yes 11 (6%) 24 (14%) 2.69 (1.22, 5.92) 0.01 Anthropometric measurements Mean (SD) Mean (SD) Weight-for-age z-score -0.46 (0.97) -0.61 (1.04) 0.86 (0.68, 1.09) 0.21 Height-for-age z-score -0.90 (0.96) -0.91 (1.27) 0.99 (0.80, 1.24) 0.96 1Odds ratios use the trial population as the reference group and are adjusted for clustering. 2mv denotes number of individuals with missing values in EMaBS and Community Survey groups, respectively. Levels of both maternal and paternal education were, on average, higher amongst trial children (Table 1). There was no significant difference in parental health or primary carer between the two groups (data not shown). In crude analysis mothers in the trial were, on average, significantly older than those in the community, but this difference was no longer significant after adjusting for tribe and education (Table 2). There was no significant difference in the mean paternal age. There were significant differences in maternal employment and income. Mothers of trial children were more than twice as likely to be employed, and subsequently tended to have a higher income, although income was no longer significantly different between the two groups once maternal employment was taken into account.Table 2 Characteristics showing differences between EMaBS children and community survey children after multivariable analysis Sociodemographic variablesAdjusted OR (95% CI)1 P 1 Maternal tribe Muganda 1 0.03 Munyankole/Mutoro/Munyarwanda 0.31 (0.14, 0.71) Musoga 0.44 (0.14, 1.32) Other 0.76 (0.36, 1.61) Maternal education None 5.89 (2.28, 15.17) <0.001 Primary 1 Secondary 2.07 (1.21, 3.55) Tertiary 0.97 (0.31, 3.03) Paternal education 1 <0.001 Secondary 0.64 (0.33, 1.23) Tertiary 0.18 (0.08, 0.40) Maternal employment Unemployed 1 Employed 0.21 (0.12, 0.37) <0.001 Crowding <=3 people/room 1 >3 people/room 0.54 (0.30, 0.97) 0.04 Walls Bricks 1 Mud/metal 0.28 (0.11, 0.73) 0.01 Barefoot exposure Never/rarely 1 Often 17.91 (6.82, 47.07) <0.001 Child sleeps under net Always 1 0.02 Sometimes 2.09 (0.88, 4.98) Never 3.66 (1.42, 9.47) Household nets treated? None 1 Some/all 0.07 (0.03, 0.17) <0.001 Measles No 1 Yes 0.23 (0.07, 0.80) 0.02 1Odds ratios use the trial population as the reference group and are adjusted for clustering. Results for tribe, maternal education and paternal education adjusted for each other; results for maternal employment adjusted for tribe and parental education; results for crowding and walls adjusted for tribe, education, employment and each other; results for barefoot exposure, child bed-net usage and household net treatment adjusted for all other factors except measles; results for measles adjusted for all other factors. Trial children were more likely to live in houses with more than three people per room and more likely to live in houses with metal or mud walls than community survey children (Table 2). In crude analysis, there was a significant difference in fuel source between the two groups, but this was no longer significant after adjustment for other household characteristics (P = 0.33). There were no significant differences in roofing materials, electricity or water source between the two groups (Table 1). There were several differences in disease risk factors between the community and trial children (Tables 1, 2). Reported mosquito net usage was markedly higher in the trial group, with an increased likelihood of the net being treated. Reported frequent barefoot exposure was higher in the community and lake exposure was similar in the two groups (Table 1). There was no significant difference between reported disease measures of malaria, slide-proven malaria or pneumonia (Table 1). Reported diarrhoea was more common among trial children, although this difference was not significant after adjusting for other parental and household factors (P = 0.26). Reported measles, although rare, was more common in community children (Table 2). BODY.DISCUSSION: We have presented a community survey as a novel method for assessing aspects of the external validity of a randomized controlled trial. We found that EMaBS trial participants were on average, more likely to have parents with higher levels of education and who were in employment, more likely to come from non-local tribal groups, more likely to sleep under a bed-net and less likely to have barefoot exposure (a risk factor for helminths) than children in the target population for the trial. However, we found no differences in reported episodes of common childhood diseases between trial participants and children in the community survey. We estimate that 31% of eligible children in the community were enrolled in the trial, and although it was not possible to assess all trial exclusion criteria in the community survey, refusal and exclusion criteria are unlikely to account for the lack of enrolment of all the remaining children. Under a fifth of children in the community had been enrolled in the trial. Approximately 22% of children were ineligible on the basis of non-residency at the time of birth, and these children may have differed in important ways from those who had not recently migrated. This is an unavoidable factor when considering the non-representativeness of trials to their community. It is possible that migration into a trial area, particularly if large and sustained, may account for a significant proportion of any non-representativeness and this highlights the need to interpret the results of trials carefully in the light of shifting demographic patterns. A similar proportion of children were ineligible because their mothers received antenatal care outside of the hospital setting, a group previously noted to differ from those who did receive hospital-based antenatal care in this area [37]. Our survey was not powered to assess whether those children excluded in this way were significantly different from those eligible for the trial. However, the relatively high level of hospital-based antenatal care is reassuring and provides support for the recruitment strategy used in this trial, hence lending support to its external validity. Approximately 60% of mothers of children in the community survey would have been eligible for recruitment into EMaBS on the basis of residence and of antenatal care in Entebbe Hospital, and 31% of these were enrolled in the trial. Non-participation could have been secondary to refusal, exclusion on the basis of trial exclusion criteria or not having been approached for recruitment into the trial. In comparison, data from the EMaBS trial itself (Figure 2) shows that of 11,783 mothers initially assessed for inclusion in the trial, 5,388 (46%) were resident in and obtained antenatal care in Entebbe, planned on delivering in Entebbe hospital, and did not already have a baby in the trial. Of these, 2515 (47%) were subsequently enrolled [18]. However, the discrepancy between the figures of 31% and 47% implies a failure to fully assess all potentially eligible mothers at the antenatal clinic, most likely as a result of the heavy patient burden at the clinic. It is possible that this could have introduced bias due to differences in characteristics of women who would be able or willing to wait for the research procedures after receiving their standard antenatal care. This would be a potential threat to this trial’s external validity if a systematic bias occurred. Figure 2 also shows the reasons for which mothers were excluded from the EMaBS trial that could not be assessed in the community survey. It is possible that sociodemographic characteristics of women with these exclusion criteria differed from characteristics of those who did not meet them. If so then this could be an explanation for the different characteristics seen between the trial and community survey participants.Figure 2 Extract from CONSORT diagram for EMaBS showing number assessed for eligibility, numbers not enrolled (with reasons) and number randomized. Data from the EMaBS trial (Figure 2) give an estimated eligibility fraction (proportion eligible of those screened) of 23% and an estimated enrolment fraction (proportion randomized of those eligible) of 98%, yielding a recruitment fraction of 22%. As far as we are aware, there is no clear consensus on an acceptable level of trial participation of eligible persons when reviewed at the community level, with most studies focusing on recruitment ratios at the level of the point of recruitment or primary care [22, 25–27]. It is therefore difficult to assess the uptake rate in this trial in context and this is an area in need of further study. Whilst the majority maternal tribe in both groups was the local Baganda, the proportion within the trial participants was 15% less than within the community at large. The difference was largely made up by a higher proportion of mothers from the Banyankole and Luo tribes. The Banyankole are a Western Ugandan tribe and the Luo are from Northern Uganda [38]. The possibility of differences between the trial and community children, based on differences due to ethnic origin, therefore exists. Representation of minority ethnic groups in clinical trials is usually an issue of under-representation, with consequent difficulty extrapolating results to these groups [21, 35, 36, 39–41]. It is therefore reassuring that none of the minority tribes for this region were under-represented in the trial compared to the community. Children enrolled in the trial were more likely to be from families of higher socioeconomic status. Maternal employment status and income were higher, as was the level of both maternal and paternal education. There was a difference in crowding status, with trial children more likely to live in crowded circumstances. It is suggested that increased crowding may be a function of increased wealth in some circumstances. In particular it may be the case that families with increased income remain in their current property, but members of extended family or friends may move into the residence. This is consistent with findings from other settings, where in general, trial participation is more likely amongst those of higher socioeconomic status [22, 24, 33, 35, 40]. These differences might have implications for the trial results because worm burden is directly related to poverty [42], and in this study population, the mother’s education was associated with a lower prevalence for every infection in mothers at enrolment [43]. Hence if, as seems possible, the helminth burden in the trial population was lower than that in the general community, the effect of the anthelminthic treatment intervention on trial outcomes would have been attenuated in the trial population compared to the theoretical effect of such an intervention applied to the whole community. In general, disease exposure risks were fewer amongst the trial children with more bed-net use and less barefoot exposure. This difference persisted after adjustment for parental socioeconomic status. It may be the case that a systemic bias in selection for children in the trial resulted in selection of children with lower risk exposure status (not mediated by parental socioeconomic status). Alternatively, health education as part of the trial may have led to less exposure prone behaviour in the families of the trial children. However, importantly, with the exception of measles, there were no differences between the two groups in terms of reported episodes of infectious diseases, one of the EMaBS primary outcomes. This suggests that the sociodemographic differences we have observed between trial and community survey participants would not have led to biased intervention effect estimates on this primary outcome in the EMaBS trial. BODY.STRENGTHS AND LIMITATIONS: We were unable to select participants at the level of the child due to the lack of sampling frame and therefore used a multi-stage sampling survey approach. This was done using a predefined random sampling approach and was taken into account in the analysis, however, it is possible that our sampling strategy will have over-represented children in less populated areas. There may be important differences between these children and children in more populous areas, hence potentially biasing our results. The fact that we used a random sampling approach should have reduced bias in the sample, however the clustered nature of our design means we may have misrepresented variables which are geographically confined. For example, there was one sampled area (a military barracks) which we were not permitted to enter, whereas children enrolled in the trial do live in this area. Indeed, this may explain the higher proportion of abodes constructed from metal amongst the trial children, as this is the predominant material used in military barracks in Entebbe. The high response rate in the community survey means that selection bias should be minimized, and data completeness for the community survey was high. For the trial children we used several different sources of information, meaning the completeness of the data for each variable differed. In particular, a number of variables that were assessed from the supplementary forms for EMaBS children are limited because out of the 199 children assessed, only 128 supplementary forms were completed. There is no reason to believe this introduced bias since characteristics of the children who completed forms were similar to those of the children who did not, however, it limited the study’s power to detect differences between the groups for these variables. It is possible that responses to the questions may have been systematically different between the trial and community-surveyed groups. This may have occurred because the interviewers in the trial clinic and the community were different throughout. Also, the parents or guardians of the non-trial children may have responded to questions on recent childhood illnesses differently to the parents or guardians of the trial children, for instance, participation in the trial may have sensitized them to keeping a more accurate record of their child’s illnesses. We included trial children in our community survey (five children provided data to both the trial and community survey during the study period) and whilst this was intended in the survey design on the basis that they are part of their community and thus not as such a limitation, it could have led to a slight underestimation of the differences between the groups. BODY.CONCLUSIONS: Recruitment at the level of the antenatal clinic did not achieve enrolment of all eligible participants into this trial and this was unlikely to be fully explained by refusal or exclusion criteria. The study population was significantly different from the community at large on the basis of ethnic composition and socioeconomic status. There appeared to be increased disease risk factors in the community survey group but little difference in terms of reported disease episodes. To our knowledge, this is the first study of its kind using a community survey to assess the external validity of a randomized controlled trial. External validity is a very important component of the assessment of trials and this approach offers a cost-effective, practical and robust method of assessing the validity of a trial.

TITLE: An explanatory randomised controlled trial of a nurse-led, consultation-based intervention to support patients with adherence to taking glucose lowering medication for type 2 diabetes ABSTRACT.BACKGROUND: Failure to take medication reduces the effectiveness of treatment leading to increased morbidity and mortality. We evaluated the efficacy of a consultation-based intervention to support objectively-assessed adherence to oral glucose lowering medication (OGLM) compared to usual care among people with type 2 diabetes. ABSTRACT.METHODS: This was a parallel group randomised trial in adult patients with type 2 diabetes and HbA1c≥7.5% (58 mmol/mol), prescribed at least one OGLM. Participants were allocated to a clinic nurse delivered, innovative consultation-based intervention to strengthen patient motivation to take OGLM regularly and support medicine taking through action-plans, or to usual care. The primary outcome was the percentage of days on which the prescribed dose of medication was taken, measured objectively over 12 weeks with an electronic medication-monitoring device (TrackCap, Aardex, Switzerland). The primary analysis was intention-to-treat. ABSTRACT.RESULTS: 211 patients were randomised between July 1, 2006 and November 30, 2008 in 13 British general practices (primary care clinics). Primary outcome data were available for 194 participants (91.9%). Mean (sd) percentage of adherent days was 77.4% (26.3) in the intervention group and 69.0% (30.8) in standard care (mean difference between groups 8.4%, 95% confidence interval 0.2% to 16.7%, p = 0.044). There was no significant adverse impact on functional status or treatment satisfaction. ABSTRACT.CONCLUSIONS: This well-specified, theory based intervention delivered in a single session of 30 min in primary care increased objectively measured medication adherence, with no adverse effect on treatment satisfaction. These findings justify a definitive trial of this approach to improving medication adherence over a longer period of time, with clinical and cost-effectiveness outcomes to inform clinical practice. ABSTRACT.TRIAL REGISTRATION: Current Controlled Trials ISRCTN30522359 BODY.BACKGROUND: Between a third and a half of medicines prescribed for long-term conditions are not taken as prescribed [1]. This applies equally to patients with type 2 diabetes who are managed with multiple medications to control cardiovascular risk factors and blood glucose [2]. Up to 37% of patients with diabetes have discontinued oral hypoglycaemic drugs within one year of initiating treatment [3], with adherence to medication falling as dosage frequency rises [4]. For those who persist with therapy, it is estimated that about 70-80% of doses are taken as prescribed [5]. Failure to take medication reduces the effectiveness of the treatment, and wastes healthcare resources in prescribed medicines not taken, extra consultations, referrals, investigations and hospital admissions [6,7]. The availability of an effective consultation-based intervention to support patients with long-term, progressive disorders in taking their medication regularly would make a major contribution to human health. A variety of approaches to help patients take their medication regularly have been tested for efficacy [8]. However, there are only a few rigorous trials, and these suggest that interventions with multiple components are most effective in improving clinical outcomes [8]. A greater focus on the determinants of non-adherence may provide a basis for improved effectiveness, as interventions should address the principal causes of sub-optimal adherence. Since causes are many and vary between individuals, the intervention may need to be tailored to the individual. The majority of studies have used measures of adherence that are imprecise, often relying on self-report [8]. This can lead to biased estimation of intervention effects, both within and between patient groups. Electronic measurement of adherence is increasingly used in intervention studies, but there few trials of its use among patients with type 2 diabetes have used it to date. We have therefore drawn on psychological evidence and theory about hypothesised causes of non-adherence relating to weak motivation (intentional non-adherence) and forgetting (non-intentional non-adherence) [9], to develop a nurse-led consultation-based intervention (the “Support and Advice for Medication Study”; SAMS) targeting these hypothesised determinants through elicitation of personal beliefs. The intervention aims to increase patients’ motivation to take their tablets regularly by reinforcing positive beliefs and facilitating problem-solving around negative beliefs and to help patients translate motivation into action by asking them to form and write down specific action plans [10-12]. Both components are delivered in a single, brief intervention, although, if effective, future work could explore delivery over a longer period of time. We evaluated this new intervention in an explanatory randomised controlled trial to establish the short-term efficacy of the intervention on tablet taking behaviour and to inform estimates of the sample size for a pragmatic and definitive trial with glycaemic control as the outcome. The trial was carried out in a primary care setting among adults with type 2 diabetes taking oral glucose lowering medication (OGLM), with electronic measurement of medication taking. BODY.METHODS: BODY.TRIAL DESIGN: This trial formed part of a programme of work to evaluate the efficacy of a new intervention, and obtain information to refine its delivery and determine parameters for future trial evaluation. We used a parallel group trial design to evaluate a two-component intervention targeting motivation and using action planning in comparison with a control “standard care” intervention (Figure 1). An unbalanced (3:2) randomisation was used to provide more data from patients exposed to the new intervention, with little loss of power. The trial was carried out in weeks 9 to 20 of a 20-week study in which there was an initial, randomised evaluation of the impact of electronic medication measurement on adherence. In the initial phase of this study (weeks 1 to 8), patients were randomised to test the extent to which prior exposure to use of an electronic medication container might affect the results of the subsequent trial. Randomisation to the intervention and standard care groups of the efficacy trial took place at week 9 (see diagram). Figure 1 Trial Profile. The trial statistician randomly allocated patients independently of trial co-ordination and intervention delivery teams. A partial minimisation procedure was used to dynamically adjust randomisation probabilities to balance the baseline stratification variables. These included self-reported adherence, the baseline allocation to use or non-use of the electronic medicines measure and the baseline HbA1c. The London multi-centre research ethics committee reviewed and approved the protocol (06/MRE02/3). BODY.SETTING AND PATIENTS: Patients were recruited from 13 general practices (primary care clinics) in Oxfordshire, Buckinghamshire, Suffolk, Essex and Huntingdonshire (UK). Patients were eligible for inclusion if aged 18 years or over with type 2 diabetes of at least three months duration, able to give informed consent, currently taking any oral glucose-lowering agent and with a HbA1c ≥7.5% (58mmol/mol). Patients were not excluded if taking insulin. Those approached were deemed by their general practitioner to be appropriate for tight glycaemic control and independent in medication taking. BODY.TRIAL MEASURES: The primary outcome was the percentage of days over a twelve-week period on which the correct number of doses of main glucose lowering medication was taken each day as prescribed. It was measured using a validated measuring device [13], a container with a lid incorporating an electronic device that recorded the occurrence and timing of opening (TrackCap, Aardex, Zurich, Switzerland). A single treatment was tracked for each patient during the period of the trial, with metformin the preferred medication. Secondary outcomes included: functional status measured with the 12-item Short Form Medical Outcomes Study health survey questionnaire (SF-12) [14], treatment satisfaction measured with the Diabetes Treatment Satisfaction Questionnaire (DTSQ) [15], satisfaction with communication with the nurse delivering the intervention and the Medication Adherence Report Scale (MARS) [16]. The MARS scale assesses adherence to medication with a five-item self report scale each with item responses scored on a five point scale. Scores are summed to give a score ranging from 5 to 25 with a higher score indicating higher levels of reported adherence. In addition, the number of medications taken was recorded and HbA1c was measured in a central laboratory. The measures are fully described in the trial protocol [10]. BODY.TRIAL INTERVENTION: Eight weeks after recruitment, patients were invited to the intervention visit to record and review their medication and randomised to either an intervention to support medication adherence or a standard care visit in which trial measurements were taken. The intervention had been developed and piloted after a detailed study to identify beliefs held by patients about diabetes and medication taking [17]. The intervention was delivered by a clinic nurse in each practice. A clinical psychologist and intervention facilitator provided initial training for the clinic nurses at a day meeting supported by a detailed manual [10]. The nurses used protocols to standardise delivery of both the intervention and the standard care visit. The psychologist and intervention facilitators provided coaching and feedback to the nurses to ensure that the intervention and standard care were delivered as planned and to ensure intervention fidelity. This addressed possible sources of contamination in intervention delivery including the need to avoid (i) delivering the intervention to standard care patients, (ii) discussing motivational strategies and action planning with other members of the primary care team and (iii) using intervention strategies not specified in the protocol. Delivery of protocols was monitored by formal assessment of audio-taped consultations with all intervention participants and a sample of standard care participants [10]. In the first, motivational component of the intervention, the nurse elicited patients` beliefs relevant to their intention to take medication regularly as prescribed using a series of questions based on the Theory of Planned Behaviour [11]. These included perceived benefits and harms of taking medicines, views of other people who were important to them and factors that may facilitate or inhibit taking medicines regularly as prescribed. Positive beliefs were reinforced verbally and non-verbally through provision of tailored information and problem solving was facilitated around negative beliefs. In the second, action planning component, the nurse asked patients to generate and write down the exact circumstances in which they would take their medication (using an “if-then” formulation to elicit where, when and how this would occur) [12]. In the standard care visit, delivered by the same clinic nurses, none of the above techniques were applied. BODY.STUDY PROCEDURES: The clinic nurse identified eligible patients registered with the practice. Eligible patients were sent a letter from the practice giving details of the trial, and a questionnaire asking about basic demographics, medication regimen, medication adherence and beliefs about taking diabetes medicines. Responders were telephoned by the clinic nurse to arrange a recruitment visit to the full twenty week study period. Patients eligible and willing to take part were randomly allocated in advance of their recruitment visit to receive their medication in a medication monitoring device or in standard packaging. At the 40-min trial recruitment visit with the clinic nurse, patients gave informed consent, including consent for tape-recording interviews for the purposes of training and assessment of fidelity of intervention delivery. Clinical data were collected, blood was taken, and questionnaires completed. For those patients allocated to the electronic medication-monitoring device, its use was explained, and the practice dispenser or pharmacist dispensed the patient’s usual prescription for metformin or alternative oral glucose lowering agent in the device. For those allocated to standard packaging, the practice dispenser or pharmacist provided medication in standard blister-packs. A follow-up and intervention visit was arranged after eight weeks. In advance of the intervention visit, patients were sent a questionnaire from the coordinating centre. Also in advance of the intervention visit, patients were centrally randomised to the intervention or standard care to allow the clinic nurse to prepare to follow the allocated intervention schedule. At the visit, patients allocated to the intervention took part in a consultation, intended to last 50 min, with the clinic nurse that included the intervention (approximately 30 min) and data collection (approximately 20 min). The standard care visit lasted approximately 20 min during which study data were collected. At the intervention visit, blood samples were taken from all patients and enquiries were made about any possible adverse events including hypoglycaemia. All patients were dispensed their usual prescription for metformin or alternative oral glucose lowering agent in a medication monitoring device. A postal questionnaire was completed one week after the intervention visit. Final follow up for all patients at 20 weeks involved a visit to the clinic nurse and included retrieval of the medication monitoring devices, a blood sample for measurement of HbA1c and a final questionnaire. Resource use data were collected on the time taken by clinic nurses to deliver the intervention and collect clinical samples at the intervention visit (preparation time, duration of visit and other input) and, in the case of standard care, the time taken to collect clinical information. Data were also collected on the time taken by intervention facilitators to train the nurses and to provide feedback. BODY.ANALYSIS METHODS: The trial was planned to follow up 200 patients, providing 80% power at the 5% significance level to detect a difference in means between randomised groups of 5% (1.5 days per month difference) in the percentage of days on which the correct number of doses was recorded as being taken. This was based on an estimate of the standard deviation of this measure of 13.5% in a pilot study for the trial conducted in 2001 in Newmarket, Cambridgeshire [10]. Analysis was by intention to treat and continuous outcomes were analysed adjusting for their corresponding baseline value, where this was measured, to improve precision. Where applicable, the missing indicator method was used [18], so that patients with a missing baseline value could be incorporated. Laboratory measurements and medication monitoring data were analysed blind to treatment allocation. The primary outcome was adherence, defined as the percentage of days over 12 weeks on which the correct number of doses was taken. It was calculated from medication monitoring data recorded from the day after the intervention visit (week 9) following randomisation through to the day of the last visit (week 20). Mean adherence was compared between the intervention and control groups using the non-parametric percentile bootstrap method to derive the difference in means with a 95% confidence interval. Subgroup analyses were carried out to explore the impact on the intervention effect of pre-specified baseline subgroup variables: HbA1c, age, gender, number of medications, self-reported adherence, and prior randomization to the electronic medication-monitoring device. These were assessed by testing the effect on the primary outcome of the interaction between each subgroup variable and the randomised group. For this purpose, continuous subgroup variables were dichotomised at the median. An additional analysis was carried out to explore the extent to which prior use of the medication-monitoring device affected nine to 20 week adherence. BODY.RESULTS: In the 13 participating practices, 797 registered patients with type 2 diabetes potentially meeting the inclusion criteria were identified, of whom 273 responded as eligible, and 211 were confirmed as eligible on assessment and randomised with 92% follow up (Figure 1). Recruitment began in July 2006 and follow up was completed by November 2008. Trial participants in intervention and standard care groups were similar, in their early 60s with diabetes for less than 10 years and taking on average 6 medications daily and reporting a score of ≥24 on the MARS scale indicating high medication adherence. A slightly greater proportion of men were allocated to standard care (Table 1). In 9 of the 13 trial practices comparison of trial participants and non-responders suggested that trial participants had a shorter duration of diabetes and were more likely to be prescribed metformin (Table 2). Table 1Baseline characteristics of trial participants  Intervention armStandard Care armAll participants        Socio demographic N=126 N=85 N=211 % male (N) 61.9% (78) 70.6% (60) 65.4% (138) Age (years) 62.5 (11.0) 64.1 (10.3) 63.2 (10.7) IMD Deprivation rank (0-100) 10.2 (6.4) 10.4 (6.8) 10.3 (6.8) Health-related       SF12 Physical [3] (norm 50 43.3 (11.2) 45.5 (10.3) 44.2 (10.9) range 0-100)       SF12 Mental [3] (norm 50 48.0 (10.6) 50.0 (9.6) 48.8 (10.2) range 0-100)       Duration of diabetes (years) 6.7 (4.8) 6.9 (5.3) 6.8 (5.0) Weight (kg) 97.4 (21.7) 94.5 (19.6) 96.2 (20.9) Systolic blood pressure (mmHg) 137.4 (16.2) 136.2 (15.9) 136.9 (16.0) Diastolic blood pressure (mmHg) 78.3 (8.9) 78.1 (9.1) 78.2 (9.0) HbA1c (%) [1] 8.37 (1.25) 8.28 (1.22) 8.33 (1.24) HbA1c (mmol/mol) 68.0 (2.7 67.0 (2.4) 67.5 (2.6) Medication-related       % treated with metformin (N) [4] 86.7% (104) 87.9% (73) 87.2% (177) Metformin daily dose (mg) * [4] 1450 (795) 1525 (780) 1480 (788) Total number of medications taken/day 5.7 (2.4) 5.9 (2.6) 5.8 (2.5) Adherence (MARS) [2] (range 5-25) 23.6 (2.3) 23.6 (2.8) 23.6 (2.5) Values are mean (SD) unless otherwise stated: missing data: [1] 17; [2] 23; [3] 2; [4] 8. * for those treated with metformin. MARS Medication Adherence Report Scale. Table 2Comparison of non-responders with trial participants*  Participants*Non-Responders  Mean (SD) Mean (SD)   N = 133* N = 413 % male (N) 65.4 (87) 62.7 (259) Age (years) 62.7 (10.7) 64.7 (12.8) HbA1c (%) 8.3 (1.2) [1] 8.7 (1.5) [2] HbA1c (mmol/mol) 67 (13) 72 (16) Duration of diabetes (years) 6.5 (4.9) 8.1 (7.1) [3] ** % prescribed metformin (N) 87.8 (115) [4] 76.0 (307) [5] ** Values are mean (SD) unless otherwise stated. Missing data: [1]n = 9; [2]n = 1; [3]n = 5; [4]n = 2; [5]n = 5. *Those participants from the nine practices in which non-responder data were collected. ** P < 0.05. Patients allocated to the intervention took their prescribed number of doses of medication on a significantly higher percentage of days compared to the standard care group (Table 3). The mean difference between groups in percentage of days that the correct number of doses of medication was taken as prescribed was 8.4% (95% confidence interval 0.2% to 16.7%, p = 0.044). No patients were identified as stopping their medication during the period of twelve weeks. There were no significant differences between groups for secondary outcomes including self report medication adherence (MARS), SF-12, diabetes treatment satisfaction, HbA1c, satisfaction with communication with the nurse, or hypoglycaemia (Table 3). Table 3Outcomes twelve weeks after randomisation to intervention or standard care  Intervention armStandard care armIntervention effect (95% C.I)P-value  Baseline Final visit Baseline Final visit     Primary outcome             Days correct dose taken (SD) (%)* - 77.4 (26.3)a   69.0 (30.8)b 8.4 (0.2, 16.7) c 0.044 Secondary outcomes             SF12 Physical (SD) † 43.3 (11.2) r 44.6 (11.1) c 45.5 (10.3) r 46.3 (9.0) l -0.7 (-2.7, 1.4) s 0.52 SF12 Mental (SD) † 48.0 (10.6) 49.5 (10.4) c 50.0 (9.63) t 52.6 (8.8)l -1.6 (-3.9, 0.6) s 0.15 Diabetes treatment satisfaction (SD) - 30.6 (5.4) c - 31.3 (4.6) f -0.7 (-2.2, 0.7) u 0.32 HbA1c % (SD) † 8.34 (1.26) k 8.34 (1.24) k 8.29 (1.23) l 8.21 (1.32) f 0.06 (-0.19, 0.32) m 0.64 HbA1c mmol/l (SD) 67.7 (13.8) 67.7 (13.6) 67.1(13.4) 66.2 (14.4) 0.7 (- 2.1, 3.5)   Medication-related             MARS Self report adherence (SD) † 23.6 (2.3) d 23.6 (2.6) e 23.6 (2.8) f 24.1 (1.6) g -0.4 (-1.0, 0.2) h 0.20 Satisfaction with communication (median, IQR)**   4 (2,5)   4 (2,5)   0.13 % reporting hypoglycaemia (N)***   1.6% (2)   0,0 (0)   0.52* Values are mean (SD) unless otherwise stated. Missing values: a = 13, b = 4, c = 17, d = 15, e = 27, f = 8, g = 12, h = 39, k = 16, l = 9, m = 24, r = 1, s = 26, t = 2, u = 25. * - Estimate of intervention effect adjusted for baseline where available and 95% confidence interval derived from the non-parametric bootstrap percentile method with 50,000 replications. † = In the analysis of the final visit measure adjusting for a baseline that has occasional missing data, the missing indicator method is used. ** p value from Mann–Whitney test., *** p value from Fisher’s exact test. MARS Medication Adherence Report Scale. There were no significant interactions to indicate that the effect of the intervention on the medication adherence outcome varied greatly by gender, number of medications, self-reported adherence (Table 4). However, taking into account the reported confidence intervals, the intervention effect may have been larger in those with better glycaemic control (p = 0.07), older age (p = 0.19) and higher self-reported adherence at baseline (p = 0.11). There was no effect of prior allocation of patients to the electronic medication-monitoring device in the first phase of the study with a mean (95% confidence intervals) difference of 6.0 (-10.5, 22.7) in percentage of days of medication not taken between groups (p = 0.48) (Table 4). In addition there was not an effect of prior allocation to the electronic medication monitoring device on overall nine to 20 week adherence (p = 0.84). Table 4Subgroup analysis of the intervention effect on the percentage of days adherence to prescribed medication Sub-group variableSub-group category (L = Low or H = High)N per sub groupIntervention armStandard care armIntervention- Control (SE)*Difference (H-L) (95% C.I.)P interactionHbA1c % (mmol/mol) 7.5 – 7.9 (58-63) 85 80.4 (24.5) 62.6 (34.2) 17.7 (6.4)       8.0 - 12.4 (64-112) 108 74.8 (27.7) 72.8 (27.8) 2.0 (5.3) -15.8 (-32.9, 1.2) 0.07 Age (years) 37 – 64 94 75.0 (25.61) 72.3 (27.3) 2.7 (5.6)       65 and over 100 80.0 (27.1) 66.4 (33.2) 13.5 (6.0) 10.8 (-5.4, 27.2) 0.19 Total number of medications 0 – 5 89 80.2 (23.3) 73.0 (27.0) 7.2 (5.3)       6 and over 105 75.1 (28.5) 65 .2 (33.7) 10.0 (6.1) 2.8 (-13.5, 19.1) 0.74 Self reported adherence at baseline 0-23 102 75.0 (26.4) 70.9 (27.2) 4.1 (5.5)       24 or 25 70 85.9 (21.3) 68.0 (33.9) 17.9 (6.8) 13.8 (-3.2, 30.0) 0.11 Prior randomisation to medication monitoring device No 102 76.6 (28.7) 71.0 (29.3) 5.6 (5.8)       Yes 92 78.2 (23.7) 66.65 (32.6) 11.6 (5.9) 6.0 (-10.5, 22.7) 0.48 Gender Male 127 77.0 (28.3) 66.4 (31.5) 10.6 (5.3)       Female 67 78.0 (22.9) 74.8 (28.8) 3.3 (6.4) -7.3 (-24.3, 9.6) 0.39 Effect estimates, confidence intervals and p-values from non-parametric bootstrap percentile method (50,000 replications). * Estimated intervention effects are positive within each subgroup. The mean total time (95% confidence interval) spent in delivering the intervention and associated clinical care data collection was 74 min (68 to 79) for the intervention group and 42 min (39 to 47) for the standard care group; a mean difference of 31 min (95% confidence interval 25 to 37). Intervention facilitators spent, on average, 2.3 h per patient listening to tape recordings, training nurses, and providing feedback in the intervention group, compared with 1.2 h in standard care. BODY.DISCUSSION: A theoretically based, single session intervention delivered to patients with type 2 diabetes in primary care consultations by clinic nurses was effective in improving objectively measured glucose lowering medication adherence compared with standard care. The effect was seen consistently over the 12 weeks of the study. The intervention had no adverse effect on measures of functional status, satisfaction, communication or hypoglycaemia. There was no effect on glycaemia measured by HbA1c, but the power and time frame of our trial were not designed to test for this effect. This study addressed key weaknesses in previous studies that have limited the quality of the evidence concerning medication adherence. The most effective interventions to improve medication adherence and clinical outcomes have been complex, multi-component and intensive, but few studies have been designed to allow exploration of the reasons for success or failure of interventions and their delivery [19]. Target groups have often been poorly defined and characterised and trial participants also tend to be unusually adherent, limiting discovery of effects that would be important in general populations [20-22]. The most commonly used measures are self report and these are often associated with larger effects than objective measurement [23,24]. Trial designs themselves have often been weak with lack of attention to central randomisation and sources of bias. Not surprisingly, previous studies in this field have shown inconsistent effects. We addressed these issues in the following ways. We developed the intervention systematically from psychological evidence and theory [25]. It is predicated on addressing weak motivation associated with ambivalence to medication taking [26], and the gap between intention and action that may be bridged by making specific action plans [11,12]. We ensured the intervention was feasible to deliver in a health service context and addressed quality assurance. Nurses were trained to deliver the intervention in workshops using scripts and feedback, and consultations were audio-taped to assure delivery as planned and to support a consistent approach to delivery across nurses over time. We also identified a patient group with the potential to benefit from improving their adherence to medication. They comprised a well characterised population which reflected the kind of patients with diabetes seen in primary care every day: in their sixties, with established diabetes of seven years mean duration, and prescribed an average six of medications daily, including metformin, without having reached optimum glycaemic control. In addition we obtained an acceptable rate of participation from eligible patients, and were able to demonstrate that the characteristics of these individuals were similar to the wider population from which they were recruited. We measured the primary outcome objectively using a validated electronic medication monitor [13] that allows a day-by-day description of adherence as well as providing summary measures. In an initial, randomised evaluation of the impact of electronic medication measurement on adherence we found that prior use of an electronic medication-monitoring device had no statistically significant effect either on adherence or in modifying the intervention effect on adherence for the primary outcome. The study design was rigorous with central randomisation and blinding of group allocation from those assessing outcome. Randomly allocated groups were well matched on the measured variables. There are number of limitations to this study. The participation rate was not high, although attempts were made to mitigate this by anonymised collection of data on non-participants. We restricted our intervention to glucose lowering medication and excluded those in whom tight glucose control was inappropriate, although support for taking other medications might be appropriate for this group of patients. Detailed work was undertaken to minimise the possibility of contamination between intervention and usual care groups, although any failure of the procedures put in place would reduce the size of effect observed. Self reported adherence, as measured by the MARS self-report questionnaire, did not differ between intervention and usual care groups. MARS attempts to capture awareness of non-adherence due to forgetting, altering, stopping, missing or taking less medication than prescribed. Moreover, estimates of adherence were close to maximal in both groups as has been found elsewhere [17,27]. Thus, while the MARS results did not confirm our principal outcome of electronic monitoring, it may be because the latter is measuring a different component of adherence, being more sensitive to unconscious non-adherence, and also that it is less constrained by ceiling effects. Self-report measures, when used in trials may also be difficult to interpret, as they are more susceptible to outcome preference bias. Our aim in this short-term explanatory study was to estimate the efficacy of the intervention on the behaviour of taking medication and the trial was therefore not powered to evaluate glycaemic effects. However, we anticipated that with a large effect on tablet taking, we might see some indication of an effect on HbA1c. Efficacy studies with similar time periods and doses of metformin (1500g per day) have demonstrated improvements in HbA1c of around 1.5% compared with placebo among patients with very poorly controlled diabetes at baseline [28], but smaller effects among patients under better control [29]. However, the impact of the improved adherence of about one week over the three month period in our intervention group was not sufficient to alter overall glycaemia. BODY.CONCLUSIONS: We have demonstrated that a well-specified and reproducible consultation-based intervention delivered in a single session by clinic nurses in primary care can increase objectively measured medication adherence with no adverse effect on treatment satisfaction. Application of this approach offers the potential for reducing the burden of disease in diabetes managed by long-term medication. A larger pragmatic trial, with further development of intervention components, designed to sustain effect, with longer follow up, and powered to evaluate the effect of the intervention on clinical outcomes is justified. BODY.COMPETING INTERESTS: No potential conflicts of interest have been reported for this paper. BODY.AUTHORS’ CONTRIBUTIONS: A.F. wrote the article, contributed to discussion and researched data. A-L.K. wrote the article, contributed to discussion and researched data. W.H. contributed to discussion, researched data and reviewed and edited the article. S.G. contributed to discussion, researched data and reviewed and edited the article. A.T.P. contributed to discussion, researched data and reviewed and edited the article. S.S. contributed to discussion, researched data and reviewed and edited the article. D.H. contributed to discussion, researched data and reviewed and edited the article. A.C. researched data and reviewed and edited the article. S.B. contributed to discussion, researched data and reviewed and edited the article. J.G. contributed to discussion, researched data and reviewed and edited the article. I.K. contributed to discussion, researched data and reviewed and edited the article. J.O. researched data. M.S. researched data and reviewed and edited the article. Y.K. researched data and reviewed and edited the article. All authors approved the final draft of the manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2296/13/30/prepub

TITLE: Safety and tolerability of the novel non-steroidal mineralocorticoid receptor antagonist BAY 94-8862 in patients with chronic heart failure and mild or moderate chronic kidney disease: a randomized, double-blind trial ABSTRACT.AIMS: Mineralocorticoid receptor antagonists (MRAs) improve outcomes in patients with heart failure and reduced left ventricular ejection fraction (HFrEF), but their use is limited by hyperkalaemia and/or worsening renal function (WRF). BAY 94-8862 is a highly selective and strongly potent non-steroidal MRA. We investigated its safety and tolerability in patients with HFrEF associated with mild or moderate chronic kidney disease (CKD). ABSTRACT.METHODS AND RESULTS: This randomized, controlled, phase II trial consisted of two parts. In part A, the safety and tolerability of oral BAY 94-8862 [2.5, 5, or 10 mg once daily (q.d.)] was assessed in 65 patients with HFrEF and mild CKD. In part B, BAY 94-8862 (2.5, 5, or 10 mg q.d., or 5 mg twice daily) was compared with placebo and open-label spironolactone (25 or 50 mg/day) in 392 patients with HFrEF and moderate CKD. BAY 94-8862 was associated with significantly smaller mean increases in serum potassium concentration than spironolactone (0.04–0.30 and 0.45 mmol/L, respectively, P < 0.0001–0.0107) and lower incidences of hyperkalaemia (5.3 and 12.7%, respectively, P = 0.048) and WRF. BAY 94-8862 decreased the levels of B-type natriuretic peptide (BNP), amino-terminal proBNP, and albuminuria at least as much as spironolactone. Adverse events related to BAY 94-8862 were infrequent and mostly mild. ABSTRACT.CONCLUSION: In patients with HFrEF and moderate CKD, BAY 94-8862 5–10 mg/day was at least as effective as spironolactone 25 or 50 mg/day in decreasing biomarkers of haemodynamic stress, but it was associated with lower incidences of hyperkalaemia and WRF. See page 2426 for the editorial comment on this article (doi:10.1093/eurheartj/eht235) BODY.INTRODUCTION: The mineralocorticoid receptor antagonists (MRAs) spironolactone and eplerenone have been shown to be effective in reducing cardiovascular mortality and hospitalizations for heart failure (HF), as well as total mortality, in patients with chronic HF and a reduced left ventricular ejection fraction (HFrEF).1,2 However, despite their proven benefits in large-scale, prospective, double-blind, randomized trials and recommendations for their use included in international guidelines,3,4 they remain under-utilized, in large part owing to the risk of hyperkalaemia and renal dysfunction. BAY 94-8662 is a next-generation non-steroidal MRA that has shown improved selectivity for the mineralocorticoid receptor (MR) over other steroid hormone receptors compared with spironolactone and improved affinity for the MR compared with eplerenone in pre-clinical studies.5 In comparative studies using pre-clinical models of hypertension-driven HF and renal dysfunction, BAY 94-8862 has been found to confer more pronounced cardiorenal end-organ protection than the steroidal MRAs.6,7 The minerAlocorticoid Receptor Antagonist Tolerability Study (ARTS) was designed to assess the safety and tolerability of BAY 94-8862 in patients with HFrEF and mild or moderate chronic kidney disease (CKD), and to select doses for further study in phase III clinical trials. BODY.METHODS: BODY.STUDY DESIGN: ARTS was a multicentre, randomized, parallel-group, phase II study, with double-blind placebo and open-label spironolactone comparator arms, conducted in patients with HFrEF (left ventricular ejection fraction, ≤40%) and mild or moderate CKD [estimated glomerular filtration rate (eGFR) using the modification of diet in renal disease (MDRD) formula, 60 to <90 mL/min/1.73 m2 and 30 to 60 mL/min/1.73 m2, respectively] at 55 centres in 10 countries worldwide. The design of the trial has been published in detail elsewhere.8 The investigation conforms to the Declaration of Helsinki and was conducted in keeping with applicable local laws and regulations. Documented approval was obtained from appropriate independent ethics committees or institutional review boards for all participating centres before the start of the study. All patients gave written informed consent to participate in the study. In part A of the study, the safety, tolerability, and renal effects of oral BAY 94-8862 [2.5, 5, or 10 mg once daily (q.d.)] compared with placebo were investigated in patients with HFrEF and mild CKD. The effects on serum potassium concentration, eGFR, and albuminuria were assessed. An independent data monitoring committee (DMC) reviewed data from part A to confirm the safety and tolerability of BAY 94-8862 in patients with HFrEF and mild CKD before the initiation of part B. In part B, the primary endpoint was the change in serum potassium concentration after treatment with oral BAY 94-8862 or placebo in patients with HFrEF and moderate CKD, measured at visits 6 and 7 [i.e. the mean of the changes from baseline to visits 6 (day 22 ± 2) and 7 (day 29 ± 2)] to reduce the variability associated with isolated measurements. The timing of the primary endpoint assessment was based on observations from the PEARL-HF trial, in which serum potassium concentrations increased in the first 7 days after treatment initialization or up-titration of spironolactone and remained nearly stable thereafter.9 The effect of oral BAY 94-8862 on serum potassium concentration was also compared with that of oral open-label spironolactone at a dose of 25 mg q.d. up-titrated to 50 mg q.d. as a secondary objective. Other secondary objectives were the safety and tolerability of BAY 94-8862 and its effects on systolic blood pressure (SBP) and levels of serum aldosterone, B-type natriuretic peptide (BNP) and amino-terminal proBNP (NT-proBNP), eGFR, and albuminuria measured at visit 4 (day 15 ± 1, i.e. before dose up-titration in the spironolactone group) and visit 7 (day 29 ± 2). Because all secondary endpoints were only exploratory, results at the end of the treatment period were assessed (i.e. visit 7 only rather than visits 6 and 7 combined). BODY.PATIENTS: Inclusion and exclusion criteria for the study are provided in detail elsewhere.8 Briefly, adult males and females without childbearing potential were eligible for inclusion if they had a clinical diagnosis of HFrEF [New York Heart Association (NYHA) class II–III and left ventricular ejection fraction ≤40%] and were treated with evidence-based therapy for HFrEF; if their serum potassium concentration was ≤4.8 mmol/L at the screening visit; and if their eGFR was 60 to <90 mL/min/1.73 m2 (part A) or 30 to 60 mL/min/1.73 m2 (part B) according to the MDRD formula. BODY.STUDY MEDICATION: In both parts of the study, patients received study drug for 4 weeks, starting within 14 days of the initial screening visit. The randomization list was generated using a validated automated system that assigned treatment groups to randomization numbers. In part A, patients were randomized 1:1:1:1 to receive oral BAY 94-8862 at doses of 2.5, 5, or 10 mg q.d., or placebo. In part B, patients were randomized 1:1:1:1:1:1 to receive oral BAY 94-8862 at doses of 2.5, 5, or 10 mg q.d., or 5 mg twice daily (b.i.d.), placebo, or open-label oral spironolactone, which was given at an initial dose of 25 mg q.d. and up-titrated to 50 mg q.d. on day 15 ± 1 if serum potassium concentration remained below or equal to 4.8 mmol/L. BODY.INVESTIGATIONS: Patients were assessed at the screening visit, at baseline/day 1, day 4 ± 1 and day 8 ± 1, and then weekly until the end of the study and at a follow-up visit 14 days after the last intake of study drug. Patients who terminated the study early were assessed as soon as possible after discontinuation of study drug. For patients who received spironolactone at a dose of 50 mg q.d., an additional assessment visit was made at day 18 ± 1. Details of assessments have been published elsewhere.8 Briefly, adverse events, vital signs, blood and urinary biomarkers, standard haematology, clinical chemistry, and urinalysis were assessed at each visit. BODY.STATISTICAL CONSIDERATIONS: Statistical analyses were performed using SAS version 9.2 (SAS Institute, Inc., Cary, NC, USA). Analyses are presented for two sets: the safety set (all patients who had received at least one dose of study drug) and the full analysis set (all patients from the safety set of part B with baseline and one or more post-baseline serum potassium measurements). Unless otherwise specified in the text, missing values were not imputed. For part A, all variables were analysed descriptively in the safety set. For part B, the full analysis set was used for the primary analysis. Five different dose–response models were fitted to the serum potassium data from the placebo and BAY 94-8862 groups,8 but none was considered to provide a better model than analysis of covariance (adjusted for centre and baseline serum potassium concentration). Analysis of covariance was therefore used for the primary analysis, as well as for assessing changes in secondary measurements in the full analysis set. Other safety variables were analysed in the safety set. In part A, no formal sample size calculation was performed; 15 patients per dose group were considered sufficient to assess the safety and tolerability of BAY 94-8862 in patients with HFrEF and mild CKD, before investigating its effects in a higher risk population, i.e. patients with HFrEF and moderate CKD, in part B. In part B, based on a planned sample size of 60 patients per group and the assumption that the common standard deviation for the change in serum potassium concentration would be 0.50 mmol/L, each comparison between the BAY 94-8862 groups and the spironolactone or placebo group had 80% power to detect a difference in serum potassium concentration of at least 0.26 mmol/L, using two-sided tests at alpha = 0.05.8 Significance levels for secondary variables were not pre-specified, and no adjustment was made for multiple comparisons; the P-values for secondary variables should therefore be considered descriptive rather than confirmatory. BODY.RESULTS: BODY.DEMOGRAPHICS: In total, 782 patients were enrolled and 458 randomized [65 (80% male) in part A; 393 (79% male) in part B] (Figure 1). One randomized patient in part B did not receive any study drug and was therefore excluded from the safety analysis set. In the safety sets of parts A and B, the mean ages were 66.3 ± 8.9 and 72.1 ± 7.8 years, respectively. At enrolment, the majority of patients (95.4% in part A and 81.6% in part B) were in NYHA functional class II and the remaining patients were in functional class III (Table 1). All randomized patients in part A were naïve to MRA therapy; 56 patients in part B had received MRA therapy previously (stopped 30 days prior to randomization). Further details of study population characteristics are available as Supplementary material online. Table 1Demographics and baseline characteristics of patients enrolled in ARTS Part A (n = 65)Part B (n = 392)Males, n (%) 52 (80.0) 312 (79.6) Mean age (range), years 66.3 (42–85) 72.1 (40–89) Mean BMI (range), kg/m2 28.6 (21.5–41.4) 28.8 (18.1–46.9) Mean systolic blood pressure (range), mmHg 133.8 (83–169) 127.3 (81–180) NYHA functional class, n (%)  II 62 (95.4) 320 (81.6)  III 3 (4.6) 72 (18.4) Medical history, n (%)  Ischaemic heart disease 24 (36.9) 251 (64.0)  Atrial fibrillation 24 (36.9) 177 (45.2)  Congestive cardiomyopathy 8 (12.3) 36 (9.2)  Arterial hypertension 28 (43.1) 261 (66.6)  Diabetes mellitus 9 (13.8) 134 (34.2)   Treated with metformin 5 (7.7) 51 (13.0) Concomitant medication, n (%)  Agents acting on renin–angiotensin system 64 (98.5) 372 (94.9)  Beta-blockers 63 (96.9) 366 (93.4)  Diuretics 46 (70.8) 349 (89.0) Baseline laboratory values  Mean serum potassium ± SD, mmol/L 4.23 ± 0.33 4.29 ± 0.42  Median serum creatinine (range), mg/dL 1.000 (0.70–1.30) 1.400 (0.80–3.10)  Mean eGFR (MDRD) ± SD, mL/min/1.73 m2 69.1 ± 8.43 47.0 ± 10.0  Geometric mean UACR (geometric SD), mg/g 13.67 (3.20) 21.33 (4.87)  Median BNP (range), pg/mL – 270.0 (10–6382)  Median NT-proBNP (range), pg/mL – 1381.45 (22.7–32 349.1)  Median serum aldosterone (range), pmol/L – 279.100 (<LLOQ–2557.70) The dash indicates data not recorded; BMI, body mass index; BNP, B-type natriuretic peptide; eGFR, estimated glomerular filtration rate; LLOQ, lower limit of quantification (for serum aldosterone, LLOQ = 7.35 pmol/L); MDRD, modification of diet in renal disease; NT-proBNP, amino-terminal pro-B-type natriuretic peptide; NYHA, New York Heart Association; SD, standard deviation; UACR, urinary albumin:creatinine ratio. Figure 1Disposition of patients in part A (A) and part B (B) of ARTS. b.i.d., twice daily; q.d., once daily. BODY.PART A: The safety and tolerability of different doses of BAY 94-8862 in patients with HFrEF and mild CKD were confirmed after analysis of data from part A of the study (Table 2) by the independent DMC. As a result of this positive outcome, part B was initiated to assess safety and efficacy of BAY 94-8862 in patients with HFrEF and moderate CKD in September 2011. Table 2Treatment-emergent adverse events, including serious adverse events, in parts A and B of ARTS BAY 94-8862 (2.5 mg q.d.)BAY 94-8862 (5 mg q.d.)BAY 94-8862 (10 mg q.d.)BAY 94-8862 (5 mg b.i.d.) (part B only)Spironolactone (25 or 50 mg q.d.) (part B only)PlaceboPart A  Total patients, N 16 16 17 – – 16  Patients with at least one TEAE, n (%) 10 (62.5) 6 (37.5) 5 (29.4) – – 6 (37.5)  Cardiac disorders, n (%) 1 (6.3) 1 (6.3) 0 – – 0   Angina pectoris 0 1 (6.3)a 0 0   Sinus tachycardia 1 (6.3) 0 0 0  Gastrointestinal disorders, n (%) 0 1 (6.3) 2 (11.8) – – 0   Constipation 0 1 (6.3) 0 0   Flatulence 0 0 1 (5.9) 0   Nausea 0 0 1 (5.9) 0  Investigations needed, n (%) 1 (6.3) 0 0 – – 2 (12.5)   Blood CPK level increasedb 1c (6.3) 0 0 1 (6.3)   Blood glucose level increasedb 0 0 0 1d (6.3)  Metabolism and nutrition disorders, n (%) 1 (6.3) 0 1 (5.9) – – 0   Diabetes mellitus 1 (6.3) 0 0 0   Hyperkalaemiab 0 0 1 (5.9) 0  Nervous system disorders, n (%) 1 (6.3) 0 0 – – 2 (12.5)   Dizziness 0 0 0 1 (6.3)   Headache 1 (6.3) 0 0 1 (6.3)  Renal disorders, n (%) 1 (6.3) 0 0 – – 0   Pollakiuria 1 (6.3) 0 0 0  Vascular disorders, n (%) 0 0 1 (5.9) – – 0   Hypotension 0 0 1 (5.9) 0 Part B  Total patients, N 66 67 67 64 63 65  Patients with at least one TEAE, n (%) 31 (47.0) 36 (53.7) 34 (50.7) 34 (53.1) 50 (79.4) 33 (50.8)   Withdrawal 7 (10.6) 3 (4.5) 4 (6.0) 6 (9.4) 11 (17.5) 6 (9.2)  Cardiac failuree, n (%) 0 2 (3.0) 3 (4.5) 1 (1.6) 2 (3.2) 3 (4.6)   Withdrawal 0 1 (1.5) 1 (1.5) 0 0 1 (1.5)  Hyperkalaemia/blood K+ level increasedf, n (%) 3 (4.5) 1 (1.5) 3 (4.5) 5 (7.8) 7 (11.1) 1 (1.5)   Withdrawal 2 (3.0) 0 0 2 (3.1) 2 (3.2) 0  Worsening of renal functiong, n (%) 1 (1.5) 3 (4.5) 7 (10.4) 4 (6.3) 24 (38.1) 6 (9.2)   Withdrawal 0 0 1 (1.5) 1 (1.6) 5 (7.9) 1 (1.5)  Hypotension, n (%) 0 2 (3.0) 1 (1.5) 7 (10.4) 4 (6.3) 0   Withdrawal 0 0 0 1 (1.5) 1 (1.6) 0 b.i.d., twice daily; CPK, creatine phosphokinase; K, potassium; q.d., once daily; TEAE, treatment-emergent adverse event. aThis was a serious adverse event that led to discontinuation of study drug. bInvestigator-reported events. cPatient with blood CPK concentration of 606 U/L at visit 4 (day 15 ± 1), moderate adverse event, study drug discontinued. dPatient with blood glucose concentration of 128 mg/dL at visit 4, mild adverse event, study drug continued. eIncludes cardiac failure, cardiac failure chronic, and cardiac failure congestive. fAny event reported as ‘hyperkalaemia’ or ‘blood potassium increased’. gAny increase in serum creatinine by ≥0.3 mg/dL from baseline and/or decrease in estimated glomerular filtration rate by ≥25% from baseline; includes renal failure chronic, renal injury, and renal impairment. BODY.PART B: The full analysis set comprised 389 patients. Of the 63 patients assigned to receive spironolactone, the dose was up-titrated from 25 to 50 mg q.d. on visit 4 (day 15 ± 1) for 30 (47.6%) patients, resulting in a mean dose of 37 mg/day for this group at visit 7 (day 29 ± 2). BODY.SERUM POTASSIUM CONCENTRATIONS: Of the 389 patients in the full analysis set, 356, 342, and 336 had serum potassium data at visits 4, 6, and 7, respectively. Patients receiving BAY 94-8862 at doses of 10 mg q.d. and 5 mg b.i.d. showed significantly greater mean increases in serum potassium concentration from baseline at the study endpoint [i.e. the mean of changes at visits 6 (day 22 ± 2) and 7 (day 29 ± 2)] than the placebo group (P = 0.0243 and P = 0.0003, respectively). However, in the 5 and 2.5 mg q.d. groups, the mean increases in serum potassium concentration were not significantly different from those in the placebo group at visit 4 (day 15 ± 1) or at the study endpoint (P = 0.1623 and P = 0.5745, respectively) (Figure 2). The mean increases in serum potassium concentration were significantly smaller in all four BAY 94-8862 dose groups than in the spironolactone group (P < 0.0001 for 2.5, 5, and 10 mg q.d. and P = 0.0107 for 5 m.g. b.i.d.) (Figure 2). Missing data were few (∼12%), and a secondary analysis using the ‘last observation carried forward’ approach to impute missing data did not alter the conclusions of the original analysis. In a descriptive sub-analysis of patients aged >75 years (n = 123), the mean increases in serum potassium concentration from baseline to visit 6/7 remained highest in the spironolactone group. A descriptive sub-analysis comparing patients in NYHA class II and III found no difference in serum potassium results between the two groups. Serum potassium results also showed no marked difference between MRA-naïve and MRA-non-naïve patients. Figure 2Mean change from baseline to visit 4 (day 15 ± 1) and the mean of visit 6 (day 22) and visit 7 (day 29 ± 2) in serum potassium concentration in patients receiving BAY 94-8862, placebo, or spironolactone in the full analysis set of part B of ARTS. *P < 0.05 between the BAY 94-8862 dose group and the placebo group at visit 6/7; †P < 0.05 between the BAY 94-8862 dose group and the spironolactone group at visit 6/7. Significance of visit 4 data was not analysed. b.i.d., twice daily; q.d., once daily. BODY.ESTIMATED GLOMERULAR FILTRATION RATE: Figure 3 shows the mean change in eGFR from baseline to visits 4 and 7 in part B of ARTS. There was a decrease in eGFR in all BAY 94-8862 groups and the spironolactone group, compared with a small increase in the placebo group. However, the decrease in the spironolactone group was significantly greater than in all BAY 94-8862 groups (P = 0.0002–0.0133 at visit 7). A similar trend was observed in the subset of patients aged >75 years. In all treatment groups except the group receiving BAY 94-8862 2.5 mg q.d., patients in NHYA class III showed a greater decrease in eGFR than those in NYHA class II; however, the greatest decrease was still observed in the spironolactone arm. Figure 3Mean change from baseline to visit 4 (day 15 ± 1) and visit 7 (day 29 ± 2) in the estimated glomerular filtration rate (eGFR), as calculated using the modification of diet in renal disease formula, in patients receiving BAY 94-8862, placebo, or spironolactone in the safety analysis set of part B of ARTS. *P < 0.05 compared with the placebo group at visit 6/7; †P < 0.05 compared with the spironolactone group at visit 6/7. Significance of visit 4 data was not analysed. b.i.d., twice daily; q.d., once daily; SD, standard deviation. BODY.SYSTOLIC BLOOD PRESSURE: In part B of the study, high variability was observed in SBP (determined by cuff measurement) in all groups. However, spironolactone significantly decreased SBP between baseline and visit 7 compared with either placebo (P = 0.0104) or all doses of BAY 94-8862 (P = 0.0023–0.0255) (Figure 4). Changes in SBP in the BAY 94-8862 groups were comparable with those seen in patients receiving placebo. Figure 4Mean change from baseline to visits 4 (day 15 ± 1) and 7 (day 29 ± 2) in systolic blood pressure in the safety analysis set of part B of ARTS. *P < 0.05 compared with the placebo group at visit 6/7; †P < 0.05 compared with the spironolactone group at visit 6/7. Significance of visit 4 data was not analysed. b.i.d., twice daily; q.d., once daily; SD, standard deviation. BODY.SERUM B-TYPE NATRIURETIC PEPTIDE, AMINO-TERMINAL-PRO-B-TYPE NATRIURETIC PEPTIDE, AND URINARY ALBUMIN:CREATININE RATIO: Data from part B of the study showed no significant overall treatment effect on BNP, NT-proBNP, or urinary albumin:creatinine ratio (UACR) (P > 0.05), so P-values for individual treatment group comparisons were not calculated, and the treatment groups were analysed only descriptively. At visit 4, median concentrations of BNP and NT-proBNP decreased from baseline in all BAY 94-8862 dose groups (Figure 5A and B). At visit 7, median BNP decreased from baseline in patients receiving BAY 94-8862 10 mg q.d. and 5 mg b.i.d. and showed a small increase from baseline in those receiving BAY 94-8862 2.5 and 5 mg q.d.; median NT-proBNP decreased from baseline in patients receiving BAY 94-8862 ≥5 mg q.d. and increased from baseline in those receiving BAY 94-8862 2.5 mg q.d. In patients receiving spironolactone, median BNP and NT-proBNP decreased from baseline at visits 4 and 7. Median BNP and NT-proBNP concentrations showed a small increase from baseline in the placebo group. The decreases in patients receiving BAY 94-8862 were at least comparable with those recorded in the spironolactone group, with a trend towards greater decreases in the BAY 94-8862 5 mg q.d., 10 mg q.d., and 5 mg b.i.d. dose groups compared with patients receiving spironolactone 25 mg q.d. at visit 4, and in the BAY 94-8862 10 mg q.d. dose group compared with patients receiving spironolactone 25 or 50 mg q.d. (mean 37 mg) at visit 7. Mean UACRs decreased in all BAY 94-8862 q.d. dose groups and in the spironolactone group, compared with a small increase in the placebo group (Figure 5C). Figure 5Change from baseline to visits 4 (day 15 ± 1) and 7 (day 29 ± 2) in serum BNP (A: median change), NT-proBNP (B: median change), and UACR (C: geometric mean change) in the safety analysis set of part B of ARTS. b.i.d., twice daily; BNP, B-type natriuretic peptide; IQR, inter-quartile range; NT-proBNP, amino-terminal pro-B-type natriuretic peptide; q.d., once daily; SD, standard deviation; UACR, urinary albumin:creatinine ratio. BODY.SERUM ALDOSTERONE CONCENTRATIONS: Figure 6 shows the change in serum aldosterone levels from baseline to visits 4 and 7 in part B. The change from baseline was higher in the BAY 94-8862 groups than in the placebo group. The largest increase from baseline was seen in the patients receiving spironolactone (P < 0.0001 compared with placebo and each BAY 94-8862 dose group at visit 7). Figure 6Mean change from baseline to visit 4 (day 15 ± 1) and visit 7 (day 29 ± 2) in serum aldosterone levels in the safety analysis set of part B of ARTS. *P < 0.05 compared with the placebo group at visit 6/7; †P < 0.05 compared with the spironolactone group at visit 6/7. Significance of visit 4 data was not analysed. b.i.d., twice daily; q.d., once daily; SD, standard deviation. BODY.ADVERSE EVENTS: In part A of the study, treatment-emergent adverse events (TEAEs) were mostly mild and considered unrelated to study drug (Table 2). The number of serious TEAEs was low (occurring in 2 of 65 patients; 3.1%). There was one case of investigator-reported hyperkalaemia in the BAY 94-8862 10 mg q.d. group at visit 5 (day 22 ± 2) (serum potassium concentration of 5.8 mmol/L as measured by the central laboratory, and 5.2 mmol/L as measured by the local laboratory). Study drug was not withdrawn and no action was taken in this case. Serum potassium concentration stayed within the normal range in this patient throughout the rest of the study. In part B, TEAEs were mostly mild (Table 2). The number of serious TEAEs was also low (occurring in 23 of 392 patients; 5.9%). The highest proportion of serious TEAEs considered to be drug-related was in the spironolactone group (occurring in 5 of 63 patients; 7.9%). The highest proportion of TEAEs leading to discontinuation of study drug was also in the spironolactone group (occurring in 11 of 65 patients; 17.5%). Considering only TEAEs that occurred before visit 4, i.e. before the spironolactone dose was up-titrated, the spironolactone group had the highest proportion of TEAEs (57.1% compared with 29.7–40.3% in the BAY 94-8862 groups) and TEAEs leading to discontinuation of study drug (9.5% compared with 1.5–6.3% in the BAY 94-8862 groups). In a post hoc analysis, data from part B were pooled. Compared with the placebo group, the pooled BAY 94-8862 groups had higher incidences of investigator-reported hyperkalaemia/increased blood potassium levels (5.3 vs. 1.5%, P = 0.3195) and renal failure (1.5 vs. 0%, P = 1.0000) and a lower incidence of renal impairment (3.8 vs. 9.2%, P = 0.0996). The incidence of each of these adverse events was significantly lower in the BAY 94-8862 groups than in the spironolactone group (hyperkalaemia/increased blood potassium levels: 5.3 vs. 12.7%, P = 0.048; renal failure: 1.5 vs. 7.9%, P = 0.0153; renal impairment: 3.8 vs. 28.6%, P < 0.0001). When pooling data from the BAY 94-8862 5 and 10 mg q.d. groups only, the incidence of hyperkalaemia/increased blood potassium levels, renal failure, and renal impairment remained significantly lower in the BAY 94-8862 groups than in the spironolactone group (3.7 vs. 12.7%, P = 0.0284; 1.5 vs. 7.9%, P = 0.0352; and 6.0 vs. 28.6%, P < 0.0001, respectively). BODY.DISCUSSION: ARTS is the first randomized clinical trial of BAY 94-8862, with double-blind placebo and open-label spironolactone comparator arms, to be conducted in patients with HFrEF and mild or moderate CKD. BAY 94-8862 at doses of 5 and 10 mg q.d. decreased the levels of BNP, NT-proBNP, and albuminuria to at least the same, if not a greater degree than spironolactone 25 or 50 mg q.d. Of particular interest is the finding that although these doses of BAY 94-8862 raised serum potassium concentrations as expected, this increase and the incidence of hyperkalaemia associated with these doses were significantly smaller than those recorded for the spironolactone 25 or 50 mg q.d. group. Furthermore, decreases in eGFR were smaller and the incidence of worsening of renal function was lower in all groups of patients receiving BAY 94-8862 than in those receiving spironolactone. Interestingly, the reduction in SBP and the rise in serum aldosterone levels in patients receiving BAY 94-8862 5 and 10 mg q.d. were smaller than observed in patients receiving spironolactone, which could be an additional benefit for patients with HFrEF whose blood pressure is already well controlled by baseline medications (e.g. angiotensin-converting enzyme inhibitors, beta-blockers, diuretics)10,11 or whose blood pressure is low due to worsening HF.4 In other populations, such as those with resistant hypertension, the greater blood pressure-lowering effect of spironolactone would be an advantage.12 The SBP, BNP, and NT-proBNP findings of the present study are consistent with the findings of a head-to-head comparison of BAY 94-8862 and eplerenone in a pre-clinical model of hypertension-driven HF.6 The incidence of adverse events related to study drug was low, with the highest proportion of serious adverse events considered to be drug-related occurring in the spironolactone group. Worsening of renal function was reported as an adverse event in 38% of patients treated with spironolactone, whereas the frequencies in the BAY 94-8862 groups were comparable with the event rate in the placebo group. Analysis of pooled data from part B of the study showed that the incidence of hyperkalaemia was significantly higher in the spironolactone group than in the BAY 94-8862 groups. Of interest, the incidences of adverse and serious adverse events even at the highest doses of BAY 94–8662, 5 and 10 mg q.d., were less than those observed with spironolactone. The effect of eplerenone was not studied in ARTS, and is difficult to predict because of the general lack of direct comparative data for spironolactone and eplerenone in populations with HF. BAY 94-8862 will be compared with eplerenone in a separate clinical trial (ARTS-HF; ClinicalTrials.gov identifier: NCT01807221). The trend towards a greater reduction in BNP and NT-proBNP levels with BAY 94-8862 5 mg b.i.d. and 10 mg q.d. compared with spironolactone 25 or 50 mg suggests that BAY 94-8862 may exert greater effects on cardiac function and haemodynamic stress at these doses than spironolactone. Studies in rodents have demonstrated that the concentrations of spironolactone and eplerenone in renal tissue are at least six-fold and three-fold higher, respectively, than in the myocardium.13 In contrast, BAY 94-8862 distributes equally to the kidney and the heart in rats (unpublished data). This suggests that BAY 94-8862 may have cardiac effects even at relatively low dosages and may in part explain the lower incidence of hyperkalaemia with BAY 94-8862 compared with spironolactone. Furthermore, pre-clinical observations have demonstrated that BAY 94-8862 has more pronounced cardiac and renal anti-remodelling effects than the steroidal MRA eplerenone.6,7,14 The plasma half-life of BAY 94-8862 is ∼2 h in healthy humans15 compared with >12 h for spironolactone in healthy volunteers (>24 h in patients with HF) owing to the generation of active metabolites.13 Although the half-life of BAY 94-8862 is relatively short, there does not seem to be any advantage in twice-daily administration in the present study. The relatively long duration of action of BAY 94-8862 despite its relatively short plasma half-life is similar to that of eplerenone, which has a plasma half-life of 4–6 h in patients with HF13 but is effective in reducing cardiovascular mortality and hospitalizations for HF at doses of 25 or 50 mg q.d.16,17 The duration of the effect of an MRA may depend on the biological half-life of the MR in producing its downstream effects in different tissues, such as sodium retention in the kidney or stimulation of pro-inflammatory gene expression, rather than the plasma elimination half-life of the MRA itself.13 Moreover, the effects of an MRA on pharmacological downstream targets in the kidney, such as the epithelial sodium channel (for sodium excretion) and the renal outer medullary potassium channel (for potassium retention), may take place over longer time frames. As a consequence of receptor blockade by an antagonist, the serum concentrations of the receptor ligand are expected to rise. The magnitude of this rise is usually associated with the degree of receptor blockade. In ARTS, serum concentrations of the MR ligand aldosterone increased as expected in all BAY 94-8862 groups, and to a greater extent in the spironolactone group. Levels of BNP, NT-proBNP, and albuminuria were reduced by BAY 94-8862 5 and 10 mg q.d. to at least the same degree as spironolactone 25 or 50 mg q.d., despite the lower compensatory rises in aldosterone induced by BAY 94-8862. The finding that spironolactone 25 or 50 mg q.d. resulted in a greater increase in serum aldosterone levels than any dose of BAY 94-8862 may have several reasons (all of which will require further investigation), including: differences in plasma half-life; the effect of the differences in serum potassium concentration on the release of aldosterone from the adrenal gland; and differences in the tissue distribution of the non-steroidal MRA BAY 94-8862 and the steroidal MRA spironolactone. BAY 94-8862 is highly selective and has a high affinity for the MR, whereas spironolactone also has a high affinity for the MR but is less selective. This accounts for the well-known anti-androgenic and progestonal side effects of spironolactone, which limit patient adherence to therapy. However, the number of patients in this study and the duration of their exposure to BAY 94-8862 are inadequate to provide any definitive information on the relative incidence of these side effects in patients receiving BAY 94-8862. Further pre-clinical and clinical studies will be required to gain greater insight into the reasons for the apparent differences in cardiac and renal efficacy of BAY 94-8862 5 and 10 mg q.d. compared with spironolactone 25 or 50 mg q.d. However, the results of the present study suggest that, in comparison with the steroidal MRA spironolactone 25 or 50 mg q.d., the non-steroidal MRA BAY 94-8862 5 and 10 mg q.d. may be at least as effective in reducing ventricular remodelling (reflected by a decrease in BNP and NT-proBNP levels), despite exerting less effect on SBP, serum potassium levels, and eGFR, as well as being associated with lower incidences of hyperkalaemia and renal adverse events in patients with HFrEF and moderate CKD. The results of the present study provide a strong impetus for further clinical evaluation of BAY 94-8862 in patients with HFrEF and concomitant CKD. BODY.SUPPLEMENTARY MATERIAL: Supplementary material is available at European Heart Journal online. BODY.FUNDING: This work was supported by Bayer Pharma AG. Editorial work (by C.C. of Oxford PharmaGenesis™ Ltd) was funded by Bayer Pharma AG. Conflict of interest: B.P. has received consulting fees from Amorcyte, Aurasence, Bayer, BG-Medicine, Cytopherx, Gambro, Lilly, Mesoblast, Novartis, Pfizer, scTherapeutics, Sticares InterACT, and Takeda; stock options from Aurasence, BG-Medicine, and scTherapeutics; and travel support from Bayer, Gambro, Mesoblast, and Pfizer. B.P. also has a patent pending (site-specific delivery of eplerenone to the heart). L.K. has received payment from Servier for speaking at a symposium. P.P. has received consulting fees/honoraria and support for travel from Bayer and Pfizer, payment for consultancy from Novartis, Johnson & Johnson, BMS, and Coridea, and payment for lectures from Novartis and BMS. P.P.'s institution has received grants from Vifor Pharma. M.G. has received consulting fees/honoraria and support for travel from the following [the asterisk indicates significant (>$10 000) support]: Abbott Laboratories, Astellas, AstraZeneca, Bayer Schering Pharma AG*, Cardiorentis Ltd, CorThera, Cytokinetics, CytoPherx, Inc., DebioPharm S.A.*, Errekappa Terapeutici, GlaxoSmithKline, Ikaria, Intersection Medical, Inc., Johnson & Johnson, Medtronic*, Merck, Novartis Pharma AG*, Ono Pharmaceuticals USA, Otsuka Pharmaceuticals*, Palatin Technologies, Pericor Therapeutics, Protein Design Laboratories, Sanofi-Aventis, Sigma Tau*, Solvay Pharmaceuticals*, Sticares InterACT*, Takeda Pharmaceuticals North America, Inc.*, and Trevena Therapeutics. G.F. has received consulting fees from Bayer and payment for lectures from Menarini. G.F.'s institution has received consulting fees from Bayer and Corthera, and grants from the European Union. H.K. has received consulting fees from Bayer. C.N. is an employee of Bayer Pharma AG. P.K. is an employee of Bayer HealthCare Pharmaceuticals and a patent holder (co-inventor of BAY 94-8862). S.-Y.K. is an employee of Bayer Vital GmbH. F.Z. has received consultant honoraria from Bayer, Biotronik, Boston Scientific, Gambro, Janssen, Novartis, Pfizer, Resmed, Servier, and Takeda, payment for lectures from Pfizer and AstraZeneca, and support for travel from Bayer. F.Z.'s institution has received grants from Roche Diagnostics. BODY.SUPPLEMENTARY MATERIAL: BODY.SUPPLEMENTARY DATA:

TITLE: Comparative analysis of corneal morphological changes after transversal and torsional phacoemulsification through 2.2 mm corneal incision ABSTRACT.PURPOSE: This paper compares and evaluates the corneal morphological changes occurring after cataract surgery through a 2.2 mm corneal incision. We use two platforms for comparison and evaluation, transversal and torsional phacoemulsification. ABSTRACT.PATIENTS AND METHODS: This study includes 139 consecutive cataractous eyes (nuclear color 2–4, according to the Lens Opacities Classification System III [LOCSIII]) of 82 patients undergoing cataract surgery through a 2.2 mm corneal incision. Two different phacoemulsification platforms were used and assigned randomly: we used the WhiteStar Signature® system with the Ellips™ FX transversal continuous ultrasound (US) mode for group I (mean age: 65.33 ± 6.97 years), and we used the Infiniti® system with the OZil® Intelligent Phaco (IP) torsional US mode for group II (mean age: 64.02 ± 7.55 years). The corneal endothelium and pachymetry were evaluated preoperatively and at 1 month postoperatively. Incision size changes were also evaluated. ABSTRACT.RESULTS: All surgeries were uneventful. Before intraocular lens implantation, the mean incision size was 2.24 ± 0.06 mm in both groups (P = 0.75). In terms of corneal endothelial cell density, neither preoperative (I vs II: 2304.1 ± 122.5 cell/mm2 vs 2315.6 ± 83.1 cell/mm2, P = 0.80) nor postoperative (I vs II: 2264.1 ± 124.3 cell/mm2 vs 2270.3 ± 89.9 cell/mm2, P = 0.98) differences between the groups were statistically significant. The mean endothelial cell density loss was 1.7% ± 1.6% and 2.0% ± 1.4% in groups I and II, respectively. Furthermore, no significant differences between groups I and II were found preoperatively (P = 0.40) and postoperatively (P = 0.68) in central pachymetry. With surgery, the mean increase in central pachymetry was 28.1 ± 23.6 μm and 24.0 ± 24.0 μm in groups I and II, respectively (P = 0.1). ABSTRACT.CONCLUSION: Ellips™ FX transversal and OZil® IP torsional phacoemulsification modes are safe for performing cataract surgery, inducing minimal corneal thickness and endothelial changes. BODY.INTRODUCTION: The main objectives of modern phacoemulsification (phaco) technology are the reduction of ultrasound (US) power and improved efficiency.1 Interrupted phaco modes, improved pump systems, chopping techniques, and vacuum-assisted phaco have reduced the amount of energy needed to remove a cataract and, therefore, the potential associated risks.1,2 However, US power required for traditional or longitudinal phaco continues to be a risk factor for endothelial cell loss and tissue damage.3 Through the use of improved phaco technologies, recent studies on cataract surgery have demonstrated a reduction in corneal endothelial cell loss after phaco.4–11 In an attempt to increase US efficiency during phaco cataract surgery, torsional phaco technology was introduced in 2005. In traditional phaco, the longitudinal movement of the phaco tip pushes nuclear fragments away with each forward stroke, although interrupted US has the supposed advantage of allowing retraction of the pushed nuclear fragments back toward the phaco tip. Furthermore, due to the jackhammer effect, the emulsifying power of longitudinal phaco is only effective during the forward movement of the tip. Conversely, the lateral tip movement of torsional phaco shears the lens material while moving in both directions without a repellent force. It also uses a lower frequency (32 MHz) than traditional phaco (40 MHz), allowing for 20% energy conservation. As such, torsional technology represents a significant improvement in the emulsifying efficiency of US during phaco.12,13 Torsional US can also be mixed, intermittently, with a quantum of longitudinal phaco. This is commercially known as OZil® Intelligent Phaco (IP) (Alcon Laboratories, Fort Worth, TX, USA), whereby the quantum is liberated at a threshold vacuum in a duration ranging from 10 to 20 milliseconds. IP aims to maximize torsional US efficacy by keeping the nuclear fragments in the ideal shearing plane.14 In addition to torsional technology, transversal US technology is a new form of phaco. The transversal US model, Ellips™ (Abbott Medical Optics, Santa Ana, CA, USA), was developed to reduce the repulsion that commonly occurs with longitudinal phaco. Ellips™ simultaneously generates lateral and longitudinal motions at a working frequency of 28 kHz; thus, the phaco tip moves in an elliptical motion, promoting an optimization of efficiency by emulsifying lens matter in more than one direction. Moreover, the accompanying longitudinal component adds the jackhammering effect to transversal US efficiency, constantly maintaining the nuclear fragments at the ideal shearing plane in front of the phaco tip.15 The transversal technology can also be used with a straight or curved tip, and the Ellips™ model can work in a continuous or hyperpulse mode with different duty cycles.16 Recently, Ellips™ FX (Abbott Medical Optics), a further hardware and software modification on the previous version, has been developed for a working frequency of 38 kHz (a 45% increase on the previous version) and a stroke length three times larger (longitudinal and lateral stroke length ratio is 1:1) than the previous version. Existing studies have experimentally compared torsional and transversal phaco systems in terms of vacuum, surge, and thermal effect.15–17 Furthermore, Christakis and Braga-Mele5 recently compared the clinical outcomes obtained after the same surgeon performed cataract surgery with phaco, using three different systems: WhiteStar Signature® Ellips™ FX (transversal), Infiniti® OZil® IP (torsional), and Stellaris® (Bausch and Lomb, Rochester, NY, USA) (longitudinal). These authors evaluated the phaco needle time, chatter, followability, the anterior chamber stability, the level of corneal edema, and the 1-day postoperative visual acuity. However, to date, no comparisons between torsional and transversal phaco have been reported in terms of corneal endothelial changes. Hence, the aim of this study was to compare and evaluate the corneal morphological changes, including endothelial modifications, as an indicator of safety for cataract surgery through a 2.2 mm corneal incision. In this study, we compared and evaluated the transversal and torsional phaco platforms. BODY.MATERIALS AND METHODS: BODY.PATIENTS: This study included 139 consecutive cataractous eyes of 82 patients undergoing cataract surgery through a 2.2 mm corneal incision. The inclusion criteria were patients of 45 years or older and the presence of a senile cataract graded as nuclear color (NC) 2–4, according to the Lens Opacities Classification System III (LOCSIII).18 The exclusion criteria were patients with glaucoma, corneal opacities, cornea guttata, abnormal iris, macular degeneration or retinopathy, previous posterior segment surgery, a corneal endothelial cell density (ECD) of less than 1900 cell/mm2, neuro-ophthalmic disease, or a history of ocular inflammation. All volunteers were adequately informed and signed a consent form. The study adhered to the tenets of the Declaration of Helsinki, and the study was approved by the local ethical committee. One of the two different phaco platforms was assigned to be used with each eye included in this study. Random assignment was performed by coin toss. Accordingly, two groups were differentiated: group I included eyes undergoing phaco via the WhiteStar Signature® platform using the Ellips™ FX transversal continuous ultrasound mode with the 0.9 mm curved microtip, and group II included eyes undergoing phaco via the Infiniti® platform using the OZil® IP torsional phaco with the 0.9 mm/45° Kelman tip. BODY.EXAMINATION PROTOCOL: Preoperatively, all patients had a full ophthalmological examination, including evaluation of the refractive status, uncorrected and corrected distance visual acuity testing (Snellen optotypes and decimal notation), slit lamp examination, and Goldmann applanation tonometry and fundoscopy. Besides these basic clinical tests, corneal endothelial cell analysis and central corneal thickness (CCT) measurements were performed (EM-3000; Tomey, Erlangen, Germany). Corneal endothelial analysis included the measurement and recording of the ECD, the coefficient of variation (CV), and the hexagonality. Patients were also evaluated during the follow-up at 1 day and 1 month after surgery. At 1 day after surgery, intraocular pressure and the integrity of the anterior segment were evaluated. The corneal endothelium and pachymetry were evaluated one month after surgery. BODY.SURGERY: All surgeries were performed by the same surgeon (AA), using a standard mini-incision of the sutureless micro-coaxial phaco technique. In all cases, a corneal incision of 2.2 mm was planned, followed by a paracentesis of 1.2 mm. After injection of the cohesive ophthalmic viscoelastic device (Healon® GV; Abbott Medical Optics) in the anterior chamber, a curvilinear rhexis of 5–5.5 mm was performed. After hydrodissection, nucleus disassembly was started using the quick-chop technique, impaling the curved phaco-tip in the central part of the nucleus (1–1.5 mm). This was followed by vertical chopping of the nucleus with the Tobias Neuhann chopper (Geuder GmbH, Heidelberg, Germany). The incision size change was tested using the Tsuneoka micro-coaxial incision gauge (Asico, Westmont, IL, USA) after completion of the cortical cleaning and prior to intraocular lens (IOL) implantation. One-piece acrylic IOLs (Tecnis® 1 ZCB00; Abbott Medical Optics) were implanted in all cases. The wound seal was confirmed at the end of the surgery by a negative Seidel test. Settings for the Infiniti® platform (torsional) were as follows: the power was set at 100% linear torsional continuous US amplitude; the IP power delivery option was activated to produce a 10 millisecond longitudinal phaco pulse when 95% vacuum was reached; the flow rate was 28 mL/min, with dynamic rise of +1; the vacuum was set at 350 mmHg; and the bottle height was 108 cm. Settings for the WhiteStar Signature® platform (transversal) were as follows: the power of the Ellips™ FX was set at 100% linear continuous transversal US; the aspiration rate was set at 28 and 26 mL/min before and after occlusion, respectively; vacuum parameters were set at a maximum of 320 mmHg; the chamber stabilization environment (CASE) vacuum was set at 280 mmHg; CASE time was set at 500 milliseconds for NC 2 and at 800–1000 milliseconds for NC 3 and 4; and the bottle height was 67 cm. The same postoperative treatment was administered to all patients. This included: using moxifloxacin HCl 0.5% (Vigamox®; Alcon Laboratories) eye drops three times per day for two weeks; rimexolone 1% (Vexol®; Alcon Laboratories) eye drops three times per day for 1 week, then twice per day for the following 2 weeks; tobramycin-dexamethasone (TobraDex®; Alcon Laboratories) eye ointment once at bedtime for 2 weeks. BODY.STATISTICAL ANALYSIS: SPSS statistics software package (v 19.0) for Windows (IBM Corporation, Armonk, NY, USA) was used for statistical analysis. Normality of all data samples was first evaluated by means of the Kolmogorov-Smirnov test. When parametric analysis was possible, the Student t-test for paired data was used for comparisons between preoperative and postoperative examinations, and the Student t-test for unpaired data was used for the comparison between groups. When parametric analysis was not possible, the Wilcoxon rank-sum test was applied to assess the significance of differences between preoperative and postoperative data, and the Mann–Whitney test was applied for the comparison between groups, in all cases using the same level of significance (P < 0.05). Before the start of the study and according to our previous experience, we estimated that a sample size of 64 eyes per group would be required to detect a mean difference of 10 cell/mm2 between both groups, with a standard deviation of 20. Power of the test was set to 0.8 with a significance level of 0.05. We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research. BODY.RESULTS: The total number of eyes included in this study was 150 of 88 patients (group I, 85 eyes; group II, 65 eyes). Six patients (11 eyes: six in group I and five in group II) did not complete the postoperative follow-up examinations and were excluded from the study. Therefore, the data used for the statistical analysis included 139 eyes of 82 patients: 79 eyes in group I and 60 eyes in group II. The mean age ± SD was 65.3 ± 7.0 and 64.0 ± 7.6 years in groups I and II, respectively. No statistically significant differences in age were detected between groups (Mann–Whitney test: P = 0.29). According to the LOCSIII, in group I, 29 eyes presented an NC 2 cataract, 41 eyes presented an NC 3 cataract, and nine eyes presented an NC 4 cataract. In group II, a similar distribution of cataract types, according to the LOCSIII, was found: 23 eyes with an NC 2 cataract, 30 eyes with an NC 3 cataract, and seven eyes with an NC 4 cataract. BODY.INTRAOPERATIVE AND EARLY POSTOPERATIVE CHANGES: All surgeries were uneventful. Intraoperatively, we observed improved followability with less chattering of free nuclear fragments in both systems. No incisional corneal burns were observed. Before IOL implantation, the mean size of the main incision was 2.24 ± 0.06 mm in both groups, with no statistically significant differences between them (Mann–Whitney test: P = 0.75). On the first postoperative day, the slit lamp examination showed mild stromal corneal folds in both groups with complete resolution at an average of 7 days, ranging from 4 to 10 days. BODY.CORNEAL ENDOTHELIAL CHANGES: Table 1 summarizes the corneal endothelial changes for groups I and II, occurring 1 month after surgery. As shown, we found significant changes in the three parameters analyzed (ECD, CV, and hexagonality) in both groups (Wilcoxon test: P ≤ 0.02) 1 month after surgery, except for the hexagonality in group II. The mean postoperative decrease in ECD was 40.0 ± 37.5 cell/mm2 and 45.3 ± 31.7 cell/mm2 in groups I and II, respectively. No statistically significant differences in ECD were found between the groups preoperatively (Mann–Whitney test: P = 0.80) and postoperatively (Mann–Whitney test: P = 0.98). In relation to preoperative values (Figure 1), group I showed a mean endothelial cell loss of 1.7% ± 1.6%, whereas group II showed a loss of 2.0% ± 1.4% (Mann–Whitney test: P = 0.29). Regarding CV, no statistically significant differences were found between groups preoperatively (Mann–Whitney test: P = 0.82) and postoperatively (Mann–Whitney test: P = 0.96). Likewise, hexagonality did not differ significantly between groups before (Mann–Whitney test: P = 0.35) and after (Mann–Whitney test: P = 0.97) surgery. BODY.CORNEAL THICKNESS CHANGES: The preoperative CCT was 534.5 ± 29.3 μm and 536.9 ± 26.2 μm in groups I and II, respectively (Mann–Whitney test: P = 0.40). The postoperative CCT was 562.6 ± 33.7 μm (increased by 28.1 ± 23.6 μm) and 560.9 ± 29.9 μm (increased by 24.0 ± 24 μm) in groups I and II, respectively (Mann–Whitney test: P = 0.68). Thus, the postoperative increase in CCT was similar in both groups (Mann–Whitney test: P = 0.10). The central pachymetry change with surgery was statistically significant in both groups (Wilcoxon test: P < 0.01). BODY.DISCUSSION: Technological advances in phaco have made cataract removal safer and more efficient by reducing phaco energy and duration. Torsional and transversal phaco modes are the results of these technological advances. Several studies have confirmed and reported the superiority of torsional phaco over longitudinal phaco in terms of needle time, chatter, followability, corneal edema, and corneal endothelium.1,5,7–12,19,20 However, to date, only one study has compared longitudinal, torsional, and transversal phaco.5 In that study, the clinical outcomes with three different phaco platforms were evaluated: WhiteStar Signature® Ellips™ FX (transversal), Infiniti® OZil® IP (torsional), and Stellaris® (longitudinal). Those authors found that the torsional machine had less phaco needle time, less chatter, better followability, and yielded less central corneal edema than the transversal or longitudinal machines. However, they did not compare the effect of using the platforms in terms of the corneal endothelium. The aim of the current study was to compare and evaluate the corneal morphological changes, including endothelial modifications, as an indicator of safety for cataract surgery through a 2.2 mm corneal incision. This study compared and evaluated the transversal and torsional phaco platforms. Because of different algorithms adopted by the two platforms, the current comparative analysis did not measure the effective phaco time, cumulative dissipated energy, phaco time, or balanced salt solution consumption. Georgescu et al17 demonstrated that a comparable unoccluded flow vacuum of 60 mL/min would correspond with approximately 220 mmHg and 120 mmHg for the Infiniti® and WhiteStar Signature® platforms, respectively. Our comparative analysis preferred the use of independent and more clinically significant parameters, such as wound stretch, corneal edema, endothelial cell loss, pleomorphism, and central pachymetry. The function of followability is to bring free nuclear fragments close to the phaco tip for continuous and smoothly progressive emulsification and aspiration. Fluidic models have shown that the longitudinal movement repels lens material from the tip, whereas the rotational motion of torsional US reduces the repulsion of the lens fragments, thus improving followability of nuclear material into the phaco tip.20 Longitudinal phaco tip movement repels lens matter with every stroke. Conversely, lateral movement reduces repulsion of the lens fragments, thus improving followability during phaco. In transversal phaco, nuclear fragments are exposed to shearing stress and the jackhammering effect caused by the transversal and longitudinal motion, respectively, but without the repulsion seen in longitudinal phaco. Furthermore, with the Ellips™ FX, the longitudinal US could not be separated from the transversal component. With the OZil® IP, the longitudinal component might be switched off or set to an automatic activation when a preset threshold vacuum was reached. For this reason, a better followability was expected in the torsional group than the transversal group. However, both systems showed improved and comparable followability. Georgescu et al17 demonstrated that the postocclusion surge (POS) was significantly worse for the Infiniti® platform than for the WhiteStar Signature® platform. However, this could only be clinically important in extreme experimental conditions, such as using 60 mL/min of flow, a 550 mmHg vacuum, and a bottle height of 60 cm. POS is often at its worst when dealing with a broken capsule and corneal endothelial damage.17 According to the settings used in our study, we expected the POS to be negligible. Indeed, no POS was noticed during surgery in either of our groups. Regarding corneal incision size before IOL implantation, no statistically significant differences were found between the groups. The minimal wound stretch found in group I may be attributable to the compliant nature of the sleeve and the microvoid between the phaco tip and incision. Hence, the transversal movement of the phaco tip did not induce wound stress or burn. It should be noted that the new, ultrasleeve 21G tip was used with the Ellips™ FX in our study. This use was off-label, due to its commercial unavailability at the beginning of the study. As for group II, our results were consistent with previous scientific evidence. Han and Miller19 reported that torsional phaco created less heat than longitudinal phaco. In concordance with this, Jun et al10 stated that the smaller incision phaco of torsional US may decrease the risk for wound burn in eyes with denser cataracts. Furthermore, Li et al21 have recently demonstrated that, in the context of torsional phaco, a micro-coaxial, 2.2 mm incision may cause slightly less damage than the 2.8 mm standard incision. Therefore, considering the twist movement of the phaco tip in torsional US and considering that the mini-flare tip and ultra-sleeve used in group II were designed to fit 2.2 mm and 2.4 mm incisions, the minimal wound stretch found in this group seems to be a coherent finding. Finally, the enhanced followability with the torsional phaco mode reduced the additional hand-piece movements to reacquire the nuclear fragments, minimizing the stretch in the incision.20 The mean endothelial cell loss in the current study was 1.7% and 2.0% in groups I and II, respectively. These values were better than those obtained in other studies using the torsional technology.8,9 This may be attributable to the use of an improved technology, the different surgical technique, the ophthalmic viscoelastic device used, and the different classification system adopted (LOCS III vs LOCS II). One study found an endothelial cell loss of 7.2% ± 4.6% using the torsional phaco technology through a 2.75 mm clear corneal incision,9 yet, in our study, no significant differences between groups were observed in endothelial cell loss, CV, and hexagonality, confirming the presence of similar postoperative levels of polymegathism and pleomorphism in both groups. Therefore, the same minimal level of endothelial damage seems to be induced with both phaco technologies. It has been postulated that a longer phaco time22,23 and higher US power22 are associated with endothelial cell loss, although some studies failed to prove these correlations.9,24,25 Reuschel et al9 found a statistically significant difference in US energy and time between the torsional and longitudinal phaco, but there was no significant correlation between these parameters and endothelial cell loss. In addition, the cell loss was not significantly different between the two groups.9 In the current study, a more significant endothelial cell loss would have been expected in the Ellips™ FX group, since the longitudinal movement component of the tip was constantly present, yet this finding was not obtained. This suggests that we have approached the critical value for safe phaco modality with the currently available torsional and transversal phaco technology, beyond which no significant improvements in endothelial cell protection can be observed. Future studies are required to confirm this hypothesis. We also found a minimal, but statistically significant increase of 28.1 and 24.0 μm in CCT in groups I and II, respectively. The difference between the groups in this minimal level of corneal edema did not reach statistical significance. However, Christakis and Braga-Mele5 found that torsional technology yielded less central corneal edema than the transversal and longitudinal technologies, corresponding to a smaller increase in the mean corneal thickness (torsional, 5%; transversal, 10%; longitudinal, 12%). One of the main factors accounting for this discrepancy with our findings may be the difference in the nuclear cataract grade, which was NC 2.0 ± 0.8 in Christakis and Braga-Mele’s study.5 BODY.CONCLUSION: Ellips™ FX transversal and OZil® IP torsional phaco platforms are safe for performing cataract surgery, inducing minimal corneal thickness and endothelial changes. In terms of followability, the effect on the endothelium and the corneal incision, and the induced corneal thickness increase, both technologies seem to be equivalent for extraction of an NC 2–4 (LOCS III) cataract through a 2.2 mm incision. Future optical coherence tomography studies are needed to compare the effects of torsional and transversal phaco types on the corneal incision. In addition, the potential benefits and applicability of these technologies in eyes with a compromised endothelium should be further evaluated.

TITLE: A Mobile Phone App to Stimulate Daily Physical Activity in Patients with Chronic Obstructive Pulmonary Disease: Development, Feasibility, and Pilot Studies ABSTRACT.BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) demonstrate reduced levels of daily physical activity (DPA) compared to healthy controls. This results in a higher risk of hospital admission and shorter survival. Performing regular DPA reduces these risks. ABSTRACT.OBJECTIVE: To develop an eHealth intervention that will support patients with COPD to improve or maintain their DPA after pulmonary rehabilitation. ABSTRACT.METHODS: The design process consisted of literature research and the iterative developing and piloting phases of the Medical Research Council (MRC) model for complex clinical interventions and the involvement of end users. Participants were healthy adults and persons with COPD. ABSTRACT.RESULTS: The mobile phone interface met all the set requirements. Participants found that the app was stimulating and that reaching their DPA goals was rewarding. The mean (SD) scores on a 7-point scale for usability, ease of use, ease of learning, and contentment were 3.8 (1.8), 5.1 (1.1), 6.0 (1.6), and 4.8 (1.3), respectively. The mean (SD) correlation between the mobile phone and a validated accelerometer was 0.88 (0.12) in the final test. The idea of providing their health care professional with their DPA data caused no privacy issues in the participants. Battery life lasted for an entire day with the final version, and readability and comprehensibility of text and colors were favorable. ABSTRACT.CONCLUSIONS: By employing a user-centered design approach, a mobile phone was found to be an adequate and feasible interface for an eHealth intervention. The mobile phone and app are easy to learn and use by patients with COPD. In the final test, the accuracy of the DPA measurement was good. The final version of the eHealth intervention is presently being tested by our group for efficacy in a randomized controlled trial in COPD patients. BODY.INTRODUCTION: Regular physical activity has significant health benefits and contributes to the prevention of non-communicable diseases [1]. Inactivity is estimated to cause 9% of premature mortality worldwide [2]. In older adults, there is strong evidence that regular exercise and participation in physical activity lowers mortality and morbidity [3], and has a significant impact on several psychological and cognitive parameters [4]. Moreover, physical activity has been observed as a behavioral determinant for healthy aging [5]. Physical activity is also a relevant behavioral determinant for patients with chronic diseases, such as chronic obstructive pulmonary disease (COPD), to maintain physical condition [6], and to improve health-related quality of life [7]. COPD is a disabling airway disease with variable extra-pulmonary effects that may contribute to disease severity in individual patients. It mostly affects older adults with a history of tobacco smoke exposure [8]. Patients with COPD demonstrate reduced levels of spontaneous daily physical activity (DPA) compared to healthy controls [9]. This contributes to a higher risk of hospital admission and shorter survival [10]. Pulmonary rehabilitation (PR) generally includes exercise training, education, psychosocial and behavioral interventions, nutritional therapy, and outcome assessment [11,12], and it can help to improve physical capacity. Unfortunately, this effect does not always translate into improved DPA, and when it does, it tends to fade out over time [13-15]. Taking into account the benefits of regular DPA [16], it is important for patients with COPD to improve, or at least to maintain their DPA levels after a rehabilitation program has ended. Technology-based assistance in health care (eHealth) can help support patients with COPD by improving self-management of the disease. Self-management interventions in patients with COPD have been shown to improve health-related quality of life, to lower the probability of a respiratory-related hospitalization, and to reduce dyspnea [17,18]. It has been postulated that an eHealth intervention might also be beneficial in the self-management of DPA in patients with COPD. An important element for successful implementation of an eHealth intervention is to engage users in the design process because design flaws can affect ease of use, usability, and reliability of the system, which may reduce a user’s willingness to use the intervention [19]. The objective of this study is to develop an eHealth intervention to support patients with COPD in improving or maintaining DPA after PR. We investigate what type of interface is adequate and feasible toward obtaining this objective and scored the resultant eHealth intervention in terms of usability and privacy. BODY.METHODS: BODY.RECRUITMENT: The design process was in alignment with the first two phases (developing and piloting) of the Medical Research Council (MRC) model for complex clinical interventions. The key elements of the development and evaluation process of the MRC model were taken into account throughout the design process [20] (Figure 1). This paper primarily focuses on phases A2 through C. Users were defined as persons suffering from COPD, who were aged 40 years or older, living independently, and had completed a rehabilitation program. BODY.EHEALTH INTERVENTION AND INTERFACE: Based on the literature and our own practice-based experience in the treatment of patients with COPD, the eHealth intervention that we sought to develop had to meet the following requirements: (1) non-obtrusive and easily transportable, (2) objective measurement of DPA, (3) direct feedback and personal DPA, and (4) monitoring and feedback available from a health care professional (HCP). At the time of this study (2010), several eHealth interventions for physical activity engagement in patients with COPD had been described. They were available in various forms, such as wearable sensors [21], television [22], computers [23], a manual input device [24], and mobile phones [25,26]. As an interface, a smartphone with app capabilities met all set requirements. Although the penetration rate of smartphone use among aging adults was estimated to be low at the time, they were expected to become the majority over the next few years [27,28]. Moreover, mobile touch screens are generally easy for the elderly to use [27], and mobile phones are already equipped with an accelerometer that is both accurate and reliable in measuring and quantifying physical activity in a laboratory setting [29]. Although various apps for mobile phones are available that stimulate engagement in physical activity, none of the apps met all the requirements that are needed to fully address our research goal. Therefore, we decided to develop a new app and an associated website for HCPs. This paper focuses on the development of an app for an eHealth intervention. We encountered 2 types of apps: those developed for mobile phones running on the interactive operating system (iOS) and those developed for mobiles phones running on the Android operating system. Following a comparison of these 2 operating systems, we opted for the HTC HD2 device (HTC). HTC was chosen as the preferred device based on its higher battery capacity, an absence of restrictions in distributing the app, and its affordable price. Figure 1Design process. BODY.PILOT STUDIES: After phases A and B (Figure 1), the product was tested in 3 pilot studies (C1, C2, and C3), and improved through an iterative process. The pilot studies were designed to test the usability of the interface and app, in addition to privacy concerns of the users. The associated website for HCPs was not yet employed in the pilot studies. We also sought to obtain an indication of the accuracy of DPA measurements by the app. Specific sample size recommendations for this type of development and feasibility pilot studies are scarce, as most recommendations are for pilot studies that focus on the feasibility of corresponding RCT studies [30]. The pilot studies were designed to minimize strain on patients with COPD. Therefore, we began the first pilot study with healthy volunteers who had previous mobile phone experience. A subsequent version was then tested in a subset of patients with COPD. Finally, a larger group of patients with COPD were invited to test the final version. We aimed to include 10, 3, and 10 participants in pilot study groups C1, C2, and C3, respectively [31]. The participants in pilot group C1 were recruited from a school, in pilot group C2 they were recruited from a hospital, and in pilot group C3 they were recruited from a rehabilitation center. For inclusion criteria, see Table 1. Table 1Characteristics of the three pilot studies in study phase C. Pilot study Inclusion criteria Duration of study Version application C1 Healthy persons, experience with mobile phones 1 week Figure 2 C2 Persons suffering from COPD, aged ≥40 years, living independently, and having completed a rehabilitation program 4 days Figure 3 C3 Persons suffering from COPD, aged ≥40 years, living independently, and having completed a rehabilitation program (same as pilot C2) 3 weeks Figure 4 The participants received instructions on the functionalities of the mobile phone and app, and information on the course of the study over a training session lasting 1.5 hours. Thereafter, each participant received a HTC Desire A8181 mobile phone with the app installed, and they were given the opportunity to practice, ask questions, and provide feedback. They were instructed to wear the mobile phones in pouches (with various choices for personalization) on their belts. This location was chosen because the best measurements are achieved by positioning the accelerometer as close to the center of gravity as possible [32,33]. They were also instructed to wear accelerometers (BHC0100 Sensewear PRO armband, Body Media, Pittsburgh, US) that had been previously validated in patients with COPD [34-37], on their right upper arms. The armband and mobile phone were worn during waking hours. The participants were instructed to perform their daily activities as usual. After each study, a group consultation session was held. The sessions started by asking the participants their general impression of the app followed by writing down 3 positive and 3 negative aspects. The most occurring aspects were written on a flip-over and further discussed. The following topics were each discussed for 5 minutes: wearing the mobile phone, using the app, comprehensibility, navigation, future use, and improvements to the app. Sessions were recorded and minutes were made. Afterwards, the sessions were separately summarized by 3 researchers and the main points were taken into consideration for adjustment of the app. Furthermore, the participants were asked to respond to 3 questionnaires (1) the Usefulness, Satisfaction, and Ease of use (USE) questionnaire on usability [38]; (2) the Florida State University (FSU) mobile device feedback preferences scale; and (3) the FSU physiological monitoring privacy scale (inspired by Beach et al [39] and Kwazney et al [40]). Results of the USE questionnaire were compared within and between pilot studies with independent and dependent t tests. All of the participants were required to provide signed informed consent prior to the study. Pilot studies were waived from ethics committee approval by the UMC Utrecht Medical Ethical Research Board (number research protocol 10/259). Correlations between the accelerometers on the armbands and the mobile phones were computed by calculating Pearson's correlation coefficient (r) in SPSS version 21. The distinctive characteristics of the pilot studies can be found in Table 1. An additional pilot study, C4, was performed to provide an extra check on DPA measurement accuracy. This was performed with 10 participants who wore the armband and mobile phone for 1 week. Participants met the same inclusion criteria as in pilot studies C2 and C3. These participants did not take part in a consultation round and did not fill out questionnaires since the development of the app was deemed ready at this point. In pilot study C1, participants were asked to record their daily activities in diaries, including corresponding times of day and durations. In pilot study C3, 3 randomly selected participants wore accelerometers during the first week. Figure 2In version 1, the y-axis provides a measure for activity, while the x-axis provides a measure for intensity. The DPA goal is met when the blue ball (representation of current activity status) is kept in the green circle at all times. The widget shows a current status towards reaching a DPA goal (pilot study C1). BODY.RESULTS: BODY.SETTING DESIGN REQUIREMENTS: A list of design requirements for the eHealth intervention was prepared with respect to the general requirements. Some aspects of the existing apps found during the desk research were also added as requirements. Furthermore, since COPD is inversely related to socioeconomic status and mostly affects older adults [41], special attention was paid to readability and comprehensibility. Focus was put on the mobile phone app for the users (Textbox 1). Figure 3In version 2, the left axis shows amount of steps, while the right axis gives a measure of intensity. The DPA goal is reached when the open circles (representation of current activity status) are kept in the rising green circles at all times (pilot study C2). Figure 4In version 3, the bar on the left side combines amount and intensity of steps. The DPA goal is met when the vertical stripe (representation of current activity status) is kept in the rising rectangle at all times until the green area is reached. Absolute number of steps and current advice on DPA progress are also shown (pilot study C3). The requirements for the monitoring website for the HCP can be found in Textbox 2. Feedback from HCP on these latter requirements was obtained by consulting with 10 independent respiratory nurses (in a consultation round), and 2 physiotherapists (by phone) who work with COPD patients. Additions to the requirements with regard to privacy and communication were made in response to their feedback. BODY.PRODUCT DEVELOPMENT: The app and website were created by a small business enterprise that specializes in developing health care apps. Interactive team work sessions were held during this process. The various designed versions of the app that were tested during the pilot studies are illustrated in Figures 2-4. Communication and multimedia design students from Utrecht University of Applied Sciences were employed to assist in improving the design of the app and the widget after pilot study C2. BODY.PILOT STUDIES: A total of 10 participants took part in pilot study C1, 3 in C2, and 7 in C3, of which 1 (10%), 3 (100%), and 4 (57%) were male, respectively. The mean (SD) age of the participants was 21.5 (2.84), 65 (10), and 60.4 (9.4) years in C1, C2, and C3, respectively. The participants were limited in their DPA due to having COPD and were enrolled in a PR program at the time of the study. The results from the consultation rounds are shown in Multimedia Appendix 1. In pilot study C2, 1 participant (33%, 1/3) was not interested in the intervention; therefore, the results from the consultation round of this group primarily focused on the remaining 2 participants. This participant did fill out the questionnaires. Eleven subjects were recruited to participate in pilot study C3. After the training session, 4 (36%, 4/11) declined to participate due to the degree of expected effort. On day 3 and 7 of pilot study C3, corrected apps were installed due to discovered errors in the algorithm that caused the app to measure too few or no steps. The results from the USE questionnaire are shown in Table 2. The usability scores for pilot study C1 were significantly lower than ease of use, learning, and contentment scores (P<.05 for all). For pilot study C3, usability scores were significantly lower than for ease of learning (P<.05). Ease of learning was significantly lower in patients with COPD compared with healthy participants in pilot study C1 (P<.004 for C2; P=.017 for C3). The feedback preferences questionnaire in general did not provide added insights to the consultation rounds. BODY.USERS' REQUIREMENTS OF THE MOBILE PHONE-BASED APP.: Software Reasonably accurate measurement of DPA DPA data are recorded on the mobile phone and available to the user in real time Filters out movement produced by riding a car, bus, or train Data are available for at least 12 weeks after generation (preferably even longer, such as 6 months to 1 year) Data are sent automatically to a secured website for HCP (4-6 times a day) Data are only available to users and HCP Data are saved when phone runs out of battery Data acquisition continues when the mobile phone is in standby mode or is being used for other purposes Goal achievement elicits a motivating or complimentary message Personal results can be published on social media if desired The app uses little energy An app-killer is added that can stop all apps except for the intervention The app can be used on mobile phones of different brands The app can be adjusted in the future Interface DPA is presented in duration, frequency, and intensity Data are available in graphs and numbers Visual display of progress and goal achievement on screensaver Progress is visible in numbers (and percentage until goal is reached) Progress is visible based on day, week, and month Letters and figures are easily readable (large font and high contrast) Navigation is easy and comprehendible; only a few steps are required to reach a desired location All text is formulated for persons with low literacy The app can be personalized BODY.WEBSITE REQUIREMENTS FOR THE HCP.: Software Data are available for at least 12 weeks after generation (preferably even longer, such as 6 months to 1 year) Data are only available to the HCP SMS text messages (short message service, SMS) or phone calls can be made from the website DPA goals can be adjusted from the website Goals can be set based on steps, duration, frequency, and intensity Goals are individually adjustable Interface DPA is presented in duration, frequency, and intensity Data are available in graphs and numbers Overview of the activity status of multiple patients Progress of each patient is easily visible in an overview (eg, traffic light colors) Individual page for each patient with detailed DPA information Table 2The mean (SD) scores of the USE questionnaire in the various pilot studies. Pilot study Scores, mean (SD) Usability Ease of use Ease of learning Contentment C1 3.8 (2.0) 5.4 (1.7) 6.6 (0.6) 4.8 (1.7) C2 3.9 (2.9) 5.1 (2.1) 4.1 (2.9) 5.7 (1.7) C3 3.7 (2.0) 4.8 (2.2) 5.9 (1.5) 4.4 (1.8) The correlations between the mobile phones and the armband accelerometers for steps per day are shown in Table 3. The armband of participant 1 (C1) malfunctioned. Participant 2 (C3) only wore the armband for 2 days and was excluded from analysis. The additional pilot study C4 was performed solely to provide an extra check on DPA measurement accuracy. The numbers of valid days for analysis were 8, 4, 8, and 8 for all participants in pilot study C1, C2, C3, and C4, respectively. Table 3Correlation between mobile phones and armband accelerometers. Pilot study, r a Participant 1 2 3 4 5 6 7 8 9 10 C1 N/A .94b .96b .76b .71b .76b .64 .61 97b .13 C2 .87b .54 .72b N/A N/A N/A N/A N/A N/A N/A C3 .45 N/A .67 N/A N/A N/A N/A N/A N/A N/A C4 .99b .98b .96b .90b .99b .74 .69 .69b .84b .99b aPearson correlation coefficient. bSignificant at P<.05 BODY.DISCUSSION: BODY.PRINCIPAL FINDINGS: Engaging patients with COPD in active control over their DPA can work as a preventive measure to prevent functional decline [42]. Therefore, our objective was to develop an eHealth intervention that will help patients with COPD to improve or maintain their DPA after a period of pulmonary rehabilitation. The final product consists of two components (1) a mobile phone app (the focus of this study); and (2) a website for HCPs. The app measures DPA as steps per day, measured by the accelerometer of the mobile phone, and shows this information to the patient via the display of a mobile phone. A physiotherapist can monitor the patient via a secure website where DPA measurements are accessible from all patients. DPA goals can be adjusted and text messages sent to inform and to motivate patients. Furthermore, the website of the intervention can help an HCP work in a more efficient way by monitoring all of their patients at once and enabling them to intervene early on in patients who have trouble maintaining DPA. BODY.USE: The mobile phone-based app was found to be easy to learn and use by the participants as well as the patients with COPD. Usability scores were lower than ease of use, learning, and contentment scores. This was significant in pilot study C1 and C3 (for ease of learning). This could be explained, in part, by the fact the app was still in the development phase and still contained some errors, as demonstrated in pilot study C3. Ease of use scores were lower for the patients with COPD, though not significantly. This could be because touch screen pointing performance reduces with age. It is influenced by size, spacing, and location of the target, as well as by size of the device and practice [27,28]. Older people prefer functions that support their declining functional capabilities, and enjoyability is an important determinant of adherence [27]. During the development of the app, attention was paid to all of these aspects. Ease of learning was significantly lower in patients with COPD compared with healthy participants. Proper instruction will greatly influence success in mobile phone usage [43]. Older adults take longer in learning to use mobile phones, and they commit more errors when entering information into mobile phone-based software app [44]. Efforts to overcome these behavioral and attitudinal barriers must include well-designed training that is targeted to older adults to teach mobile phone usage skills as well as creating software with an improved interface and operation [44]. BODY.DESIGN: The graphic design of the app was adjusted several times to improve use and to provide a better understanding of the DPA data, as well as to accommodate those with low technology literacy. A combination of qualitative and quantitative feedback proved the best fit. BODY.PRIVACY: The key aspect, with respect to privacy, is to give the user control over their data distribution. An important element of the intervention is that an HCP has insight into a patient’s DPA data. This did not pose a problem for the participants in the pilot studies. BODY.MEASUREMENT OF DPA: Distance travelled, cycling, strength training, and the intensity of walking stairs were not properly captured by the app. The first two activities could be added by using GPS data, but this put too much strain on battery life. The accuracy of the measurement varied greatly between participants. Possible reasons for poor correlations include the amount of time spent in a train, bus, or car (participant 7, pilot study C1), unclear diary entries as to whether the mobile phone was worn during exercise (participant 8, pilot study C1), a phone pouch that contained a magnet (participant 10, pilot study C1), using a walker (participant 2, pilot study C2), and using a mobility scooter (all participants of pilot study C3). Using a walker, mobility scooter, or other forms of assistive devices for DPA were added to the exclusion criteria for participants in the randomized controlled trial (RCT). In pilot study C3, the errors in the app in the first week probably also accounted for poor correlations. An additional pilot study (C4) showed a mean (SD) correlation between the armbands and mobile phone accelerometers of r=.88 (.12). BODY.LIMITATIONS: During pilot study C3, errors in the algorithm were discovered twice in the distributed app. Although these were swiftly corrected, this could have had a negative impact on the participants’ views of the app. In pilot study C3, there were 4 (36%, 4/11) dropout participants beforehand due to too much expected effort in learning how to use a mobile phone, and in pilot study C2 there was 1 participant (33%, 1/3) that was not interested in, and did not use, the intervention. This participant had trouble understanding how to use the mobile phone. As mentioned before, proper instruction is key in usage success. More extensive instruction might have improved understanding and prevented dropout. The results of the questionnaires in pilot study C2 may have been negatively influenced by this participant. Battery life posed a major problem while developing the app. Not all desired options, such as GPS-tracking and continuous measurement, were possible due to limited battery capacity. The “5 minutes on and off” configuration was chosen so the battery would last a whole day, which was deemed important for adherence. With the development of mobile phone technology and accompanying batteries with higher capacity, the app could be adjusted back to continuous measurement, and GPS-tracking could be added. Using a mobile phone to measure DPA is a good way to obtain objective data on this parameter; however, it is not a highly valid and reliable measurement instrument such as that used in research settings. Additional validated accelerometers would provide improved measurement accuracy of DPA, but it was reasoned that (long-term) adherence to the intervention would benefit from the least amount of devices worn. This app can be useful in obtaining an indication of a patient’s activity outside of a clinical setting. It will provide much more reliable data compared to a patient’s recall [45,46]. BODY.COMPARISON WITH PRIOR WORK: A review conducted by Bort-Roig et al [47] evaluated 10 studies that described the accuracy of physical activity data as measured by a mobile phone. The participants were mostly overweight or healthy adults. The studies reported measurement accuracy ranging from 52% to 100% in identifying certain activities and postures (eg, walking or standing). As described, there is room for improvement in DPA measurement accuracy when using a mobile phone accelerometer. This review also found that physical activity profiles, real-time feedback, social networking, expert consultation, and goal setting were identified as key features that facilitated physical activity engagement. Most of these features are also incorporated in our eHealth intervention. We found one pilot study that similarly focused on physical activity stimulation in patients with COPD [48]. Their intervention consists of a mobile phone app, website, and separate accelerometer. The participants felt encouraged to be more active. The positive effects included an awareness of DPA performance, the stimulating effect of a daily target goal, and a positive effect on self-efficacy. Motivation dropped when technical problems occurred, which is something that we also encountered in pilot study C3. BODY.CONCLUSIONS: By employing a user-centered design approach, a mobile phone was found to be an adequate and feasible interface for an eHealth intervention because it is non-obtrusive, can measure DPA objectively, and, by using an appropriate app, direct feedback on DPA can be given. Moreover, by combining the app with an appropriate and secured website, monitoring and feedback by an HCP is possible. The mobile phone and app are easy to learn and use by patients with COPD. Battery life lasted a whole day with the final version, and readability and comprehensibility of text and colors were good. The accuracy of DPA measurement was good in the final test. The idea of providing an HCP with DPA data caused no privacy issues in the participants. The final version of the eHealth intervention is presently being tested by our group for efficacy in a RCT in COPD patients.

TITLE: Exercise and postprandial lipemia: effects on vascular health in inactive adults ABSTRACT.BACKGROUND: There is evidence to suggest that postprandial lipemia are is linked to the impairment of endothelial function, which is characterized by an imbalance between the actions of vasodilators and vasoconstrictors. The aim of this study was to determine the effects of a 12-week high-intensity training (HIT) and moderate continuous training (MCT) protocol on postprandial lipemia, vascular function and arterial stiffness in inactive adults after high-fat meal (HFM) ingestion. ABSTRACT.METHODS: A randomized clinical trial was conducted in 20 healthy, inactive adults (31.6 ± 7.1 years). Participants followed the two exercise protocols for 12 weeks. To induce a state of postprandial lipemia (PPL), all subjects received a HFM. Endothelial function was measured using flow-mediated vasodilation (FMD), normalized brachial artery FMD (nFMD), aortic pulse wave velocity (PWV) and augmentation index (AIx). Plasma total cholesterol, high-density lipoprotein cholesterol (HDL-c), triglycerides and glucose were also measured. ABSTRACT.RESULTS: The effects of a HFM were evaluated in a fasted state and 60, 120, 180, and 240 min postprandially. A significant decrease in serum glucose between 0 min (fasted state) and 120 min postprandially was found in the HIT group (P = 0.035). Likewise, FMD (%) was significantly different between the fasted state and 60 min after a HFM in the HIT group (P = 0.042). The total cholesterol response expressed as area under curve (AUC)(0–240) was lower following HIT than following MCT, but no significant differences were observed (8%, P > 0.05). Similarly, triglycerides AUC(0–240) was also lower after HIT compared with MCT, which trended towards significance (24%, P = 0.076). The AUC(0–240) for the glucose response was significantly lower following HIT than MCT (10%, P = 0.008). FMD and nFMD AUC(0–240) were significantly higher following HIT than following MCT (46.9%, P = 0.021 and 67.3%, P = 0.009, respectively). PWV AUC(0–240) did not differ following between the two exercise groups (2.3%, P > 0.05). ABSTRACT.CONCLUSIONS: Supervised exercise training mitigates endothelial dysfunction and glucose response induced by PPL. Exercise intensity plays an important role in these protective effects, and medium-term HIT may be more effective than MCT in reducing postprandial glucose levels and attenuating vascular impairment. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT02738385 Date of registration: April 14, 2016. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12944-018-0719-3) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Postprandial lipemia (PPL) is defined as the elevation of circulating triglyceride-rich lipoproteins after high-fat meal (HFM) consumption. There is evidence to suggest that these exaggerated elevations in triglycerides are linked to impairment of endothelial function, characterized by an imbalance between the actions of vasodilators and vasoconstrictors [1]. Although the pathophysiology of endothelial dysfunction has not been fully clarified, reduced nitric oxide (NO) and increased oxidative stress are important contributors to the reduction of the vasodilatory response [2]. Endothelial dysfunction induced by PPL is considered an early and reversible predictor of atherosclerotic disease and cardiac events [3, 4]. As humans spend a considerable part of the day in a postprandial state, interventions that can reduce the magnitude and duration of this metabolic state may be beneficial in the prevention of cardiovascular disease (CVD). Exercise training prior to high-fat meal ingestion has been shown to have an attenuating effect on postprandial metabolism [5, 6]. In the same line, studies have reported that energy expenditure through prior exercise is related to the magnitude of this effect [7, 8]. Mestek et al. reported, however, that isocaloric sessions before a meal mitigate PPL independently of the intensity of the exercise session [9]. By contrast, other studies showed that the magnitude of PPL was influenced by prior exercise intensity [5, 10]. Thus, the effects of the intensity of the exercise undertaken on postprandial response remain contentious. With regard to postprandial endothelial function, evidence has shown that a single bout of exercise prior to HFM consumption improves fasting and postprandial endothelial function compared with a resting control condition [11–13]. Accordingly, it has been reported that acute moderate- and high-intensity exercise has transient benefits for macrovascular endothelial function in both fasting and postprandial states, and that these effects may be due to the improvement in antioxidant status [14, 15]. Nevertheless, the limited prior studies carried out to investigate the effects of exercise intensity have produced inconsistent findings [15–17]. The aforementioned studies investigating the protective effects of exercise performed a few hours before consumption of an HFM on postprandial metabolism and endothelial function have focused on the acute effects of exercise. Thus, although a 12-week training program has been established as a protocol to assess the chronic effects of exercise [18], the potential impact on postprandial metabolism and vascular function after HFM have not been previously investigated. A recent narrative review summarized the current literature on the possible contributions of medium- to long-term physical training to the reduction of the postprandial response, concluding that the data are inconclusive [18]. Interestingly, a recent systematic review and meta-analysis on cardio-metabolic health showed that performing even a short period (∼4 min) of high-intensity exercise has greater benefits than moderate-intensity exercise in terms of cardiometabolic risk factors [19]. Considering that most adults do not meet the public health recommendations of at least 150 min per week of moderate-intensity exercise and also that habitual physical activity declines during middle age [20], it is of special interest to identify how much high-intensity exercise is needed to optimize vascular function in adulthood. Thus, we Given the above, we hypothesized that medium-term exercise could attenuate the postprandial decrement in metabolism and endothelial function and that this effect would differ according to exercise intensity. On this basis, we aimed to determine the effects of a 12-week high-intensity training (HIT) or a moderate continuous training (MCT) program on postprandial metabolism and vascular function and arterial stiffness after HFM ingestion in healthy, inactive Latin-American adults. BODY.METHODS: BODY.STUDY DESIGN AND SETTING: Details of the study design and methods of the primary HIT-Heart Study trial have been described elsewhere (ClinicalTrials.gov ID: NCT02738385; April 14th, 2016) [21]. The study was performed in accordance with the Declaration of Helsinki (2000) and was approved by the local office for Medical Research Ethics Committee of The University of Santo Tomás, Colombia (ID 27–0500-2015). Postprandial biochemical and vascular function responses were assessed at baseline and over 12 weeks of training. We have previously provided an overview of the methods as per the Consolidated Standards of Reporting Trials (CONSORT) checklist [22]. BODY.PARTICIPANTS: Participants (n = 20) were recruited at the University of Rosario (Bogota, Colombia) from February 2015 to May 2016. Subjects were eligible to participate if they were located in the metropolitan region, with available time (1 h per day) to support the trial. Inclusion criteria were individuals aged 18–45 years who were inactive (< 150 min·wk.− 1 of moderate-intensity activity or 75 min·wk.− 1 of vigorous-intensity activity), had a body mass index (BMI) ≥18 and ≤ 30 kg/m2 and identified as being willing and having almost immediate availability. Individuals with a history of any medical condition identified by the American Heart Association as an absolute contraindication to exercise testing were excluded from the study [23]. Furthermore, individuals were also excluded if they presented any of the following: systemic infections, weight loss or gain of > 10% of body weight in the past 6 months for any reason, currently taking medication that suppresses or stimulates appetite, uncontrolled hypertension (systolic blood pressure 160 mmHg or diastolic blood pressure 95 mmHg), gastrointestinal disease (including self-reported chronic hepatitis or cirrhosis, any episode of alcoholic hepatitis or alcoholic pancreatitis within the past year, inflammatory bowel disease requiring treatment in the past year, recent or significant abdominal surgery e.g., gastrectomy), asthma, diagnosed diabetes (type 1 or 2), fasting impaired glucose tolerance (blood glucose ≥118 mg·dl− 1), use of any prescribed drugs, any active use of illegal or illicit drugs, or inability to participate because of a physical impairment. In addition, two exercise physiologists tested whether subjects had alterations in ventricular function and/or cardiomyopathy, measured by standard 12-lead electrocardiography (ECG) at rest and every 3 min during a maximum treadmill exercise test. All subjects remained under usual medical care and clinical follow-up (i.e., regular appointments with a physician) throughout the protocol. All participants provided written informed consent before participating in the study. BODY.BLINDING AND RANDOMIZATION: The coordinating Research Center for Physical Activity Measurement (CEMA) in Bogotá randomized the procedures with software using randomly permuted blocks. Group allocation was conducted via an online system in which the details of eligible participants were entered to obtain group assignments (i.e., 3:2 or 2:3). Assessors were blinded to study group assignments. BODY.INTERVENTIONS: After inclusion, patients performed a maximal cardiopulmonary exercise test on a maximum treadmill exercise test (Precor TRM 885, Precor Corp., Rome, Italy) following the modified Balke protocol [24]. Physiological parameters (maximal O2 consumption [VO2], heart rate [HR] and Borg ratings) from the test were used to establish the exercise intensity. Based on averaged maximum HR (HRmax) and VO2peak, the participants were classified according to normative values, referenced to age and sex. MCT and HIT interventions lasted 12 weeks, with 3 sessions per week consisting of fast walking or running on a treadmill with the deck inclined to reach the desired intensity. HR was recorded each session using an HR monitor (Polar Pacer, Polar Electro, Kempele, Finland). In addition, rating of perceived exertion (RPE) was also measured in each exercise session. BODY.MODERATE CONTINUOUS TRAINING (MCT) GROUP: Each preparatory period started with an exercise dose of 6 kcal·kg− 1·week− 1, which was increased progressively by 2 kcal·kg− 1·week− 1 until week 4 and was then maintained at 12 kcal·kg− 1·week− 1 for weeks 5 to 12, which was equivalent to 300 kcal of energy expended by the end of the training and cool-down (3 min) periods with total exercise time ranging from 45 to 55 min. Exercise training sessions were designed to elicit a response in the acceptable moderate-to-vigorous range, that is, 60–75% of heart rate reserve (HRR), and were adjusted according to ratings on the Borg scale [25].. During the supervised intervention, HR was recorded using an HR monitor (Polar Pacer) to ensure compliance with the exercise stimulus at the predetermined target HR zone. BODY.HIGH-INTENSITY TRAINING (HIT) GROUP: We calculated training energy expenditures according to participants’ age ranges and set the target energy expenditures to meet the consensus public health recommendations from the Cardiometabolic HIT-RT Study [25]. Each preparatory period started with an exercise dose of 6 kcal·kg− 1·week− 1, which was increased progressively by 2 kcal·kg− 1·week− 1 until week 4 and was then maintained at 12 kcal·kg− 1·week− 1 for weeks 5 to 12. The overall goal for the HIT group was to perform exercise sessions in 4 × 4-min intervals at 85–95% of HRR (with the target zone maintained for at least 2 min), interspersed with a 4-min recovery period at 75–85% of HRR. During each exercise session, participants adhered to the 12 kcal·kg− 1·week− 1 energy expenditure format, which was equivalent to 300 kcal of energy expended by the end of the training and cool-down (3 min) periods, with total exercise time ranging from 32 to 45 min. During the supervised intervention, HR and Borg ratings were measured as described for the MCT group. Both groups were instructed to refrain from exercise training and to avoid changing their physical activity levels outside the study. All participants reported adhering to these instructions. Although diet was not controlled, participants met with the study dietician for nutrition assessment and counseling at baseline, and an individualized iso-energetic nutrition intervention plan was developed from the baseline food intake assessment according to participant preferences. This plan was standardized at 1300–1500 kcal·day− 1 (50–55% carbohydrates, 30–35% total fat, < 7% saturated fat and 15–22% protein), distributed across 3–4 meals per day. BODY.DATA COLLECTION AND OUTCOME MEASURES: BODY.EXPERIMENTAL PROCEDURE: All measurements were performed at baseline and at the 12-week follow-up by personnel who were blinded to the treatment allocation. To control for confounding variables, we instructed the subjects to: i) fast for 10–12 h, ii) abstain from exercise for 24 h, iii) abstain from caffeine, tobacco, and vitamin supplements for 12 h, and iv) be awake between 0600 and 0700 h, all prior to each testing session. The HFM, which has been previously reported [26], consisted of a breakfast containing 1049 cal: 79 g of fat, 31 g of saturated fat, 4.5 g of trans fat, 666 mg of cholesterol, 69 g of carbohydrates, 31 g of protein, and 2.22 mg of sodium, adjusted by individual body weight. The effects of the HFM were measured in a fasted state and 60, 120, 180, and 240 min postprandially. Figure 1 represents the schedule of experimental events for each subject.Fig. 1Schedule of experimental events for each subject. HIT, high-intensity interval training; MCT, moderate-intensity continuous training; HFM, high-fat meal. Discontinuous arrows represent capillary blood samples and assessment of endothelial function BODY.BIOCHEMICAL PARAMETERS: Blood was drawn from an antecubital vein. The biochemical profile included plasma total cholesterol, high-density lipoprotein cholesterol (HDL-c), triglycerides and glucose (measured by enzymatic colorimetric methods). Inter-assay reproducibility (coefficients of variation) was determined via ten replicate analyses of five plasma pools over 15 days and was shown to be 2.0, 3.2, 2.6 and 1.5% for total cholesterol, HDL-c, triglycerides and serum glucose, respectively. All determinations were analyzed in serum using a Cardiocheck® and A1CNow + ® system. BODY.VASCULAR FUNCTION AND ARTERIAL STIFFNESS MEASURES: All subjects were tested at the same time of day and after consumption of a low nitrate diet for 48 h. Vascular function and arterial stiffness, as measured by flow-mediated vasodilation (FMD), aortic pulse wave velocity (PWV) and the augmentation index (AIx) were measured. FMD was measured as described in previous studies from our group in the Colombian population [26] using the guidelines reported by Atkinson et al. [27]. The same operator performed all Doppler ultrasound (Mindray M-9® DS USA; Mahwah, NJ) examinations using a 7.5-MHz linear array probe. Ultrasound imaging of the brachial artery was performed with the subjects in the supine position after 15 min of rest, with the arm abducted approximately 80° from the body and the forearm supinated. The ultrasound probe was positioned with a 60° insonation angle in a longitudinal plane at a site 1–3 cm proximal to the antecubital fossa to visualize the anterior and posterior lumen-intima interfaces, to measure diameter and central flow velocity (pulsed Doppler). After the baseline images were recorded, a blood pressure cuff, positioned on the arm, was inflated to 200 mmHg for 5 min; to assess FMD, images were acquired continuously for 3 min after cuff deflation, during the reactive hyperemia period. Brachial artery diameter recording was restarted at least 30 s before cuff deflation and continued for 3 min thereafter. The peak artery diameter and the time to reach this peak after cuff deflation were recorded. Images were recorded on a DVD for subsequent measurements by one observer blinded to the study design. FMD was calculated as the percent rise of peak diameter from the preceding baseline diameter and was measured every 1 min after deflation for 3 min. Normalized brachial artery FMD (FMDn) was calculated according to the allometric relationship between the baseline artery diameter and the peak diameter [27]. The intra-session coefficient of variation was ≤1% for the baseline diameter. Reliability was estimated by intra-class correlation coefficients based on four baseline measurements (n = 8 subjects), yielding values of 0.91 for baseline diameter and 0.83 for FMD (our own data). The technical error of measurement was 1.23% for baseline diameter, 1.77% for maximum diameter and 20% for %FMD. The PWV was measured by analyzing the oscillometric pressure curves registered from the upper arm. Patient data and the measured distance between the jugulum and the symphysis were registered in an arteriographic computer program (Arteriograph Software v.1.9.9.2; TensioMed, Budapest, Hungary). A tape measure was used to measure the distance between the jugulum and the symphysis, the aortic distance. The cuff was placed on the patient’s upper arm and connected to the device. The algorithm measuring blood pressure in the arteriography device has been previously validated [28]. PWV was calculated as the jugulum-to-symphysis distance (m) divided by one-half of the return time (return time/2) (s). For PWV measurements, the two recordings with the lowest standard deviations were chosen. The standard deviation was calculated on the basis of all heartbeats during a period of 8 s. The AIx was calculated as the ratio of the difference between the systolic peaks of the first pulse [1] and second pulse [2] relative to the central pulse pressure, expressed as a percentage [(pulse 2 - pulse 1/central pulse pressure) × 100]. Thus, it provides the brachial/aortic AIx without applying a transfer function. The R value, used as an estimate of the measurement errors for the repeated measurements between two sessions, was low for the arteriograph (1.18 m·s− 1). BODY.STATISTICAL ANALYSIS: To retain the data of all randomly allocated participants, we performed an intention-to-treat analysis (all randomly assigned patients). The Shapiro-Wilk test was used to verify data distribution normality. Once it was confirmed that the sample data satisfied the normality assumption, statistical analyses relevant to our main research interests were conducted. T-tests for continuous variables and chi-squared tests for categorical variables were used to investigate any possible differences in baseline characteristics between the two conditions (HIT and MCT). We used a generalized linear model to analyze the influence of the different training protocols on biochemical and vascular function outcomes with repeated measures [2 (group) × 2 (test time)]. The area under the curve (AUC), expressed in arbitrary units (au) via the trapezoidal method, was calculated and used to analyze the response to the training protocols. The effect of training on AUC measures was analyzed by two-way analysis of variance. Significant differences in AUC from 0 to 240 min after the HFM following 12 weeks of HIT or MCT were analyzed using two-way analysis of variance. A criterion alpha level of P ≤ 0.05 was used to determine statistical significance. All data are reported as the mean ± standard deviation. Statistical analyses were conducted using PASW Statistics 17 for Windows (SPSS, Inc., Chicago, IL). BODY.RESULTS: BODY.STUDY PARTICIPANTS: Additional file 1 The demographic features of the HIT and MCT groups, as well as their biochemical and endothelial function variables in the fasted state at baseline, are outlined in Table 1. No statistically significant differences (P > 0.05) in baseline characteristics between the exercise training protocols were found (t-test), confirming that participants in both groups began the trial under similar conditions.Table 1Demographic, biochemical and endothelial function variables in the fasted state across HIT and MCT groups at baseline CharacteristicsHIT (n = 11)MCT (n = 9)P valueSex, n (%)  Male 8 (40.0) 5 (55.6) 0.898  Female 3 (60.0) 4 (44.4) 0.916 Age, mean (sd), y 31.8 (7.8) 31.4 (6.4) 0.928 Biochemical parameters, mean (SD)  Total cholesterol (mg/dL) 159.4 (47.4) 170.1 (41.8) 0.301  High-density lipoprotein (mg/dL) 46.9 (9.6) 43.0 (14.1) 0.236  Triglycerides (mg/dL) 100.4 (36.8) 134.1 (82.2) 0.118  Glucose (mg/dL) 78.3 (5.6) 82.3 (13.7) 0.190 Vascular function parameters, mean (SD)  Dbase, mm 3.0 (0.6) 2.7 (0.4) 0.157  FMD, % 7.2 (3.3) 7.3 (5.6) 0.487  Dpeak mm 3.2 (0.5) 3.0 (0.5) 0.140  Ddiff 0.2 (0.5) 0.3 (0.4) 0.496  FMDn, % 6.5 (2.9) 7.4 (5.7) 0.433  PWV, m·s−1 6.7 (0.8) 7.1 (1.2) 0.204  AIx (aortic), % 41.7 (10.4) 24.5 (32.7) 0.148  AIx (brachial), % 16.5 (5.2) 25.1 (16.5) 0.152  Pulse Pressure (mmHg) 45.3 (5.2) 44.6 (4.1) 0.931  Central systolic blood pressure (mmHg) 99.6 (43.7) 108.0 (5.8) 0.493  Pulmonary artery occlusion pressure (mmHg) 40.6 (6.8) 35.6 (3.9) 0.087  Systolic blood pressure (mmHg) 116.2 (6.5) 116.8 (5.1) 0.184  Diastolic blood pressure (mmHg) 71.0 (8.7) 72.3 (7.0) 0.278  Mean blood pressure (mmHg) 86.0 (7.6) 87.3 (6.0) 0.482 HIT 4 × 4-min high -intensity interval training, MCT moderate-intensity continuous training, D diameter, FMD flow-mediated vasodilation, nFMD normalized flow-mediated vasodilation, PWV pulse wave velocity, AIx augmentation index BODY.BIOCHEMISTRY AND ENDOTHELIAL RESPONSE: Postprandial biochemical and endothelial function responses with summary measures of these responses after 12 weeks of HIT or MCT are shown in Table 2. A significant difference in glucose between 0 min (fasted state) and 120 min postprandially in the HIT group was found (P = 0.035). Likewise, %FMD was significantly different between the fasted state and 60 min after HFM in the HIT group (P = 0.042).Table 2Intent-to-treat analysis of the effect of 12 weeks of HIT or MCT on postprandial lipemia biochemical and vascular function response after HFM ingestion HITMCT0 min60 min120 min180 min240 min0 min60 min120 min180 min240 minBiochemical parameters  Total cholesterol (mg/dL) 151.3 (21.0) 159.0 (28.1) 159.0 (30.2) 163.2 (30.0) 164.8 (20.2) 153.1 (29.09) 158.6 (25.1) 165.1(25.1) 166.8 (25.0) 167.1 (29.4)  High-density lipoprotein (mg/dL) 46.0 (14.1) 47.3 (14.6) 46.1 (12.7) 48.1(16.1) 48.7(15.9) 42.1 (9.5) 44.2 (11.6) 42.5 (12.2) 41.6 (12.6) 40.5 (10.9)  Triglycerides (mg/dL) 108.1 (35.8) 118.7 (41.7) 142.1 (53.7) 177.2 (91.5) 171.7 (84.5) 117.7 (33.1) 132.4 (31.1) 196.8 (56.1) 222.7 (68.2) 222.5 (91.2)  Glucose (mg/dL) 76.4 (11.0) 78.8 (12.9) 88.5 (8.8)b 84.3 (7.8) 86.3 (5.0 85.9 (6.3) 93.6 (12.4) 94.2 (7.8) 93.3 (10.3) 89.5 (10.7) Vascular function parameters  Dbase, mm 2.7 (0.4) 2.8 (0.3) 2.7 (0.3) 2.7 (0.4) 2.8 (0.3) 3.1 (0.5) 3.2 (0.4) 3.2 (0.5) 3.3 (0.5) 3.4 (0.4)  FMD, % 13.4 (4.6) 6.3 (7.3)a 12.3 (5.3) 12.0 (5.8) 10.4 (4.6) 9.4 (4.0) 6.1 (3.9) 7.5 (3.1) 7.3 (5.7) 6.7 (4.5)  Dpeak mm 3.0 (0.3) 2.9 (0.3) 3.1 (0.3) 3.0 (0.3) 3.1 (0.4) 3.4 (0.5) 3.4 (0.5) 3.4 (0.4) 3.6 (0.5) 3.6 (0.4)  Ddiff 0.3 (0.2) 0.1 (0.1) 0.4 (0.3) 0.3 (0.2) 0.3 (0.2) 0.3 (0.2) 0.2 (0.1) 0.2 (0.1) 0.3 (0.2) 0.2 (0.1)  nFMD, % 13.5 (6.3) 13.4 (4.6) 11.7 (6.1) 11.8 (7.3) 10.1 (5.1) 8.1 (4.1) 9.4 (4.0) 6.5 (3.2) 6.3 (5.2) 5.5 (4.2)  PWV, m·s−1 6.6 (1.5) 7.0 (1.6) 6.6 (2.4) 6.7 (1.2) 6.8 (1.0) 6.7 (0.9) 6.5 (1.0) 6.7 (1.0) 6.5 (1.0) 6.7 (1.0)  AIx (aortic), % 26.3 (14.6) 14.7 (9.4) 15.9 (12.1) 18.3 (12.5) 19.1 (9.2) 38.7 (69.0) 7.6 (4.2) 3.6 (16.0) 9.5 (4.1) 11.2 (7.0)  AIx (brachial), % −22.3 (28.9) −45.2 (18.7) −42.7 (24.0) −38.1 (24.8) −36.5 (18.2) −41.7 (16.5) −59.3 (8.5) −34.6 (45.0) −55.5 (8.2) −52.0 (13.8)  PP(mmHg) 45.5 (7.7) 49.8 (7.3) 45.9 (8.5) 48.5 (10.4) 47.4 (7.4) 45.1 (7.6) 46.7 (6.4) 51.0 (8.4) 46.0 (6.6) 48.8 (7.2)  SBPao (mmHg) 107.6 (14.2) 102.4 (7.7) 102.6 (12.9) 105.8 (10.2) 106.3 (9.6) 104.3 (7.3) 103.6 (8.4) 104.5 (6.1) 105.0 (8.4) 109.3 (9.6)  PPao (mmHg) 40.6 (6.6) 39.0 (3.4) 36.9 (7.1) 39.7 (6.3) 39.3 (5.1) 30.7 (12.9) 33.8 (5.3) 38.0 (6.9) 35.0 (6.2) 36.8 (6.1)  Systolic blood pressure (mmHg) 112.5 (9.1) 113.2 (6.4) 111.5 (8.4) 114.6 (7.2) 114.3 (6.6) 113.0 (7.6) 116.4 (9.1) 118.6 (6.1) 117.0 (8.1) 121.2 (10.8)  Diastolic blood pressure (mmHg) 67.0 (10.3) 63.4 (8.8) 65.6 (11.4) 66.1 (9.1) 67.0 (8.0) 67.8 (9.4) 69.7 (6.2) 66.5 (6.6) 71.0 (11.7) 72.4 (6.8)  Mean blood pressure (mmHg) 82.0 (9.2) 80.0 (7.4) 80.8 (9.7) 82.2 (7.0) 82.9 (6.7) 82.8 (7.8) 85.3 (6.8) 83.7 (5.2) 86.2 (10.2) 88.7 (7.7) Values are participant characteristics at baseline, mean (SD). HIT4 × 4-min high-intensity interval training, MCT moderate-intensity continuous training, D diameter, FMD flow-mediated vasodilation, FMDn, normalized flow-mediated vasodilation, PWV pPulse wave velocity, AIx, augmentation index, PP pulse pressure, SBPao central systolic blood pressure. PPao, pulmonary artery occlusion pressure. Whole group repeated measures ANOVA, a = 0 min vs 60 min; b = 0 min 120 min Figure 2 shows the effects of HIT and MCT on total cholesterol, triglycerides and glucose postprandial responses, with summary measures of these responses. The total cholesterol response expressed as AUC(0–240) was lower following HIT than following MCT, but no significant differences were observed (8%, P > 0.05). Similarly, triglycerides AUC(0–240) was also lower following HIT than following MCT, with a trend toward significance (24%, P = 0.076). AUC(0–240) for the glucose response was significantly lower following HIT than MCT (10%, P = 0.008).Fig. 2Total cholesterol, triglycerides (TG) and glucose responses to PPL (left) and incremental AUC (right) after 12 weeks of HIT and MCT. Deviations from fasting were analyzed by repeated-measures ANOVA. Differences between total cholesterol, TG and glucose AUC(0–240) after 12 weeks of HIT of MCT were analyzed by two -way analysis of variance Figure 3 shows the effects of HIT and MCT on FMD, nFMD and PWV postprandial responses, with summary measures of these responses. FMD and nFMD AUC(0–240) were significantly higher following HIT than following MCT (46.9%, P = 0.021 and 67.3%, P = 0.009, respectively). PWV AUC (0–240) did not differ between HIT and MCT (2.3%, P > 0.05).Fig. 3FMD (%), FMDn (%) and PWV responses to PPL (left) and incremental AUC (right) after 12 weeks of HIT and MCT. Deviations from fasting were analyzed by repeated-measures ANOVA. Differences between FMD (%), FMDn (%) and PWV AUC(0–240) after 12 weeks of HIT of MCT were analyzed by two-way analysis of variance No adverse events were reported during this study. As we have previously reported, the average exercise-training days and total exercise time during the program were 35.5 days (SD 1.3) and 1100 min (SD 258) in the MCT group and 35.4 days (SD 0.9) and 1031 min (SD 147) in the HIT group (P = 0.043) [21]. BODY.DISCUSSION: The aim of this study was to investigate the effects of chronic MCT and HIT on postprandial lipemia and vascular function and arterial stiffness after HFM consumption in inactive adults. The main finding of this study is that a 12-week regimen of HIT reduces glucose concentrations and exerts greater post-HFM endothelial function than MCT, supporting the idea that the effect of chronic exercise on postprandial response is dependent on exercise intensity [29]. Recent evidence has shown that acute exercise increases FMD following HFM consumption [12, 30]. To the best of our knowledge, the present study is the first to demonstrate that medium-term exercise training prevents the decline in FMD induced by PPL, supporting the protective effect of regular exercise on vascular function. This is clinically relevant since endothelial function is an independent risk factor of CVD [3]. Interestingly, the higher AUC values of FMD in the HIT group after HFM (P = 0.009) indicates that this regimen may provide major vascular benefits in inactive adults. In agreement with our results, previous studies focusing on the effect of acute exercise demonstrated that FMD remained greater after HFM consumption following a single bout of HIT as compared with MCT [15, 31]. However, although it is of special interest to calculate incremental AUC values drawn from hourly measurements up to 4 h using the trapezoid rule [12, 32], most previous studies have not included these data. In contrast to our present findings, a recent study conducted in 11 physically active young men reported that FMD response did not differ between the two conditions [16]. The differences in training status between this study population (physically active) and our study cohort (inactive) might explain the inconsistent findings, since it has been shown that FMD responses after HFM consumption may differ between active and inactive subjects [33]. Thus, based on our results and previous research, it can be hypothesized that exercise attenuates the negative effects of HFM consumption on endothelial cell function in an inactive population. Further studies investigating the mechano-sensory mechanisms contributing to the effect of exercise on vascular function as well as possible interactions among molecular pathways are required [34]. The mechanism by which chronic exercise training can modulate postprandial endothelial function is unclear. Regular exercise has been proposed to decrease PPL and therefore reduce postprandial oxidative stress by maintaining low lipoprotein levels [35]. An alternative mechanistic explanation is that regular exercise might increase antioxidant capacity, leading to maintenance of endothelial function [15]. Indeed, a substantial increase in exercise intensity has been linked to greater protection of vascular function against oxidative stress, supporting the possibility that HIT might trigger larger vascular effects at the cellular and molecular levels [15]. Likewise, exercise might exert a positive effect on endothelial function by stimulating the production and bioavailability of NO, as physical activity induces the activity of endothelial NO synthase (eNOS), increases the capacity of the cellular antioxidant system and diminishes the formation of reactive oxygen species (ROS) [36]. In addition, it has been demonstrated that a single session of exercise increased circulating and intramuscular free radical levels [37], which may lead to inactivation of NO with consequences for endothelium-mediated vasodilation [38]. It seems that acute exercise mediates the oxidant-antioxidant balance in favor of antioxidants, resulting in the maintenance of vascular function, and a similar effect is observed from the co-ingestion of antioxidants [39]. Thus, it is tempting to speculate that the effects of different intensities of exercise on postprandial FMD are related to changes in antioxidant status. We also found that medium-term HIT decreased the glucose response over the postprandial observation period by 10% compared with MCT (P = 0.008), indicating that the 7magnitude of postprandial glucose response was dependent on exercise intensity. This result contrasts with previous reports that failed to find differences between the two training protocols regarding postprandial glucose levels [10, 15, 16]. However, it should be noted that these studies only examined the effect of postprandial glucose level after acute exercise. Thus, it is possible that only medium or long-term training has a significant effect on postprandial glucose response. We found similar total cholesterol, HDL-c and triglyceride responses after HFM consumption in both training regimens, suggesting that medium-term exercise training might not play an important role in the postprandial decrement in lipid responses. In previous studies focusing on acute exercise, significant differences between HIT and MCT were found for triglycerides, but not for total cholesterol or HDL levels [10, 15, 16]. In this context, results from preliminary studies have suggested that the positive effect of exercise training on PPL might be short lived, demonstrating variations in the effect sizes for exercise training performed within 24 h prior to HFM ingestion and for exercise training performed more than 24 h pre-prandial [8, 40]. Thus, we hypothesize that postprandial triglyceride response might be short lived, showing a relevant effect only after acute exercise. BODY.STUDY LIMITATIONS: This study has some limitations. Due to the high sensitivity of endothelium to nutritional changes, it would be ideal to administer isocaloric meals to participants at least three days before the measurement of endothelial function. In this study, although diet was not controlled, a dietician provided an individualized iso-energetic nutrition intervention plan. Second, since endothelial function is well known to be affected by age and training status and our study cohort comprised healthy, inactive mature adults, our findings may not be generalizable to other populations with different characteristics. A final possible limitation is that we did not examine other factors such as antioxidant status, NO, IL-6 and TNF-α levels that might ameliorate postprandial response, and this should be studied in future research. The main strength of our study is that, to our knowledge, this is the first randomized clinical trial on the effect of exercise-training intensity on biochemical parameters and endothelial functional responses to HFM consumption in inactive adults from the Latin-American population. In addition, we provide measurements of these PPL responses at multiple time points to better describe their time course after chronic exercise. BODY.CONCLUSION: In summary, the novel finding of this study was that medium-term supervised physical training may mitigate endothelial dysfunction and glucose response induced by PPL. Exercise intensity seems to play an important role in these protective effects, suggesting that HIT might be the more effective in reducing postprandial glucose levels and attenuating vascular impairments. Therefore, medium-term HIT is an effective strategy to reduce CVD. BODY.ADDITIONAL FILE: Additional file 1:Figure S1. CONSORT guidelines flow diagram for enrolment and randomization. (TIFF 1515 kb)

TITLE: The impact of individual Cognitive Stimulation Therapy (iCST) on cognition, quality of life, caregiver health, and family relationships in dementia: A randomised controlled trialIndividual Cognitive Stimulation Therapy (iCST) for dementia ABSTRACT.BACKGROUND: Cognitive stimulation therapy (CST) is a well-established group psychosocial intervention for people with dementia. There is evidence that home-based programmes of cognitive stimulation delivered by family caregivers may benefit both the person and the caregiver. However, no previous studies have evaluated caregiver-delivered CST. This study aimed to evaluate the effectiveness of a home-based, caregiver-led individual cognitive stimulation therapy (iCST) program in (i) improving cognition and quality of life (QoL) for the person with dementia and (ii) mental and physical health (well-being) for the caregiver. ABSTRACT.METHODS AND FINDINGS: A single-blind, pragmatic randomised controlled trial (RCT) was conducted at eight study sites across the United Kingdom. The intervention and blinded assessment of outcomes were conducted in participants’ homes. Three hundred fifty-six people with mild to moderate dementia and their caregivers were recruited from memory services and community mental health teams (CMHTs). Participants were randomly assigned to iCST (75, 30-min sessions) or treatment as usual (TAU) control over 25 wk. iCST sessions consisted of themed activities designed to be mentally stimulating and enjoyable. Caregivers delivering iCST received training and support from an unblind researcher. Primary outcomes were cognition (Alzheimer’s Disease Assessment Scale–cognitive [ADAS-Cog]) and self-reported QoL (Quality of Life Alzheimer’s Disease [QoL-AD]) for the person with dementia and general health status (Short Form-12 health survey [SF-12]) for the caregiver. Secondary outcomes included quality of the caregiving relationship from the perspectives of the person and of the caregiver (Quality of the Carer Patient Relationship Scale) and health-related QoL (European Quality of Life–5 Dimensions [EQ-5D]) for the caregiver. Intention to treat (ITT) analyses were conducted. At the post-test (26 wk), there were no differences between the iCST and TAU groups in the outcomes of cognition (mean difference [MD] = −0.55, 95% CI −2.00–0.90; p = 0.45) and self-reported QoL (MD = −0.02, 95% CI −1.22–0.82; p = 0.97) for people with dementia, or caregivers’ general health status (MD = 0.13, 95% CI −1.65–1.91; p = 0.89). However, people with dementia receiving iCST rated the relationship with their caregiver more positively (MD = 1.77, 95% CI 0.26–3.28; p = 0.02), and iCST improved QoL for caregivers (EQ-5D, MD = 0.06, 95% CI 0.02–0.10; p = 0.01). Forty percent (72/180) of dyads allocated to iCST completed at least two sessions per week, with 22% (39/180) completing no sessions at all. Study limitations include low adherence to the intervention. ABSTRACT.CONCLUSIONS: There was no evidence that iCST has an effect on cognition or QoL for people with dementia. However, participating in iCST appeared to enhance the quality of the caregiving relationship and caregivers’ QoL. ABSTRACT.TRIAL REGISTRATION: The iCST trial is registered with the ISRCTN registry (identified ISRCTN 65945963, URL: DOI 10.1186/ISRCTN65945963). In a randomized controlled trial, Martin Orrell and colleagues investigate the impact of individual cognitive stimulation therapy (iCST) on cognition, quality of life, caregiver health, and family relationships for people with dementia. ABSTRACT.AUTHOR SUMMARY: ABSTRACT.WHY WAS THIS STUDY DONE?: Cognitive stimulation therapy (CST) is a structured group activity programme for people with dementia that has been shown to improve quality of life (QOL) and cognition. This therapy is recommended by organisations such as the Alzheimer’s Disease International (ADI) and the UK National Institute for Health and Care Excellence. Although CST is becoming more widely available both in the UK and internationally, some people may not have access to groups because groups are not available near their home, they are not able to get to centres offering groups because of transport, health, or mobility problems, or they would prefer not to do group activities. This study aimed to look at the potential use and benefits of an adapted version of CST called individual CST (iCST) delivered by a family carer or friend at the person with dementia’s home for 30 min ideally two or three times a week. ABSTRACT.WHAT DID THE RESEARCHERS DO AND FIND?: The research team produced the iCST programme (including a manual, activity workbook, and materials such as maps and dominoes) in collaboration with people with dementia, carers, health care professionals, and experts. In a randomised controlled trial (RCT), 356 pairs of people with dementia and carers were split between two groups; 180 pairs received iCST, and 176 pairs continued with activities, treatments, and services offered as part of usual care but did not receive iCST, to test whether iCST benefits cognition and QOL for people with dementia and mental and physical health for carers. The study found that people with dementia receiving iCST did not benefit in terms of cognition or QOL, and neither was there evidence to suggest iCST improved carers’ mental or physical health. However, people with dementia in the iCST group reported better relationship quality with their family carer at 26 wk, and carers delivering iCST had better QOL at 26 wk. ABSTRACT.WHAT DO THESE FINDINGS MEAN?: We did not find that iCST improves cognition or QOL for people with dementia. Given that iCST appears to have a positive effect on the caregiving relationship and carer well-being, the programme might be a useful part of personally tailored home care packages. BODY.INTRODUCTION: There are an estimated 5.3 million individuals with dementia in the United States, with the number of cases rising each year as the population ages [1]. Family caregivers are an essential source of care with an estimated economic value of $217 billion a year [2]. Dementia caregiving poses unique challenges, and whilst there may be positive aspects, often this role is stressful and can adversely affect the physical and mental health of the caregiver [3,4]. The stress-health model indicates the experience of psychological, behavioural, and physical symptoms associated with dementia are stressful and can reduce quality of life (QoL) for the person and their caregiver [4–6]. In addition, the person’s increasing dependence on others to fulfill basic needs, restructuring of the established relationship, and apathy can all reduce the quality of the relationship between the caregiver and recipient [7–9]. Conflict in the caregiving relationship is a risk factor for deterioration of functioning in the person with dementia and presentation to services [10], and there is evidence to suggest that maintenance of this relationship may facilitate a good QOL, slow the progression of cognitive and functional decline, and delay institutionalisation [11,12]. There is growing recognition that psychological interventions can improve QOL and should be more widely available. Amongst those that enhance the QOL of people with dementia, cognitive stimulation therapy (CST) has a robust evidence base [13–15] and has been shown to improve patient QoL and cognition and also to be cost-effective [16]. An extended programme of maintenance CST (CST plus an additional 24 weekly sessions) was found to improve QoL [17]. Cognitive stimulation is based on the theory of “use it or lose it,” whereby mental stimulation may counter or slow cognitive decline, and evidence that activation of neurons may enhance neuronal function and survival [18,19]. CST sessions are designed to provide general stimulation of a range of cognitive skills through enjoyable activities in a social setting, although language appears to be particularly affected [20]. Further investigation of CST’s impact on QoL indicates that the domains of energy level, memory, ability to do chores, and relationship with caregiver are most responsive to improvement, and that improvements in QoL may be mediated by improvements in cognition [21]. Typically, CST is delivered in day centres or residential care facilities, without the family caregiver. The stress-health model suggests that improvements in QOL and cognition from CST may improve caregiver outcomes, but few studies have examined this [5]. Many of the therapies currently available are directed at either caregivers or people with dementia, but a meta-analysis of psychological interventions for caregivers suggests that interventions are less efficacious when they target caregivers alone [22]. Home-based, multi-component dyadic interventions, engaging both the caregiver and the person with dementia, have been found to yield a range of benefits, including reduction in behavioural symptoms [23], reduction in negative caregiver reactions [24], and reduction in nursing home admissions [25]. The current evidence on both caregiver-focused and dyadic interventions also suggests that delivery one to one is more effective than in a group [6,22]. This suggests that a home-based, one-to-one version of CST led by a family caregiver may yield benefits for both the person and the caregiver. Few studies have focused on the use of cognitive stimulation–based programmes delivered in the home, and CST has never been directly adapted for use in this context. However, a small study of home-based memory management by family caregivers with psychoeducation improved memory in the person with dementia, improved caregiver well-being, and reduced care home admissions by 18 mo follow-up [26]. Similar benefits in cognition in people with dementia and caregiver well-being have been reported in other studies [27,28]. A further potential benefit of developing a home-based version of CST would be increasing the accessibility of the intervention for people unable to get to groups due to health/mobility problems, lack of groups in the local area, or preference not to participate in group activities [29]. The aim of the individual CST (iCST) trial was to investigate the primary outcomes of whether family caregiver-delivered CST improves (i) cognition and QoL of people with dementia and (ii) mental and physical health of caregivers. We hypothesised iCST may elicit cognitive benefits for the following reasons: (i) the programme provides mental stimulation through multisensory activities exercising a range of cognitive skills (e.g., memory, communication) in an environment that supports learning [30]; (ii) like CST, iCST focuses on implicit memory, which tends to be maintained longer than explicit memory and, moreover, responds to stimulation [31]; (iii) discussion of new thoughts and ideas, and making associations (key principle of iCST) stimulates language [30]. The existing evidence on group CST and other individual family-led cognitive interventions [13,26–28] also supports the choice of cognitive change as a primary outcome in this trial. QoL was also chosen as a primary outcome because (i) improvements in cognitive function appear to mediate improvements in QoL; therefore, if iCST benefitted people cognitively as predicted, we expected to observe an associated positive impact on QoL, and (ii) cognitive stimulation has consistently been found to improve QoL [15]. Secondary outcomes for people with dementia included behavioural and psychological symptoms, activities of daily living, depressive symptoms, and the quality of the caregiving relationship. We posited that iCST may improve the caregiving relationship because (i) improving cognition may help people communicate more effectively with their caregiver, which is associated with higher relationship satisfaction [11], and (ii) the programme provides an opportunity for people and their caregivers to participate in enjoyable activities together [32,33]. In addition, enhancing the quality of the caregiving relationship may also improve QoL for the person with dementia [11], supporting selection of QoL as a primary outcome in this trial. For caregivers, health-related QoL, mood symptoms, resilience, and relationship quality were secondary outcomes. We hypothesised that participating in activities together may help caregivers develop or maintain a closer relationship with the person they are caring for, and focusing these kinds of positive aspects of caregiving may improve well-being and reduce stress and burden [34]. Experience of enjoyment through caregiving could serve as a coping resource; therefore, we anticipated participating in iCST may positively impact resilience [34]. BODY.METHODS: BODY.ETHICS STATEMENT: Ethical approval was obtained through the East London 3 Research Ethics Committee (ref no. 10/H0701/71). The study was registered as a clinical trial (ISRCTN 65945963). Participants gave informed consent in accordance with the UK Mental Capacity Act (2005) [35]. BODY.TRIAL DESIGN AND SETTING: We conducted a single-blind, two-group pragmatic randomised trial of iCST over 25 wk against treatment as usual (TAU) over 25 wk (Fig 1). The full protocol is described elsewhere [29]. The trial operated from eight centres across the UK (London, Bangor, Dorset, Devon, Hull, Lincolnshire, Manchester, and Norfolk & Suffolk). From April 2012 to July 2013, recruitment took place in a variety of community settings including National Health Service (NHS) memory clinics, community mental health teams (CMHTs) for older people, and associated outpatient clinics. 10.1371/journal.pmed.1002269.g001Fig 1BODY.PARTICIPANT FLOW THROUGH THE TRIAL.: “Withdrawn” indicates participants’ withdrawal from trial and all associated research activities. “Did not complete” indicates participants who missed 13-wk follow up assessment but returned for the 26-wk post test. BODY.RECRUITMENT: Participants were recruited in the community from memory clinics, CMHTs, outpatient clinics, day centres, and voluntary organisations such as the Alzheimer’s Society. The aim of the project was briefly described to potential participants by members of the research and clinical team, and permission for them to be contacted by local researchers was obtained prior to further contact. Research assistants discussed the project and provided full details to participants, answered any questions related to the project, and, if participants agreed, undertook written informed consent. BODY.PARTICIPANTS: Participants met the criteria for dementia of the Diagnostic and Statistical Manual of Mental Disorders [36], had dementia of mild to moderate severity (Mini Mental State Examination [MMSE] score ≥10) [37], had some ability to communicate and understand, and were able to see and hear well enough to participate in activities. In addition, each participant lived in the community, had no major illness affecting their participation, and had a caregiver (relative or friend) able to deliver the intervention and act as an informant for the assessments. If caregivers were not able to support the person in the delivery of iCST sessions, they were not recruited into the trial. BODY.SAMPLE SIZE: Based on previous studies [13] and the Cochrane Review of cognitive stimulation [15], we conservatively powered the trial to detect a mean difference (MD) between iCST and TAU of 0.35 on the Alzheimer’s Disease Assessment Scale–Cognitive Subscale (ADAS-Cog) [38]. To yield 80% power when using a t test with a two-sided 5% significance level, and assuming 15% attrition, we needed a sample size of 306 dyads. As actual attrition was 23% (83/356), we more than compensated by increasing the target to 356 dyads during the trial. One hundred eighty dyads were allocated to iCST, and 176 received TAU. BODY.RANDOMISATION AND BLINDING: After baseline (BL) assessment, we allocated dyads (people with dementia and caregivers) at random between iCST and TAU groups in the ratio 1:1. To prevent subversion, we used dynamic allocation [39] and the web-based randomisation service managed by North Wales Organisation for Randomised Trials in Health, an accredited UK Clinical Trials Unit. We stratified participants by centre and use of cholinesterase inhibitors. We concealed allocations from researchers conducting 13-wk mid-point and post-test (26 wk) assessments. To assess the success of blinding, these researchers recorded the perceived allocation of each participant at each assessment. BODY.INTERVENTION: The iCST program was delivered at home by a caregiver in regular contact with the person with dementia (see Text A in S1 Appendix). The iCST package included a manual containing guidance on sessions, key principles of iCST, and ideas for activities; an activity workbook with paper resources for activities; and a toolkit of additional items such as playing cards, dominoes, sound activity compact discs (CDs), and maps. Dyads engaged in up to three, 30-min sessions of structured cognitive stimulation through themed activities (e.g., word games, current affairs, being creative, see Table A in S1 Appendix for all iCST themes) per week over 25 wk (maximum of 75 sessions). A sample session is shown in Box 1 with details of the procedure and content of the first session of the iCST program: “My Life (Life History) Part I.” All sessions follow the same general structure, and a selection of activities is offered for each theme with two levels of difficulty; Level A activities were intended to be less cognitively demanding and more discussion based, whilst Level B activities were more cognitively challenging (see Box 1 for examples). BODY.BOX 1. SAMPLE ICST SESSION: MY LIFE (LIFE HISTORY) PART I: BODY.INTRODUCTION/WARM-UP (5–10 MIN): Discuss orientation information such as the day, date, and weather using aids such as the newspaper and surroundings (e.g., looking out of the window to prompt comments about the weather). The purpose of this introduction is to orientate the person to the here and now. Talk about something currently happening; this could be national or local news, events in the community, or personal events such as birthdays and special occasions (e.g., birth of a new child in the family). Discussion can be prompted using newspapers, online news articles, and documents such as invitations, etc. BODY.MAIN ACTIVITY (20–25 MIN): Level A: Look at old and recent family photographs of family and friends. Prompt with questions about shared traits of family members, interesting stories about them, tips to maintain good relationships, or advice you would give about having a family. Level B: Record family history in a family tree. Add details for each generation such as relationships, birthdays, and place of birth. Share stories about the family and talk about how people’s lives have changed over the years. Materials from iCST activity workbook: An example of a family tree and a template to give dyads a starting point for this activity, which can be used for the Level B option. No materials are provided for Level A, as this activity requires dyads to provide photographs from their own personal collection. Materials from the iCST toolkit: The colour pencils could be used to design the family tree, the UK and world maps could be used to prompt discussion about places of birth and location of family members, and the magnifying card could be used to see finer details on photographs or written records such as birth certificates, which may be used as cues in the activity. The development of the programme was rigorous, adhering to the UK Medical Research Council framework [40]. The program was based on a modified CST manual, the recent Cochrane review of cognitive stimulation [15], an individual manualised programme of reality orientation [28], and consultation with caregivers, people with dementia, and professionals in dementia care in a series of focus groups, consensus work, and field testing [41]. BODY.TREATMENT ADHERENCE, CAREGIVER TRAINING, AND SUPPORT: We followed previous studies [42] applying the treatment integrity model developed and expanded on by Lichstein, Riedel, and Grieve [43]. Intervention pairs were visited at home by a dementia researcher as soon as possible after randomisation to provide them with the iCST materials and train them in the iCST approach. Dementia researchers included mental health nurses, clinical psychologists, occupational therapists, and research assistants. All researchers who provided support to family caregivers received standardised training and followed a treatment protocol. The standardised training package researchers delivered to caregivers taught them how to use the iCST manual and activity workbook and implement the key principles of iCST. Clips from the group CST training DVD, “Making a Difference 2” [44], were shown to demonstrate good practice. After learning about the iCST approach, the caregiver delivered the first session with support from the researcher, who provided assistance and feedback. Typically training visits lasted between 60 and 90 min. Caregivers also received up to 10 h of support from the dementia researcher during the trial, including telephone support (initially weekly) and two scheduled monitoring home visits (monitoring visit 1 [MV1] and monitoring visit 2 [MV2]). If the key family caregiver was unable to continue delivering iCST, an appropriate caregiver was substituted if possible. BODY.TAU: As the trial examined the effects of adding iCST to TAU, the regular services offered were the same in both groups. Not surprisingly, TAU varied between and within centres and over time (see Text B in S1 Appendix for more information). Standard best practice methods around pragmatic RCTs were followed, and it was expected that both the iCST and TAU groups had access to a similar range and similar types of mentally stimulating activities outside the research trial, for instance non CST-based group activities offered by day centres, hobbies, gardening, support groups, or visits to places of interest. In terms of use of CST, participants who had attended CST groups in the 3 mo before recruitment into the trial were considered ineligible. Sites were asked to record any instances of engagement in CST or other activities offered by local services during the trial. As far as we were aware, participants were unlikely to have access to any comparable individual cognitive stimulation interventions, as this type of structured therapy is generally not available in the UK. General service use and medication were recorded in both groups. BODY.ASSESSMENT AND MEASURES: We completed primary and secondary measures at BL, 13 wk after BL (mid-point), and 26 wk after BL (post-test and primary endpoint). BODY.OUTCOME MEASURES FOR THE PERSON WITH DEMENTIA: The ADAS-Cog was selected to measure the primary outcome of cognition and consists of 11 tasks assessing memory, language, praxis, attention, and other cognitive abilities [38]. The scale is widely used and psychometrically sound, with good reliability and validity. The measure we selected to assess QoL was the Quality of Life Alzheimer Disease Scale (QoL-AD), which has good validity and reliability [45]. Secondary outcomes included dementia-specific QoL (Dementia Quality of Life [DEMQOL]) [46], neuropsychiatric symptoms (Neuropsychiatric Inventory [NPI]) [47], functional ability (Bristol Activities of Daily Living Scale [BADLS]) [48], and depressive symptoms (Geriatric Depression Scale [GDS-15]) [49]. Quality of the carer–patient relationship (Quality of the Carer–Patient Relationship Scale [QCPR]) was an additional measure included in response to data from the field-testing phase of the trial, which indicated that the caregiving relationship may benefit as a result of participating in the intervention [50,51]. The QCPR is split into two subscales: criticism and warmth. As a covariate, we graded severity of dementia using the Clinical Dementia Rating Scale (CDR) [52]. BODY.OUTCOME MEASURES FOR THE CAREGIVER: The primary outcome for caregivers was mental and physical health (well-being) measured by the Short Form-12 Health Survey (SF-12) [53]. Secondary outcomes were anxiety and depressive symptoms (Hospital Anxiety and Depression Scale [HADS]) [54], health-related QOL (European Quality of Life–5 Dimensions [EQ-5D]) [55] to which we applied societal weights [56], resilience (Resilience Scale [RS-14]) [57], and quality of the caregiving relationship (QCPR) [50]. BODY.CAREGIVER ADHERENCE MEASURES: Caregivers in the iCST group completed self-report questionnaires at the set-up visit, MV1, and MV2, which required them to rate their confidence (4-point scale: very little, fair, good, very confident) in delivering iCST, quality of support (5-point scale A: excellent, very good, good, fair, poor), knowledge of iCST (see 5-point scale A), successful engagement (5-point scale B: all of the time, most of the time, some of the time, a little of the time, none of the time), and application of specific techniques (opinions rather than facts, developing ideas in a sensitive manner, incorporating person’s interests into programme, adapting sessions for the person) and skills in delivering the sessions (see 5-point scale B). The questionnaire was developed specifically for use in this trial to measure treatment integrity and whether the intervention was carried out as intended [58]. BODY.ANTICIPATED RISKS: As there are no documented harmful side effects from participating in CST, we expected few adverse events in this trial [13]. Sites recorded and reported serious adverse events (SAEs) to the Chief Investigator. BODY.STATISTICAL ANALYSIS: We analysed all available data by treatment allocated, following the principles of Intention to Treat (ITT). Statisticians performing the main analysis were blind to randomised intervention assignment. We used analysis of covariance (ANCOVA) to estimate the differences between iCST and TAU groups for people with dementia in primary and secondary outcomes at the 13-wk mid-point and post-test (26 wk). The model adjusted for covariates expected to influence outcome variables, including BL score on the outcome measures, the age of the person with dementia, and relationship with the caregiver. The fixed factors were gender, marital status, and use of anti-cholinesterase inhibitors, and centre was a random factor. We used a similar ANCOVA for primary and secondary outcomes for caregivers at the 13-wk mid-point and post-test with covariates of BL scores, age of caregiver, and relationship with the person with dementia, fixed factors of gender and marital status, and random factor of centre. Effect sizes were calculated using Cohen’s d. BODY.ADHERENCE ANALYSES: Carer adherence data were collected and paired t test analysis performed to compare the differences between the set-up visit, MV1, and MV2. BODY.EXPLORATORY ANALYSES: To analyse adherence, linear regression was used to assess the relationship between the follow-up outcome measures and the number of iCST sessions attended after adjusting for BL outcome measures. This method was considered more efficient for an exploratory analysis than either defining an average number of sessions to complete a week or predefining a number of sessions to be “enough” of the therapy. Any participants who did not provide any data post-test or at the 13-wk mid-point were not included in the analysis. If a participant had less than 20% of the items missing for a scale, then we prorated the scores for that measure [59]. This left fewer than ten total scores missing, and for these we then used multiple imputation based on a linear regression method. The number of imputations created was based on the percentage of missing data. BODY.RESULTS: BODY.PRELIMINARY ANALYSES: There were no differences between the two groups at BL on clinical and demographic factors (Table 1). Three hundred and fifty-six pairs participated in the trial. Recruitment was complete by July 2013, with the final post-test assessments complete by February 2014. Analysis by treatment allocated included 134 iCST and 139 TAU dyads. Twenty-three percent (83/356) of the total sample (75/356, 21% excluding deaths) dropped out by post-test. Rates of attrition in the iCST (46/180, 26%) and TAU (37/176, 21%) groups were not significantly different. Average BL MMSE scores were similar (iCST = 21.12, standard deviation [SD] = 4.48; TAU = 21.33, SD = 4.11). Characteristics of completers and noncompleters are provided in Table B in S1 Appendix, which shows no differences at BL. Reasons for dropouts are shown in Table C in S1 Appendix. Out of the 46 withdrawals in the iCST group, 18 did not wish to continue (sometimes noting they were too busy), and 28 were unable to participate largely because of ill health or having relocated. Six from the TAU group withdrew because they were not allocated to iCST, amounting to only one in 30 TAU participants. Seventy percent of the sample had mild dementia (CDR score = 1). In relation to other activities, there was no difference between the intervention and TAU groups at BL or post-test in terms of day centre attendance, lunch club attendance, or education classes. 10.1371/journal.pmed.1002269.t001Table 1BODY.BASELINE CHARACTERISTICS OF PERSON WITH DEMENTIA AND CAREGIVER.: CharacteristicPerson with dementiaTotal (%)(n = 356)iCST (%)(n = 180)TAU (%)(n = 176)Female 165/356 (46) 83/180 (46) 82/176 (47) Ethnicity: white 331/356 (93) 164/180 (91) 167/176 (95) Marital status: married/cohabiting/civil partnership 252/356 (71) 125/180 (69) 127/176 (72) Lives with spouse/partner 225/356 (63) 113/180 (63) 112/176 (64) Highest level of education school leaver (14–16 y) 213/356 (60) 113/180 (63) 100/176 (57) Anti-cholinesterase inhibitors 270/356 (76) 136/180 (76) 134/176 (76) Caregiver Female 261/356 (73) 135/180 (75) 126/176 (72) Ethnicity: white 329/356 (92) 164/180 (91) 166/176 (94) Marital status: married/cohabiting/civil partnership 297/356 (83) 149/180 (83) 148/176 (84) Highest level of education school leaver (14–16 y) 156/356 (44) 79/180 (44) 80/176 (45) iCST, individual cognitive stimulation therapy; TAU, treatment as usual. BODY.RESEARCHER RATINGS OF PERCEIVED GROUP ALLOCATION: The response rate for the researcher perceived group allocation questionnaires (see Table D and Table E in S1 Appendix) was high at the 13-wk mid-point (92%, 264/288) and post-test (93%, 255/273). At both assessment time points, most blinded researchers were not able to identify whether dyads were receiving iCST or TAU (60%, 160/264 at mid-point and 57%, 145/255 post-test). Overall, again at both time points, only 23% were able to accurately predict which group the dyads had been allocated to, with the remainder judging incorrectly. BODY.OUTCOMES FOR PERSON WITH DEMENTIA: The primary outcomes of cognition and QOL (ADAS-Cog, QoL-AD) were not statistically significant at the 5% level between iCST group and TAU group at the 13-wk mid-point or the primary end point post-test (Table 2). However, there was a significant improvement in QCPR total score for the iCST group relative to the TAU group, with an MD of 1.77 (95% CI 0.26–3.28; p = 0.02) and effect size of 0.32. No significant differences between groups were detected for activities of daily living, depression, or behavioural and psychological symptoms. There were no differences in primary or secondary outcomes at the 13-wk mid-point (Table 3). Summaries of outcomes and change from BL scores are provided in Tables F and G in S1 Appendix, respectively. 10.1371/journal.pmed.1002269.t002Table 2BODY.OUTCOME MEASURES AT 26-WK POST-TEST BY ICST VERSUS TAU: COMPLETE CASE ANALYSIS, ADJUSTING FOR BL OUTCOME MEASURES, MARITAL STATUS, CENTRE, AGE, AND ANTICHOLINESTERASE INHIBITORS.: 26-wk post-test person with dementiaiCST (n = 134)TAU (n = 139)MD95% CI of MDp valueADAS-Cog 20.03 20.58 −0.55 (−2.00–0.90) 0.45 QoL-AD 37.90 37.92 −0.02 (−1.04–1.00) 0.97 DEMQoL 94.45 94.14 0.31 (−1.62–2.22) 0.79 NPI [P] 8.10 8.42 −0.32 (−2.78–2.12) 0.79 GDS-15 3.29 3.31 −0.02 (−0.51–0.47) 0.94 QCPR Total* 57.42 55.65 1.77 (0.26–3.28) 0.02* MMSE 19.63 20.10 −0.47 (−1.26–0.30) 0.23 BADLS [P] 11.91 12.57 −0.66 (−2.07–0.75) 0.36 QoL-AD [P] 32.45 32.00 0.45 (−0.71–1.60) 0.45 DEMQoL [P] 99.67 97.94 1.73 (−0.61–4.07) 0.15 Caregiver SF-12 Physical component 49.57 49.11 0.46 (−1.21–2.13) 0.59 SF-12 Mental component 48.44 48.31 0.13 (−1.65–1.91) 0.89 HADS Anxiety 6.09 6.30 −0.21 (−0.94–0.52) 0.57 HADS Depression 4.16 4.67 −0.51 (−1.09–0.08) 0.09 EQ-5D health state today 78.20 76.99 1.21 (−2.14–4.57) 0.48 EQ-5D calculated utility value* 0.82 0.76 0.06 (0.01–0.10) 0.01* RS-14 83.42 81.85 1.58 (−0.37–3.52) 0.11 NPI Carer distress 3.13 3.22 −0.09 (-0.55–0.37) 0.70 QCPR total 59.65 60.21 −0.56 (−1.93–0.82) 0.43 Note: Complete case data are presented owing to little difference between this and imputed data results. * Significant difference. ADAS-Cog, Alzheimer’s Disease Assessment Scale–Cognitive; BADLS, Bristol Activities of Daily Living Scale; DEMQoL, Dementia Quality of Life, EQ-5D, European Quality of Life–5 Dimensions; GDS, Geriatric Depression Scale; HADS, Hospital Anxiety and Depression Scale; iCST, individual cognitive stimulation therapy; MD, mean difference; MMSE, Mini Mental State Examination; NPI, Neuropsychiatric Inventory; [P], Proxy rated measure; QCPR, Quality of the Carer–Patient Relationship; QoL-AD, Quality of Life Alzheimer Disease; RS-14, Resilience Scale; SF-12, Short Form-12 Health Survey; TAU, treatment as usual. 10.1371/journal.pmed.1002269.t003Table 3BODY.THE MEANS (AND 95% CIS) COMPARING THE ICST AND TAU FOR PERSON WITH DEMENTIA OUTCOME MEASURES AT 13-WK MID-POINT AFTER ADJUSTING FOR MARITAL STATUS, CENTRE, AGE, AND ANTICHOLINESTERASE INHIBITORS.: (Complete case data are presented due to little difference between this and imputed data results). 13-wk mid-point person with dementiaMissingiCST (n = 142)TAU (n = 146)MD95% CI of MDp valueADAS-Cog 10 22.00 21.71 0.29 (−1.10–1.68) 0.68 QoL-AD 4 38.40 38.54 −0.14 (−1.12–0.84) 0.78 DEMQoL 11 91.72 92.05 −0.33 (−2.31–1.65) 0.74 NPI [P] 2 12.27 13.72 −1.45 (−3.68–0.76) 0.20 GDS-15 12 3.27 3.36 −0.09 (−0.56–0.38) 0.71 QCPR total 7 56.62 55.52 1.10 (−0.15–2.35) 0.09 MMSE 3 20.32 20.16 0.16 (−0.60–0.92) 0.69 BADLS [P] 1 12.73 12.93 −0.20 (−1.44–1.04) 0.75 QoL-AD [P] 3 32.66 31.91 0.75 (−0.27–1.77) 0.15 DEMQoL [P] 3 99.28 98.73 0.55 (−1.70–2.80) 0.64 Caregiver SF-12 Physical component 0 50.51 50.57 −0.06 (−1.45–1.33) 0.93 SF-12 Mental component 0 47.59 48.30 −0.71 (−2.34–0.92) 0.39 HADS Anxiety 1 10.47 10.31 0.16 (−0.81–1.15) 0.74 HADS Depression 1 6.34 6.05 0.29 (−0.35–0.91) 0.37 EQ-5D health state today 1 4.13 4.27 −0.14 (−0.67–0.39) 0.60 EQ-5D calculated utility value* 1 77.55 77.00 0.55 (−2.59–3.69) 0.73 RS-14 1 0.81 0.79 0.02 (−0.02–0.06) 0.19 NPI Carer distress 0 83.35 83.41 −0.06 (−1.63–1.51) 0.94 QCPR total 2 3.16 3.15 0.01 (−0.43–0.43) 0.99 * Significant difference at 5% level. ADAS-Cog, Alzheimer’s Disease Assessment Scale–Cognitive; BADLS, Bristol Activities of Daily Living Scale; DEMQoL, Dementia Quality of Life, EQ-5D, European Quality of Life–5 Dimensions; GDS, Geriatric Depression Scale; HADS, Hospital Anxiety and Depression Scale; iCST, individual cognitive stimulation therapy; MD, mean difference; MMSE, Mini Mental State Examination; NPI, Neuropsychiatric Inventory; [P], Proxy rated measure; QCPR, Quality of the Carer–Patient Relationship; QoL-AD, Quality of Life Alzheimer Disease; RS-14, Resilience Scale; SF-12, Short Form-12 Health Survey; TAU, treatment as usual. BODY.CAREGIVER OUTCOMES: There were no differences in the primary outcome of functional health status (well-being) on the SF-12 (Table 2). The EQ-5D calculated utility value for the caregiver was significantly better post-test for the iCST group, with an MD of 0.06 (95% CI 0.01–0.10, p = 0.014) and effect size of 0.25. Reduced HADS depression score in the iCST group at post-test (−0.51, 95% CI −1.09–0.08, p = 0.09) did not reach significance. No differences in any other outcomes were found at the 13-wk mid-point (Table 3). BODY.ADHERENCE ANALYSIS: One hundred and seventy-three carers completed questionnaires at setup, 141 at MV1, and 124 at MV2. Some carers did not complete the questionnaires, as they dropped out before the monitoring visit. At the set-up visit, carers scored their knowledge of iCST at 3.14 (out of 4), and it had improved to 3.58 at MV2 (MD = 0.371, 95% CI 0.285–0.457, p ≤ 0.001). In addition, carers’ confidence in delivering iCST improved from 2.98 (out of 4) at MV1 to 3.23 at MV2 (MD = 0.25, 95% CI 0.173–0.327, p ≤ 0.001). Overall, carers stated they felt that they had very good abilities to apply iCST key principles and skills related to the intervention, with scores ranging from 3.76 to 3.96 at MV1 and improving between MV1 and MV2. These included “focusing on opinions rather than facts” (MD = 0.89, 95% CI 0.038–0.139, p ≤ 0.001), “developing ideas in a sensitive manner” (MD = 0.145, 95% CI 0.082–0.208, p ≤ 0.001), “incorporating their relative’s personal interests in the activities” (MD = 0.153, 95% CI 0.078–0.229, p ≤ 0.001), and “adapting the sessions to accommodate their relative’s abilities (MD = 0.089, 95% CI 0.017–0.160, p = 0.016). At setup, 71% (122/173) carers anticipated they would need little or no support in delivering the intervention. Set-up training, telephone support, and monitoring visits were well received by most carers, with 81% (114/141) of ratings being “good” or “excellent.” The majority of carers (83%, 144/173) felt they would be able to engage in iCST with the person with dementia most or all of the time. However, from the carer’s perspective, there appeared to be no significant differences in the person with dementia’s level of engagement from MV1 to MV2 (MD = −0.016, 95% CI −0.067–0.034, p = 0.529). In terms of number of sessions completed, only 40% (72/180) of dyads allocated to iCST completed at least two sessions per week, in line with the expected minimum for effectiveness, with 22% (39/180) completing no sessions at all. BODY.EXPLORATORY ANALYSES: BODY.OUTCOMES FOR PEOPLE WITH DEMENTIA: Twenty-two percent of the sample (39/180) in the iCST group did not complete any of the sessions, but 51% (91/180) were able to complete more than 30 sessions over 25 wk. Forty percent completed two to three sessions per week. When the linear regression model was fitted, there was no relationship between the number of sessions attended and the primary outcomes at any time point. However, the total number of sessions was associated with a significant improvement in the quality of the caregiving relationship from the person with dementia’s viewpoint (QCPR total, p = 0.003; QCPR criticism, p = 0.001). This result was consistent for QCPR total after regression analysis with imputed data. The imputation was not conducted for QCPR criticism at post-test, as no data were missing (Table 4). At the 13-wk mid-point, QCPR lower criticism had a significant association with higher number of sessions received (p = 0.004; results shown in Table H in S1 Appendix). 10.1371/journal.pmed.1002269.t004Table 4BODY.REGRESSION OF OUTCOME MEASURES AT 26-WK POST-TEST ON THE NUMBER OF SESSIONS OF ICST ATTENDED, ADJUSTING FOR BL OUTCOME MEASURES, MARITAL STATUS, CENTRE, AGE, AND ANTICHOLINESTERASE INHIBITORS.: Observed data, 26 wk post-testPerson with dementiacoefficient95% CIp valueCaregivercoefficient95% CIp valueADAS-Cog −.013 (−0.040, 0.015) .361 SF-12 Physical component .018 (−0.013, 0.049) .275 QoL-AD .008 (−0.011, 0.027) .402 SF-12 Mental component .017 (−0.016, 0.050) .338 DEMQoL .007 (−0.029, 0.044) .691 HADS total −.020 (−0.042, 0.001) .064 NPI total −.002 (−0.048, 0.044) .927 HADS Anxiety −.007 (−0.021, 0.006) .283 GDS-15 .001 (−0.008, 0.011) .815 HADS Depression* −.013 (−0.025, −0.003) .018* QCPR total* .043 (0.015, 0.071) .003* EQ-5D health state today .020 (−0.043, 0.083) .525 MMSE .006 (−0.009, 0.021) .455 EQ-5D calculated utility value .0007 (−0.000, 0.002 .090 BADLS [P] −.015 (−0.041, 0.011) .264 RS-14 .023 (−0.013, 0.061) .232 QoL-AD [P] .012 (−0.010, 0.034) .269 NPI Carer distress −.005 (−0.014, 0.003) .228 DEMQoL [P] .013 (−0.031, 0.058) .558 QCPR −.006 (−0.032, 0.020) .673 * Significant difference. ADAS-Cog, Alzheimer’s Disease Assessment Scale–Cognitive; BADLS, Bristol Activities of Daily Living Scale; DEMQoL, Dementia Quality of Life, EQ-5D, European Quality of Life–5 Dimensions; GDS, Geriatric Depression Scale; HADS, Hospital Anxiety and Depression Scale; iCST, individual cognitive stimulation therapy; MMSE, Mini Mental State Examination; NPI, Neuropsychiatric Inventory; [P], Proxy rated measure; QCPR, Quality of the Carer–Patient Relationship; QoL-AD, Quality of Life Alzheimer Disease; RS-14, Resilience Scale; SF-12, Short Form-12 Health Survey. BODY.OUTCOMES FOR CAREGIVERS: HADS depression scores showed a significant reduction in the iCST group post-test (p = 0.018) for caregivers who had participated in a higher number of sessions (Table 4). This was supported by the imputation analysis (p = 0.013). BODY.SAES: Twenty-five SAEs occurred in the iCST group and 26 in the TAU group, of which 44 related to people with dementia and seven involved caregivers. The most frequent reported category of SAE was “hospitalisation” (63%, 32/51), of which there were 16 instances in each group. There were more deaths in the TAU group (8/10) than the iCST group (2/10). Five SAEs categorised as “life-threatening” were recorded in total, three of which occurred in the iCST group, and four “medically significant” SAEs occurred in the iCST group. For three people with dementia, two SAEs were reported, which were hospitalisations followed by death. We judged that none were definitely, probably, or possibly related to treatment received within the trial, either iCST or TAU. BODY.DISCUSSION: We undertook a pragmatic RCT to evaluate the impact of a programme of individual, home-based CST on cognition and QoL of people with dementia and mental and physical health of caregivers. No significant differences were found between the iCST and TAU groups for the primary outcomes of cognition and QoL for people with dementia and mental and physical health for caregivers. iCST appeared to enhance the quality of the caregiving relationship from the person with dementia’s perspective. In addition, the caregivers in the intervention group benefitted in terms of improvements in QoL (EQ-5D). Analyses incorporating level of adherence to the iCST programme (number of sessions completed) revealed that people with dementia who participated in more sessions were more likely to experience gains in the relationship with their caregiver at the primary end point of the study (26 wk), and caregivers who completed more sessions had fewer depressive symptoms. The EQ-5D demonstrated improvements in QoL for caregivers, but there was no difference in general health status (SF-12). The discrepancy in findings between these two measures may be related to inherent differences in the measures or their sensitivity to change. With a total of 356 participating pairs, to our knowledge, this is the largest trial of a CST-based approach. The trial also represents an innovation in CST, as previously the intervention had been delivered only in groups without caregivers. A potential limitation of this trial is the low levels of adherence to the intervention. Because less than half of the iCST group completed at least two sessions per week (72/180, 40%) and 22% (39/180) did not complete any sessions, the power of the study to identify significant differences in outcomes between the iCST and TAU groups may have been compromised. Data from the development phase of the trial and qualitative data gathered from post-trial interviews may yield insight into the reasons for low levels of adherence. The principal reason for nonadherence in the field-testing study was difficulty fitting iCST into a busy schedule [51]. In post-trial interviews challenges that hindered adherence included difficulty engaging the person in the activities, which in some cases was due to the activities being too easy, poor health of the person with dementia, and having a negative outlook about the progressive nature of dementia, which may have affected caregiver motivation to deliver the intervention [60]. It may be that the intervention is simply not suitable for all and that there are particular characteristics of (i) the context in which the intervention is delivered (e.g., relationships, health, life events), (ii) the person delivering the intervention (e.g., motivation, personality), and (iii) the person receiving the intervention, which act as mediators of successful engagement in and adherence to the programme. More detailed investigation of these factors in order to discern their impact on adherence would be useful in future research. We were able to standardise the training and support in order to maximise treatment adherence and fidelity. However, the quality of support and training provided may have varied between sites, as it was delivered by a number of researchers with various levels of experience, skills, and qualifications. In terms of the extent to which the engagement strategies embedded into the trial (e.g., training, support, monitoring visits) were effective overall, data from the adherence questionnaires completed by caregivers at setup, MV1, and MV2 suggest the training and support provided by researchers was more than adequate; therefore, these components may not account for poor adherence. 61]. Blinding to allocation appeared successful for the most part, with the majority of researchers reporting dyads equally likely to be in the iCST or TAU groups. Dyads were reminded not to disclose their allocation to visiting researchers. However, on rare occasions, they did share this information or left iCST materials on show, which were seen by the researcher during the 13-wk mid-point or post-test assessment visit. If researchers were unblinded at the 13-wk mid-point, they were typically advised not to conduct the post-test assessment if possible. In terms of external validity, the intervention was tested in a wide diversity of urban and rural areas across eight sites in the UK. Despite this, there was a lack of variation in ethnicities of participants, which may affect scope for generalisation of results. Neither can we be sure the programme is suitable or acceptable across different cultures with the current data we have. The group CST programme has been successfully adapted for a wide range of cultures, with guidelines published to assist this process [62]; thus, iCST could be similarly tailored. Although, like CST, iCST was intended for people with mild to moderate dementia, most participants had mild dementia, and so it may be difficult to generalise the findings to people with moderate dementia. A further limitation of the trial is that the effect sizes for the significant improvements observed in relationship quality from the person with dementia’s perspective and caregiver QoL are small. Consequently, the extent to which the findings translate to tangible clinical or real-life benefits is hard to determine, particularly because investment of time and resources is necessary in order to deliver this intervention. However, there was a lot of feedback from people to indicate that the iCST activities were meaningful, enjoyable, and stimulating, suggesting that they may play a useful role as part of better care [60]. This trial contributes further to the body of knowledge of dyadic psychosocial interventions and demonstrates the benefits of relationship-centred care. The findings support the use of mentally stimulating activities as a means of improving outcomes for people with dementia and their caregivers [32,33]. Whilst CST is categorised as an intervention for people with dementia, the involvement of a family caregiver in the delivery of iCST and the observed positive impact of the sessions on both the person and the caregiver categorise iCST as a multi-component intervention. iCST provides caregivers with training and the manual with guidance and key principles that may be psychoeducational. iCST may also have elements of a support intervention, as caregivers were in regular telephone contact with researchers. Improvements in the quality of the caregiving relationship were only demonstrated from the person with dementia’s perspective. Whilst caregiver QoL improved, the person with dementia’s QoL did not. This suggests that iCST may offer mutual, caregiver-specific, and patient-specific benefits. The sessions present opportunities for joint activities between the person and the caregiver, which may contribute to the positive outcomes each experienced. Hellstrom, Nolan, and Lundh found caregivers and people with dementia identified four activities they felt “sustained couplehood”: “talking things through,” “being appreciative and affectionate,” “making the best of things,” and “keeping the peace” [32]. The iCST programme and key principles correspond to each of these needs, which may account for our findings. In terms of “talking things through” iCST activities facilitate discussion and may reinforce positive patterns of communication, which may transfer to interactions outside the sessions. Affection and appreciation may be demonstrated in the supportive and fun atmosphere that sessions are intended to create. Pairs may view participating in activities together as a source of enjoyment related to “making the best of things.” Finally, the iCST key principle of “focusing on opinions rather than facts” may contribute to “keeping the peace” by reducing criticism and celebrating success rather than dwelling on failure. Providing enjoyable activities for a person with dementia can improve caregiver well-being [63]; thus, adopting a facilitative role in iCST sessions may be the mechanism responsible for the observed improvements in caregiver QoL. This appears plausible given our finding that caregivers who delivered more iCST sessions had fewer depressive symptoms. The finding that the iCST programme did not significantly affect cognition contrasts with previous studies of group CST [13,15,26–28]. Furthermore, significant QoL benefits for people with dementia were not detected in this trial, despite being consistently associated with both short- and longer-term programmes of group CST [13,15,17]. The social setting and additional stimulation from participating in a group context may account for the difference in outcomes between iCST and group CST. Woods and colleagues suggest that the reported QOL benefits associated with CST are likely to be mediated by improvements in cognition [21]. Thus, the lack of significant cognitive change experienced by people with dementia may account for our lack of findings on QOL outcomes. Alternatively, an optimum “dose” of iCST may be necessary to impact cognition. There is evidence to suggest that two sessions of group CST per week are associated with cognitive benefits, whereas once weekly sessions are not [64]. Given 22% (39/180) of the sample did not complete any iCST sessions and adherence was generally low, people may not have received enough stimulation to benefit. The dyads who were either too busy or no longer wanted to participate amounted to just under 40% (18/46) of the total withdrawals, so in future studies, it is possible that with alternative approaches to retention and closer monitoring, some of these may have been persuaded to stay in the trial. Given some people with dementia and their family caregivers did not fully engage in iCST, the trial could be replicated with enhanced processes to support better adherence. In the development phase of this trial, delivery by paid caregivers was suggested as an alternative if family caregivers were not willing or able to deliver iCST, and field-testing with a sample of paid caregivers demonstrated this was feasible [51]. Therefore, future research should investigate how far adherence can be improved if the intervention is delivered by a health care professional or paid caregiver. Although iCST does not appear to deliver clinical benefits for cognition and QoL for people with dementia, there was evidence of improvement in terms of the caregiving relationship from the person with dementia’s perspective. There was also evidence of improvement in caregivers’ QoL and depressive symptoms for those who completed more sessions. From a clinical perspective, reduction in depressive symptoms and improved QoL of the caregiver by means of a low-cost, non-drug intervention are worthwhile outcomes. The longer-term associated effect of reducing depression may be that caregivers remain better mentally and perhaps physically (as a related consequence) for longer. This carries possible advantages such as prolonging their ability to provide care for the person with dementia, reduced instances of crises requiring intervention (e.g., emergency temporary hospitalisation), and less burden on health and social care services, as their health is maintained with less need for additional resources (e.g., medication, counselling, cognitive behavioural therapy). Enhancing the caregiving relationship through iCST may reduce the risk of presentation to services and deterioration of the person’s functioning, which has been associated with conflict in the caregiving relationship [10]. In turn, risk of institutionalisation may be reduced or delayed, which is important from a societal and cost perspective, as the cost of residential care is high [65]. This indicates that iCST may be introduced as a useful component of individually tailored home care packages, which may also help maintain people with dementia in their home situation for longer. BODY.SUPPORTING INFORMATION: S1 Appendix(DOC) Click here for additional data file. S1 CONSORT Checklist(DOC) Click here for additional data file. S1 iCST Materials list(DOC) Click here for additional data file.

TITLE: Effect of provision of home-based curative health services by public sector health-care providers on neonatal survival: a community-based cluster-randomised trial in rural Pakistan ABSTRACT.SUMMARY: ABSTRACT.BACKGROUND: Although the effectiveness of community mobilisation and promotive care delivered by community health workers in reducing perinatal and neonatal mortality is well established, evidence in support of home-based neonatal resuscitation and infection management is mixed. We assessed the effectiveness of adding training in neonatal bag and mask resuscitation and oral antibiotic therapy for suspected neonatal infections to a basic preventive and promotive interventions package delivered by public sector community-based lady health workers (LHWs) in rural Pakistan. ABSTRACT.METHODS: We did a cluster-randomised controlled trial in two subdistricts of Naushahro Feroze in rural Sindh, Pakistan, between April 15, 2009, and Dec 10, 2012. LHWs, trained in basic newborn resuscitation and in recognition and treatment (with oral amoxicillin) of suspected neonatal respiratory infections, were linked with traditional birth attendants and encouraged to attend home births. Control clusters received routine care through the existing national programme. The primary outcome was all-cause neonatal mortality. Independent data collection teams recorded data for all pregnancies and their outcomes, morbidity, mortality, and household practices related to maternal and newborn care. ABSTRACT.FINDINGS: Of the 27 randomised clusters with functional LHW programmes, 13 were allocated to the intervention group (n=242 749) and 14 to the control group (n=256 985). In the intervention group, LHWs did 80% of the planned community mobilisation sessions, but were able to attend only 1184 (14%) of 8425 deliveries and 4318 (25%) of 17 288 neonatal visits within 72 h of birth (p<0·0001 for both variables compared with the control group). The neonatal mortality rate was 42 deaths per 1000 livebirths in intervention clusters compared with 55 per 1000 in the control group (risk ratio 0·80, 95% CI 0·68–0·93; p=0·005). ABSTRACT.INTERPRETATION: The reduction in neonatal mortality in intervention clusters occurred against a background of improvements in domiciliary practices for maternal and newborn care. However, the poor reach of LHWs in accessing newborn infants at birth and in the early postnatal period underscores the limitations of tasking community health workers in public sector programmes working in similar circumstances with such complex interventions. Such community-based interventions in health systems should be accompanied by concerted efforts to improve quality of care in facilities and referral systems. ABSTRACT.FUNDING: Saving Newborn Lives, Save the Children USA. BODY.INTRODUCTION: Globally, 5·9 million children still die yearly before reaching their fifth birthday.1 2·7 million of these deaths are accounted for by neonates.2, 3 Much of the improvement in child survival over the past few decades has resulted from reductions in post-neonatal deaths from measles, pneumonia, and diarrhoea.4 Other than a reduction in deaths due to neonatal tetanus, improvements in neonatal survival in much of south Asia have lagged behind reductions in post-neonatal mortality. About two-thirds of the global burden of maternal deaths, neonatal deaths, and stillbirths is concentrated in ten large countries in sub-Saharan Africa and south Asia, of which Pakistan is one.5, 6 Two groups of conditions account for most neonatal deaths: preterm birth complications (an estimated 1·055 million deaths) and intrapartum-related events, which were formerly known as birth asphyxia (0·691 million deaths).1 A further 0·581 million neonatal deaths annually are due to sepsis, meningitis, pneumonia, and diarrhoea.1 Almost half of all stillbirths occur during labour and delivery, and nearly half of all neonatal deaths occur in the hours immediately after birth.5 Although the proportion of facility-based births is increasing in many countries, a large proportion of deaths occur at home, and combinations of community-based services, outreach services, and high-quality facility-based services are needed to make a difference.7, 8 In view of shortages of trained physicians and midwives in many settings, task shifting to a range of ancillary health workers, including community health workers, is a possible option.9 BODY.RESEARCH IN CONTEXT: Evidence before this study The role of community-based approaches and community health workers in promotion of care and delivery of health-care messages to improve perinatal and newborn care is well established. We did a systematic review of available information about community-based strategies for improving newborn care with community health workers in Pakistan, and also consulted the global literature. We searched PubMed, the Cochrane library, and regional databases of WHO and UNICEF with the terms “community health workers” or “community platforms” and “newborn”, and linked these medical subject heading terms to “Pakistan” to identify articles published in English on or before March 31, 2017. A Cochrane review included data from 26 cluster-randomised or quasi-randomised trials of a wide range of interventional packages, including two subsets from three trials. The data showed major reductions in neonatal mortality (including both early and late mortality), stillbirths, and perinatal mortality as a result of implementation of community-based interventional care packages. Although we identified strong evidence of improved household behaviours and improved care seeking in facilities, results for home-based neonatal resuscitation by either community health workers or traditional birth attendants were mixed, and results for antibiotic administration by community health workers were limited to a few efficacy trials, none of which were done in large public sector programmes. After completing a feasibility assessment, we worked with the National Program for Family Planning and Primary Care in Pakistan to assess the effect of training lady health workers (LHWs) in rural Pakistan to attend births, provide home-based bag and mask resuscitation as required, and provide oral amoxicillin to neonates with suspected pneumonia or serious infections before referral. Added value of this study Our cluster-randomised trial showed that community intervention by the LHW programme led to a 20% reduction in neonatal mortality and was associated with significant improvements in household practices and newborn care practices. However, overall effective coverage by functional LHWs was only 48%, and they were able to perform resuscitation in only 4% of potentially eligible neonates with birth asphyxia. The intervention had no effect on cause-specific neonatal mortality due to asphyxia or suspected serious infections. We also identified no effects on care seeking for facility births and stillbirths in the intervention clusters. Implications of all the available evidence Our trial supports the use of community health workers in large programmes for community mobilisation and support strategies for preventive and promotive maternal and newborn care. However, in view of the reality of large-scale public sector programmes, tasking such health workers with complex additional domiciliary care responsibilities might not be advisable. Improvement of maternal and newborn care in facilities and promotion of care seeking and transportation could prove more effective. In Pakistan, despite some improvements in coverage of antenatal care and skilled attendance, a high proportion of neonatal deaths occur at home, particularly in rural areas, where few trained professionals and skilled birth attendants are available.10, 11 Results of a 2012–13 survey of demographics and health showed large urban–rural disparities in terms of delivery (32% of deliveries in urban areas were home births vs 60% in rural areas) and neonatal mortality (47 deaths per 1000 livebirths in urban areas vs 62 per 1000 in rural areas).12 The Pakistani Government initiated a rural health programme with community health workers—so-called lady health workers (LHWs)—in 1994, with a focus on preventive and promotive strategies for maternal health, family planning, and primary care.11 More than 100 000 LHWs are deployed across rural Pakistan, but coverage is variable (ranging from 40–80%),13 and they do not routinely attend home deliveries. Each LHW is responsible for maintenance of birth records, provision of a range of promotive and preventive educational services, management of milder illnesses such as childhood diarrhoea and respiratory infections, and referral of people who need high-level care to health facilities for about 100 households in rural villages. They also provide services for family planning, basic maternal antenatal care, and oral polio vaccines during vaccination campaigns, and promote routine immunisations.14 Several community-based trials have been done in rural Pakistan to assess the potential effects of training community health workers on neonatal mortality. These trials included both public sector LHWs and community health workers supported by non-governmental organisations to deliver community mobilisation, health education through home visits,15, 16 and innovations such as the use of cord chlorhexidine by traditional birth attendants.17 We have also shown that strengthening of the LHW programme's links with the community and promotive care through community group sessions is associated with reductions in perinatal and neonatal mortality.15 Although other community-based strategies—eg, women's groups,18 promotion of preventive interventions such as exclusive breastfeeding—improved neonatal outcomes in low-income and middle-income countries,19, 20 the success of therapeutic interventions such as neonatal resuscitation and antibiotics has varied.21, 22, 23, 24 Community-based management of pneumonia and severe pneumonia in children older than 1 month by LHWs was effective in rural Pakistan,25, 26 but effectiveness in possibly infected neonates has not been fully assessed. Thus, despite long-standing recommendations to increase the range of interventions to improve neonatal survival in settings where referral is difficult or not possible,27 the effectiveness of home-based management of neonates in need of resuscitation at birth or born prematurely by front-line community health workers in programmatic settings is unclear. If effective, such strategies could have clear benefits, including reducing neonatal mortality, in populations with little access to doctors, nurses, or midwives. In collaboration with Pakistan's national programme for family planning and primary care, we did a cluster-randomised effectiveness trial of training LHWs to deliver a preventive and promotive community mobilisation and education package15 alongside recognition of possibly asphyxiated newborn infants at birth and bag and mask resuscitation as needed, and recognition and management of suspected neonatal infections. BODY.METHODS: BODY.STUDY SETTING AND DEVELOPMENT: We did a prospective cluster-randomised trial between April 15, 2009, and Dec 10, 2012, in the district Naushahro Feroze in rural Sindh. The district is located 450 km north of Karachi and has five talukas (subdistricts) and an official population of around 1·3 million. The trial was done in a subpopulation of 0·56 million in two talukas of Naushahro Feroze (Bhirya [population 0·26 million], and Naushahro Feroze [population 0·23 million]) and three talukas of Moro and Kandiaro (combined population 0·07 million). The study site is typical of most rural districts in Sindh and southern Punjab. The trial was approved by the ethics review committee of the Aga Khan University (1212-Ped/ERC). All households in the selected districts were included in the study. Community assent was obtained from village representatives for participation in the study; participating women gave verbal consent. To develop the intervention and define the clusters, we did a baseline cross-sectional household survey of the catchment population of 35 health facilities (14 basic health units, 12 government dispensaries, eight rural health centres, and the referral district headquarters hospital) between April 15 and Aug 30, 2009. We collected information about knowledge and practices relating to neonatal care from a random sample of newly delivered mothers identified in the baseline survey. We then did a formative qualitative study to develop and adapt the proposed intervention package to the local context and assess acceptability in close consultation with the federal and provincial LHW programmes and health departments. The preventive component of the package was adapted from one used in a previous trial.14 The therapeutic components of the intervention package focused on the immediate household management of intrapartum events (birth asphyxia), recognition of low birthweight and suspected serious neonatal infections, and prompt referral to public sector hospitals. We did a planned pilot trial of improved practices between July 1 and Dec 31, 2009, in the catchment population of one health facility, which was subsequently excluded from the main trial. The study tested the package, refined implementation, and assessed the feasibility of linking LHWs with traditional birth attendants so that LHWs could attend home births, and the ability of LHWs to use a bag and mask for neonatal resuscitation. On the basis of this pilot trial, the intervention package was finalised before rollout in the main trial (table 1).Table 1Description of intervention package Intervention clustersControl clustersLHWs' programme of support and training Recognition of high-risk pregnancies and neonatal danger Yes Yes Promotion of antenatal care and use of iron or folate in pregnancy Yes Yes Promotion of adequate maternal diet and rest Yes Yes Provision of clean delivery kit to pregnant women Yes No Immediate neonatal care Yes Yes Promotion of exclusive and early breastfeeding Yes Yes Cord care (dry, clean, and avoid any traditional application) Yes Yes Delayed bathing Yes Yes Recognition and domiciliary care of neonates with birth asphyxia, low birthweight, and suspected sepsis, and referral Yes No LHWs present at home births Yes No Domiciliary care with bag and mask for asphyxiated neonates and referral for aftercare Yes No Improved thermal care for low-birthweight and premature babies (frequent breastfeeding, waddling, co-bedding, early referral in case of any danger sign) Yes No Provision of first dose of amoxicillin to suspected infected neonates and referral to referral hospital; daily follow-up and provision of amoxicillin for 7 days in case of refused referral to hospital Yes No Provision of inflatable bag and mask, sucker bulb, amoxicillin, clean delivery kits, and management protocols to LHWs Yes No Support group (health education) training Exclusive training on support group methods, communication, and counselling skills for LHWs Yes No Male motivators training Yes No Incorporation of three flip charts on birth asphyxia, low birthweight, and sepsis in LHW curriculums Yes No Orientation for traditional birth attendants (dais) Basic essential neonatal care training and linkage with LHWs Yes No Health facility strengthening Health-care providers training on essential neonatal care and management of birth asphyxia, low-birthweight babies, and neonatal sepsis Yes No Health-care providers training on essential neonatal care and management of sick newborn infants according to WHO guidelines No Yes Provision of inflatable bag and mask and oral amoxicillin with management protocols Yes No LHW=lady health workers. BODY.CLUSTER DEFINITION, RANDOMISATION, AND MASKING: We defined a cluster as the catchment population of an individual functional primary care facility (basic health units and rural health centres) and their affiliated LHWs. Basic health units typically serve a population of 10 000–20 000 and have 10–20 affiliated LHWs. Rural health centres cater to a population of 25 000–50 000 and have 25–50 affiliated LHWs. Most LHWs have a catchment population of about 1000 individuals (120–200 households), are mostly resident in the same area, and are not transferred to other facilities or areas. In the original trial proposal, we anticipated the potential inclusion of 34 clusters representing the entire district. Clusters were assigned (1:1) to either the intervention or control groups. To ensure reasonable balance between the two arms, we used stratified, restricted randomisation to allocate clusters. Two strata were defined on the basis of level of health facilities: hospital or rural health centre (nine clusters) or basic health units (18 clusters). 1 million random allocation schemes were generated by the study statistician (SC), who used a computer algorithm. Acceptable schemes were those in which the total populations of each arm was restricted to within 15 000 of each other, total livebirths per year per arm to within 1000 of each other, overall neonatal mortality rates to within five per 1000 livebirths of each other, the ratio of LHWs to population to be within one per 10 000 population of each other, and overall female literacy rates to be within 5% of each other. In all, 28 476 distinct allocation schemes satisfying these restriction criteria were available, of which one was randomly chosen by the algorithm, with random allocation of one of the arms to the intervention.28 Delivery of the intervention was not blinded for practical reasons. Data collection teams were not actively informed which clusters were allocated to intervention and control arms. BODY.PROCEDURES: The trial was fully integrated and implemented within a programmatic setting. Senior faculty members of the Division of Women and Child Health of Aga Khan University (Karachi, Pakistan) held 5 days' training for LHW programme master trainers, who subsequently trained LHWs from the intervention clusters at the health facilities to which they were affiliated (an initial 3 days of training and monthly 1 day refresher sessions thereafter). Study supervisors along with LHW programme managers monitored the refresher sessions. Each intervention LHW was provided with a bag and mask for neonatal resuscitation (Laerdal Medical, Stavanger, Norway) and oral amoxicillin (125 mg/1·25 ml, to be given as 50 mg/kg per dose). LHWs were also given pictorial guides describing the management of asphyxia, thermal care, co-bedding, breastfeeding of low-birthweight babies, and recognition of suspected pneumonia and administration of oral amoxicillin before referral. As already included in the LHW programme guidance, we reinforced the importance of linkages of LHWs with traditional birth attendants in their areas. LHWs were encouraged to maintain close links with traditional birth attendants, keep records of expected births, and attend home deliveries. Clean delivery kits were provided to pregnant women in the intervention clusters during health education sessions delivered by LHWs, and the importance of provision of urgent neonatal care at birth, if needed, was emphasised. LHWs were trained as per national and project guidelines to do additional postnatal visits on days 3, 7, 14, and 28 after birth. LHWs were reimbursed additional travel costs, if any, to attend deliveries or postnatal visits, but no additional salary or other financial incentives were provided. A 3-day orientation programme in basic immediate maternal and newborn care was also organised for traditional birth attendants in the intervention arm, who were trained in the use of clean delivery kits, and strongly encouraged to inform LHWs in a timely manner to attend the home birth. No remuneration, commodities, or resuscitation training was provided to traditional birth attendants. Separate training sessions on health education and community mobilisation were held for male community mobilisers—volunteers from the villages, who were tasked with monthly mobilisation and review meetings with male elders and members of the community. These meetings aimed to promote antenatal care, postnatal care, and facility births. For people choosing to deliver at home, the importance of informing the LHW so that she could attend the childbirth was also reinforced, and volunteers were identified to escorts LHWs to attend deliveries, especially after dark. In the control clusters, the LHW programme continued to function as usual. LHWs continued to have regular monthly debriefing and refresher trainings according to the standard national LHW programme curriculum in the health facilities to which they were affiliated. However, as in the intervention arm, health-care providers from public sector facilities in the entire district received a one-time refresher training on essential neonatal care and management of sick newborn infants according to WHO guidelines29 in three separate workshops done between January and May, 2010. LHW programme supervisors monitored the delivery of the intervention package and related components and maintained their own records. An independent surveillance system was implemented, with 13 data collection teams visiting each household in the trial area quarterly. Verbal consent from heads of households and respondents was obtained for data collection. These data collectors were managed and deployed independently of the LHW programme and shuffled periodically as per previous surveillance protocols.14 They gathered standardised information from each household on all pregnancies, their outcomes, new pregnancies, neonatal morbidity and mortality, in-migrations, and out-migrations. Data collectors recorded whether the LHW was present at the time of delivery and instituted any interventions on the baby, and recorded treatments provided and referrals to hospital. Every 6 months, women reporting a livebirth since the previous surveillance visit were interviewed with a structured questionnaire to assess knowledge and practices related to neonatal care and LHW visits or actions. 30, 31 BODY.OUTCOMES: The primary outcome of the trial was all-cause neonatal mortality. The initial proposal included perinatal mortality as a primary outcome, but on the basis of feedback from the LHW programme and the range of outcomes captured therein, we principally focused on neonatal mortality instead. Secondary outcomes included cause-specific neonatal mortality due to intrapartum events, prematurity, and sepsis, and the stillbirth rate. BODY.STATISTICAL ANALYSIS: In the Hala trial,13 an almost 20% reduction in neonatal mortality was reported with LHW training in preventive care and community mobilisation for improved household practices and care seeking compared with the control population, and thereafter the LHW programme adopted the Hala package within its training programme. In view of the 45–50% reduction in mortality reported in other trials of home-based treatment,21, 32, 33 we estimated that a 40% reduction in neonatal mortality from the enhanced intervention was plausible. We assumed an average cluster size of 10 000, a crude annual birth rate of 25 per 1000, and an average neonatal mortality rate of 40 per thousand livebirths in the control arm (coefficient of variation 0·25). We estimated that inclusion of all births for 3 years would provide greater than 90% power to detect a 40% reduction in neonatal mortality rate and close to 80% power to detect a 30% reduction.28 We compared baseline characteristics of intervention and control areas by visual inspection. During the intervention period, frequency of birthing practices, LHW contacts, and neonatal morbidity rates in both groups were compared with logistic regression, with robust SEs to account for between-cluster variation, and adjusted for surveillance round and randomisation stratum. Analysis of mortality outcomes was done with generalised estimating equations, with robust SEs to account for the cluster randomisation. To obtain estimates of the risk ratio for intervention versus control clusters, binomial regression models with a log link were fitted, controlling for cluster-level log(baseline mortality, stillbirths, and early pregnancy loss), randomisation stratum, and surveillance round. The trial is registered with ClinicalTrials.gov (NCT01350765). BODY.ROLE OF THE FUNDING SOURCE: Although SW was involved in periodic review of the study progress, the funder of the study had no role in study design; data collection, analysis, or interpretation; or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. BODY.RESULTS: The trial was planned to span 36 months, but ended in Dec 10, 2012 (after quarterly surveillance round 11), because of the conclusion of activities in Pakistan by the Saving Newborn Lives programme of Save the Children, USA. Seven of the 34 clusters in the study area could not be included because no LHWs were posted therein. Thus 27 clusters, including 35 health-care facilities and their affiliated LHWs, were included (Figure 1, Figure 2). The entire population of the 27 clusters was included in the trial.Figure 1Study site and clusters Figure 2Trial profile At baseline, the study groups were balanced in terms of population and number of households (table 2). 73% of women in both groups had no education (table 2). Most households owned their homes (96%), had electricity (96%), and had access to piped water (88%; table 2). Roughly a quarter of the households in both groups had no toilets; only 19% of houses in control villages had underground waste disposal arrangements, compared with 10% in the intervention areas (table 1). According to the baseline survey, neonatal mortality and stillbirth were slightly less frequent in the intervention clusters than in the control clusters (43·7 vs 44·5 per 1000 livebirths, and 39·4 vs 42·7 per 1000 total births, respectively; table 2). The rate of early fetal death at baseline was broadly similar in both groups (table 2), as were the major causes of stillbirths and neonatal deaths (appendix).Table 2Baseline sociodemographic characteristics Control (14 clusters)Intervention (13 clusters)Population 256 985 242 749 Households 32 498 30 959 People per household 7·91 (4·30) 7·84 (4·81) Children younger than 5 years per household 1·41 (1·23) 1·32 (1·19) Female education n 51 806 49 192 No education 37 578 (73%) 36 045 (73%) Religious education only 4855 (9%) 3846 (8%) Primary and middle school 6254 (12%) 6005 (12%) Higher secondary school 2434 (5%) 2616 (5%) Secondary school graduate and above 685 (1%) 680 (1%) Finished walls (cement, stone with lime, or bricks) 8119 (25%) 7412 (24%) Finished floor (ceramic tiles, cement, or carpet) 6697 (21%) 5806 (19%) Finished roofing (ceramic tiles, cement, or roofing shingles) 10 455 (32%) 9484 (31%) Families owning their own home 31 177 (96%) 29 650 (96%) Single-room households 18 319 (56%) 16 756 (54%) Households with piped water 28 163 (87%) 27 433 (89%) Households with no toilet facility 8072 (25%) 7044 (23%) Households with underground sewerage (liquid waste) 6095 (19%) 3188 (10%) Households with electricity 31 182 (96%) 29 871 (96%) Households using solid fuels for cooking 25 796 (79%) 23 953 (77%) Birth outcomes and mortality rates Livebirths 8071 7755 Stillbirths 360 318 Early fetal deaths 1016 826 Neonatal deaths 358 339 Neonatal deaths per 1000 livebirths 44·6 43·7 Stillbirths per 1000 births 42·7 39·4 Early fetal death rate per 1000 pregnancies 107·6 92·8 Data are n, n (%), or mean (SE). Of the 63 457 households in the study area, 35 155 (55%) were included in the LHW programme registers and hence covered. Of the 272 officially appointed LHWs in the intervention clusters, only 228 (84%) were fully functional and working (18 did not engage with the project, 12 were non-residents of the area, eight had not received formal training, and six were transferred to the study areas during the study; table 3). During the intervention period, LHWs reported doing 6943 (80%) of the 8650 planned female health education sessions in intervention villages. At different periods, together representing almost a third of the overall length of the trial, LHWs were involved in local polio campaigns and mother and child activities, and were hence unavailable for trial-specific activities. 192 LHWs (71%) did more than 20 community sessions during the intervention period (table 3).Table 3LHW characteristics and programme performance n (%)LHWs in intervention clusters Transferred from other health-care facility 6 (2%) Non-resident 12 (4%) Uncooperative 18 (7%) Untrained 8 (3%) Functional 228 (84%) Health education sessions done by LHWs 0 8 (3%) 1–10 29 (11%) 11–20 43 (16%) >20 192 (71%) Neonates visited by individual LHWs 0 11 (4%) 1–33 147 (54%) 34–66 100 (37%) >66 14 (5%) N=272. Overall, 6943 health education sessions were done, and 8471 neonates were visited. LHW=lady health workers. Between February, 2010, and December, 2012, the actual period of trial intervention, 51 436 pregnancies were identified in the trial area (appendix). 2544 pregnancies (5%) ended in early fetal death (<28 weeks' gestation), 5143 women (10%) migrated out of the study area, and 5440 women (11%) were still pregnant at the end of the trial (appendix). We noted no significant differences between the intervention and control clusters in the proportions of facility births (53% vs 54%; p=0·73) and deliveries facilitated by skilled birth attendants (54% vs 56%; p=0·53; table 4). A significantly higher proportion of home deliveries were attended by LHWs in the intervention clusters than in the control clusters (14% vs 1%; p<0·0001; table 4). Clean delivery kits were used significantly more often for home deliveries in the intervention clusters than in the control clusters (p<0·0001; table 4).Table 4Post-intervention birthing practices and neonatal morbidity Control clustersIntervention clustersOR (95% CI)p valueHome births 9238/19 984 (46%) 8627/18 325 (47%) 1·04 (0·82–1·34) 0·73 Facility births 10 746/19 984 (54%) 9698/18 325 (53%) 0·96 (0·75–1·22) 0·73 Skilled birth attendant present 11 150/19 984 (56%) 9900/18 325 (54%) 0·92 (0·72–1·18) 0·53 Presence of LHW at deliveries managed by traditional birth attendants 55/8834 (1%) 1184/8425 (14%) 28·4 (14·3–56·4) <0·0001 Instrumental deliveries 3474/19 984 (17%) 3008/18 325 (16%) 0·94 (0·81–1·09) 0·42 Use of clean delivery kits at home births 1488/7723 (19%) 4236/7698 (55%) 6·16 (2·91–13·0) <0·0001 LHW postnatal visits 271/18 609 (1%) 5256/17 288 (30%) 32·3 (15·7–66·1) <0·0001 LHW early postnatal visits 188/18 609 (1%) 4318/17 288 (25%) 35·5 (17·9–70·7) <0·0001 Neonates with reported breathing problem or delayed cry at birth 2825/19 163 (15%) 2391/17 705 (14%) 0·90 (0·72–1·12) 0·34 Neonates with breathing problem resuscitated by LHW 2/2825 (<1%) 98/2391 (4%) 67·6 (14·2–320·5) <0·0001 Low-birthweight births 992/10 159 (10%) 933/10 125 (9%) 0·96 (0·76–1·22) 0·74 Neonates with reported illness 6626/19 163 (35%) 5439/17 705 (31%) 0·84 (0·61–1·14) 0·26 Care seeking for neonates with reported illness 6137/6626 (93%) 5037/5439 (93%) 1·02 (0·76–1·37) 0·89 Sick neonates visited by LHW 102/6626 (2%) 1566/5439 (29%) 27·2 (13·5–54·5) <0·0001 Neonates with possible infection 5201/19 163 (27%) 4350/17 705 (25%) 0·87 (0·66–1·16) 0·36 Neonates with possible infection seen by LHW 86/5201 (2%) 1252/4350 (29%) 25·1 (13·2–47·7) <0·0001 Neonates with possible infection managed with amoxicillin given by LHW 19/5201 (<1%) 707/4350 (16%) 54·6 (21·8–137·3) <0·0001 Data are n/N (%). OR=odds ratio. LHW=lady health worker. During the study period, rates of early fetal loss, although much lower than reported baseline values, were broadly similar in both groups (61 per 1000 pregnancies in intervention clusters vs 63 per 1000 pregnancies in control clusters; risk ratio [RR] 0·97, 95% CI 0·88–1·08; p=0·60; table 5).Table 5Summary of birth outcomes from the quarterly surveillance (rounds 1–11) by trial group Control clustersIntervention clustersMortality risk ratio*(95% CI)p valueEarly pregnancy loss n 1353 1191 .. .. Rate per 1000 known pregnancies 63 61 0·97 (0·88–1·08) 0·60 Stillbirths n 1066 830 .. .. Rate per 1000 total births 53 45 0·89 (0·76–1·04) 0·13 Early neonatal mortality n 871 610 .. .. Rate per 1000 livebirths 45 34 0·79 (0·67–0·93) 0·006 Late neonatal mortality n 179 126 .. .. Rate per 1000 livebirths 9 7 0·72 (0·62–0·85) 0·0001 Neonatal mortality n 1050 736 .. .. Rate per 1000 livebirths 55 42 0·80 (0·68–0·93) 0·005 Perinatal mortality n 1937 1440 .. .. Rate per 1000 total births 96 78 0·86 (0·75–0·99) 0·03 *Estimated with generalised estimating equations, controlling for baseline mortality, randomisation stratum, and surveillance round. Stillbirth rates were somewhat higher than those reported in the baseline survey, but again did not differ significantly between groups after baseline rates and randomisation were controlled for (RR 0·89, 95% CI 0·76–1·04; p=0·13; table 5; appendix). The neonatal mortality rate was lower in intervention clusters than in control clusters (42 vs 55 per 1000 livebirths; RR=0·80, 95% CI 0·68–0·93; p=0·005; table 5). In the first surveillance round after implementation of the intervention, neonatal mortality in both groups was similar, and thereafter consistently lower in the intervention clusters (appendix). An analysis of cluster-level summaries produced a broadly similar pattern of results, but with point estimates suggesting slightly larger intervention effects on mortality endpoints, particularly for stillbirths and perinatal mortality (appendix). Our study was not powered for cause-specific neonatal mortality outcomes and we identified no significant difference between the groups for major categories of neonatal mortality, including preterm birth complications, perinatal asphyxia, and neonatal infections, or for most causes of stillbirths, except for those related to obstructed labour and complications (p=0·003; appendix). Of all the livebirths in the intervention clusters, 2391 neonates reportedly had breathing problems or delayed cry at birth, 98 (4%) of whom were resuscitated by LHWs attending births (90 survived; appendix). 4350 neonates in intervention clusters had some features of suspected infections, including fast breathing, according to maternal reports (appendix). LHWs visited 1252 (29%) of these neonates, of whom 707 (56%) were managed with oral amoxicillin (appendix). 661 (93%) given amoxicillin survived (appendix). In control clusters, LHWs visited 82 (2%) of the 5201 neonates who had signs of suspected infections. 19 (23%) of the visited were managed with oral amoxicillin, 17 (89%) of whom survived (appendix). More women from intervention clusters (35%) than from control clusters (2%) reported visits by LHWs during the antenatal period (p<0·0001; table 6). Similar proportions of women from both groups sought antenatal care at least once during pregnancy and received two or more doses of the tetanus toxoid vaccine during the antenatal period (table 6). More women in the intervention arm than in the control arm were visited by the LHW within 3 days of delivery (29·9% versus 0·4%; p<0·0001). Breastfeeding within 1 h of birth, giving colostrum to neonates, and co-bedding or swaddling were significantly more common in intervention than in control clusters (table 6). Restriction of the analysis to functional LHWs and the covered areas only suggested that birth attendance, use of clean delivery kits, postnatal visits, and sick neonates seen were slightly higher than those in the intervention group overall, but rates of neonatal resuscitation were broadly similar (appendix). Broadly similar findings were noted in the restricted analysis comparing functional LHWs in the intervention clusters with overall findings in the intervention clusters (appendix). In this restricted analysis, 23% of neonates with possible infections were given amoxicillin (appendix).Table 6Household knowledge, attitudes, and practices around delivery at surveillance rounds 3, 5, 7, 9, and 11 Control clusters (n=10 859)Intervention clusters (n=10 118)OR (95% CI)p valueWomen attending at least one antenatal consultation 8568 (79%) 8413 (83%) 1·33 (0·96–1·82) 0·08 Women attending four or more antenatal consultations 2161 (20%) 2567 (25%) 1·41 (1·07–1·87) 0·01 Women visited by LHW during pregnancy 230 (2%) 3566 (35%) 29·0 (13·3–62·7) <0·0001 Women receiving two or more tetanus toxoid vaccination doses during pregnancy 1257 (12%) 1364 (14%) 1·15 (0·87–1·53) 0·32 Use of new blade for cutting cord 4608 (42%) 3984 (39%) 0·86 (0·69–1·09) 0·22 Use of cord clamp for tying cord 5703 (53%) 5186 (51%) 0·95 (0·76–1·19) 0·68 Cord cutting after placenta delivery 9653 (89%) 8980 (89%) 0·97 (0·63–1·50) 0·89 Dry cord care 1304 (12%) 1580 (16%) 1·35 (1·03–1·76) 0·03 Use of new cloth or clean towel for cleaning and drying neonate 6357 (59%) 6117 (61%) 1·07 (0·81–1·42) 0·61 Delayed bathing until after 6 h 6585 (61%) 6318 (62%) 1·08 (0·76–1·52) 0·68 Neonates warmed after birth 10 727 (99%) 10 021 (99%) 1·25 (0·85–1·83) 0·27 Mothers giving colostrum 6678 (61%) 7355 (73%) 1·77 (1·50–2·10) <0·0001 Mothers starting breastfeeding within 1 h 3079 (28%) 3956 (39%) 1·65 (1·26–2·16) 0·0002 Neonates receiving skin-to-skin contact with mother (co-bedding or swaddling) 4130 (38%) 4929 (49%) 1·55 (1·15–2·12) 0·005 Mothers' awareness to take appropriate* action for asphyxiated babies 1293 (12%) 1699 (17%) 1·50 (1·22–1·81) <0·0001 Mothers' awareness to seek care for low-birthweight babies 10 723 (99%) 9941 (98%) 0·74 (0·45–1·23) 0·25 Neonatal massage 10 105 (93%) 9633 (95%) 1·51 (1·10–2·08) 0·01 Mothers visited by LHW within 3 days of delivery 43 (<1%) 3022 (30%) 115·2 (57·7–230·1) <0·0001 Data are n (%). OR=odds ratio. LHW=Lady health worker. *Clean mouth and nose, rub back of baby, and give artificial respiration. BODY.DISCUSSION: In our cluster-randomised study of the effectiveness of an integrated community-based package comprising preventive and home-based immediate curative care delivered by public sector LHWs in in a programmatic setting in rural Pakistan, we noted a 20% reduction in neonatal mortality in intervention compared with control clusters. This 20% reduction is similar to the 15% reduction reported in an earlier trial, which did not include additional training of LHWs to resuscitate neonates at home as required or treat suspec15 This overall effect of the combined intervention was much smaller than we hypothesised at trial outset, and was lower than the reductions recorded in other cluster-randomised trials32, 33 in the region. However, those studies were mostly efficacy trials that directly employed, supervised, and remunerated community health workers, as opposed to true effectiveness studies done within existing public sector health systems with their inherent constraints. In the only effectiveness trial34 of scale-up of integrated management of neonatal and childhood illnesses, which was done through the public health system in India, a much smaller (9%) reduction in neonatal mortality was reported. The Newhints trial35 in Ghana, which was based on home visits by existing community-based volunteers, showed increases in the coverage of several essential newborn care behaviours, but did not significantly affect neonatal mortality. Our findings suggest limited additional benefits compared with basic promotive and preventive care of training public sector LHWs in Pakistan to resuscitate newborn infants delivered at home and treat suspected neonatal infections with oral antibiotics. They are also a stark reminder of the limitations to what busy public sector community health workers can deliver. In addition to community health workers' technical limitations, the effectiveness of such additional activities is dependent on the workload and range of other duties that such workers have in health systems. However, we think that the reduction in neonatal mortality shows the importance of community outreach services and the role of task shifting in reducing perinatal and neonatal mortality in such high-risk rural populations. Several key household behaviours related to maternal and newborn care improved in the households of the intervention clusters compared with control clusters, suggesting that community-based health promotion was effective. These findings are consistent with those noted in the previous trial of LHWs in Hala,15 and included an important increase in routine LHW visits to mothers in both the antenatal and postnatal periods and evidence of improved practices such as colostrum administration, early initiation of breastfeeding, and dry cord care. Our study design and operational plan had several strengths. First, the enhanced training programme was integrated with the district LHW programme and implemented like other regular trainings in the district health system. Training in newborn care was guided by specific learning objectives and accompanied by assessment of trainee performance and skills, which all LHWs passed. We achieved close engagement of the LHW programme through training and implementation through their trainers, monthly continuous education sessions (generally called monthly refresher trainings) at catchment facilities, and monitoring of intervention delivery by programme supervisors and district health officials. The regular refresher sessions were based on the LHW programme's standard protocols and well documented. Second, we used a detailed, independent data-collection system. We gathered data for outcomes and exposures through a quarterly active surveillance system via independent teams of data collectors. As part of the quality-assurance process, around 5% of the households were revisited by independent monitors within 3 days of the surveillance visits to corroborate findings. Trained programme managers also monitored LHW performance and activities with standard checklists and generated monthly summaries. Despite a few transfers of LHWs and some choosing not to participate, randomisation through the reporting facilities ensured no contamination between intervention and control clusters, although some sharing of messages between families and residents was inevitable. Our trial also had several limitations in view of its scale and the fact that implementation was largely dependent upon LHW functionality and availability. There was a substantial loss of working days and suspension of routine and project-specific mother and child health-care intervention activities as a result of LHWs' deployment in periodic mass polio immunisation campaigns. LHWs were frequently tasked with additional duties for immunisation activities such as measles campaigns and child health days. During the massive seasonal floods of 2010 and 2011,36 LHWs were deployed twice for several months to provide flood relief activities in the district. We estimate that almost 30% of LHW time was spent on such activities during the trial. We do not regard this as improper implementation; rather, we think it reflects the reality of busy public sector programmes and employees, who have to multitask. Attendance of childbirths by LHWs alongside traditional birth attendants, although considered feasible in the initial assessment and actively promoted, was a particular challenge. LHWs attended only a small proportion of home births. Stated barriers to attendance included the need to travel alone, restricted mobility at night, that these activities were additional to their routine activities, and the lack of remuneration by families (unlike traditional birth attendants, who get compensated directly by families). Information obtained by data collectors on household practices was based on maternal recall and not validated through direct observations, and the possibility of some respondent bias and over-reporting of recommended health-care activities cannot be ruled out. Owing to low maternal literacy and social taboos, a large proportion of mothers did not remember or report the exact date of their last menstrual period, and the possibility of differential misclassification of miscarriages and stillbirths cannot be excluded either. Our effectiveness trial included fewer newborn infants in the early neonatal period than did other efficacy trials21, 22, 29, 34 from the region, but was nonetheless associated with a significant reduction in neonatal mortality. The trial also showed that, notwithstanding several limitations, some LHWs could establish rapports with and work alongside local traditional birth attendants, including attending some births. The reduction in neonatal deaths was associated with an increase in antenatal care visits and the use of clean delivery kits at birth. However, the proportions of births attended by LHWs (14%) and potentially asphyxiated newborns resuscitated by LHWs during those visits (4%) were very small. The same was true for early postnatal home visits by LHWs (25%) and provision of amoxicillin (16%) to overtly sick neonates in the intervention clusters. Although the rates were substantially higher in intervention than in control clusters, overall intervention coverage for these morbidities by LHWs in the intervention clusters remained very low and no differences in cause-specific mortality as assessed by verbal autopsies were identified, although the study was not powered to assess such effects. We therefore cannot ascribe improvements in neonatal outcomes to individual intervention components related to home-based treatment. The choice of oral amoxicillin for treatment of suspected pneumonia based on clinical features including fast breathing was consonant with WHO guidance at the time,25, 26 although this recommendation for early neonatal pneumonia or infection has been challenged,37 and criteria for presumed serious bacterial infections in neonates have become more holistic.38 A significant difference from previous findings in Sindh15 was the lack of effect on facility births and stillbirths. Although we trained public sector staff across all facilities in the district at the beginning of the trial, notable differences from Hala were identified in terms of levels of staff motivation, quality of care, and the availability of adequate around-the-clock services in public sector facilities and the district headquarter hospital. This finding is important in view of the general benefits of referral and care seeking noted in community-based interventions for maternal and newborn care.20 Previous studies have shown that care for preterm and low-birthweight infants and newborn resuscitation can be delivered in home settings by motivated and well-trained community health workers,39 but outside of skin-to-skin care,29 few have shown effective management of preterm infants in domiciliary settings. We firmly believe that if packages of community-based maternal and newborn care are delivered through community health workers, appropriate measures to strengthen health systems, transport systems, and quality of care therein are also needed. In addition to community mobilisation and support, urgent attention is needed for the provision of adequate basic and emergency newborn care facilities in health facilities, accompanied by strengthening of quality care for preterm infants and infants with presumed serious bacterial infections.40, 41 Although the importance of community-based maternal and newborn care is well recognised, the limitations of this delivery platform in reducing neonatal mortality is also well appreciated. Growing evidence supports the provision of high-quality basic and emergency maternal and newborn care in referral facilities. Pakistan and other south Asian countries should prioritise both community-based strategies for promotive and preventive care and high-quality facility-based care as key measures to achieve universal health care and the Sustainable Development Goal 3 targets for maternal and newborn health.8

TITLE: Tourniquet use in total knee replacement does not improve fixation, but appears to reduce final range of motionA randomized RSA study involving 50 patients ABSTRACT.BACKGROUND AND PURPOSE: Although a tourniquet may reduce bleeding during total knee replacement (TKA), and thereby possibly improve fixation, it might also cause complications. Migration as measured by radiostereometric analysis (RSA) can predict future loosening. We investigated whether the use of a tourniquet influences prosthesis fixation measured with RSA. This has not been investigated previously to our knowledge. ABSTRACT.METHODS: 50 patients with osteoarthritis of the knee were randomized to cemented TKA with or without tourniquet. RSA was performed postoperatively and at 6 months, 1 year, and 2 years. Pain during hospital stay was registered with a visual analog scale (VAS) and morphine consumption was measured. Overt bleeding and blood transfusions were registered, and total bleeding was estimated by the hemoglobin dilution method. Range of motion was measured up to 2 years. ABSTRACT.RESULTS: RSA maximal total point motion (MTPM) differed by 0.01 mm (95% CI –0.13 to 0.15). Patients in the tourniquet group had less overt bleeding (317 mL vs. 615 mL), but the total bleeding estimated by hemoglobin dilution at day 4 was only slightly less (1,184 mL vs. 1,236 mL) with a mean difference of –54 mL (95% CI –256 to 152). Pain VAS measurements were lower in the non-tourniquet group (p = 0.01). There was no significant difference in morphine consumption. Range of motion was 11° more in the non-tourniquet group (p = 0.001 at 2 years). ABSTRACT.INTERPRETATION: Tourniquet use did not improve fixation but it may cause more postoperative pain and less range of motion. The use of a tourniquet during total knee replacement (TKA) is routine at many departments. It is believed to facilitate dissection and reduce peroperative bleeding, but the main argument for its use is that bleeding bone surfaces might impair the fixation of cemented prostheses, due to less cement penetration (Alcelik et al. 2011). On the other hand, a tourniquet is associated with reperfusion trauma and oxidative stress. There is also a risk of injury due to pressure on skin, muscle, nerves, and arteriosclerotic vessels, and a risk of deep vein thrombosis (Newman 1984, Irvine and Chan 1986, Silver et al. 1986, Abdel-Salam and Eyres 1995, Clarke et al. 2001, Olivecrona et al. 2006). Tourniquets reduce the surgical field, which might jeopardize sterility of the postoperative wound dressing. They are cumbersome to apply, especially in overweight patients. We have had the clinical impression that postoperative pain is to some extent due to the tourniquet. Because of these drawbacks of tourniquet use, our department stopped using tourniquets in 2003. After a short learning period, we perceived this to be more practical and to be associated with less postoperative pain. To our knowledge, a positive effect of tourniquet use on implant fixation has never been shown. However, this possibility meant that our new routine should be evaluated in a trial. Radiostereometric analysis is performed to measure early postoperative migration. It is generally believed that there is an association between early migration and the risk of late loosening (Nilsson and Karrholm 1996). This has been supported by one study (Ryd et al. 1995), and by theoretical reasoning. Thus, if a relevant difference in early migration between patients operated with or without a tourniquet could be excluded, it would be reasonable to assume that tourniquet use is not related to the quality of the fixation. No RSA data on the effect of tourniquet use are available in the literature. We performed a randomized controlled trial with blind evaluation to study the effects of tourniquet use. We used migration by RSA as the primary effect variable. BODY.PATIENTS AND METHODS: BODY.PATIENTS (:Figure 1) Figure 1.Flow diagram of participants. The study was approved by the Regional Ethics Committee in Linköping, reference number 2006/59-31. We included 50 patients (30 women) from the county of Östergötland, Sweden, who were on the waiting list for elective primary total knee surgery due to osteoarthritis. Sample size was estimated from a maximum total point motion (MTPM) mean difference of 0.2 mm with SD of 0.2 (95% CI and 80% power), which were considered relevant based on an earlier study (Hilding et. al 2000). All patients (Table 1) were operated in Motala, Sweden. Inclusion criteria were primary or secondary osteoarthritis without other severe disease (ASA 1–2). Exclusion criteria were inability to give informed consent, rheumatic arthritis, malignancy, coagulation disorder or medical treatment influencing the coagulation, liver disease, severe heart disease, or bilateral operation. The patients who declined to participate in the study were not registered. Table 1.Patient characteristi Tourniquet n = 25 No tourniquet n = 23 Men / women 10 / 15 9 / 14 Age, mean (SD) 70 (8) 71 (6) BMI, mean (SD) 29 (4.8) 28 (4.8) Preoperative ROM (°), mean (SD) 109 (17) 113 (17) Patients were allocated to treatment groups immediately before surgery by opening a sealed envelope. Randomization was stratified for sex and performed in blocks. Patients were not informed about tourniquet use, and care was taken not to disclose treatment allocation. The trial was performed in compliance with the Declaration of Helsinki. BODY.SURGERY: The operations were performed between June 2007 and April 2009 by HL in 34 cases and by 2 other surgeons in 15 cases. All operations were done under spinal anesthesia. We used the Nexgen CR all-poly tibia knee prosthesis (Zimmer), inserted after pulsed lavage, and cemented with Palacos R+G (Heraeus Medical Nordic, Sollentuna, Sweden) (40 g Palacos and 0.5 g gentamicin). 2 g cloxacillin was given intravenously just before and 3 times after the operation. Low–molecular-weight heparin (Innohep, 4,500 IE subcutaneously) was used for the first 14 postoperative days. For RSA measurements, altogether 12 tantalum beads (0.8–1 mm) were inserted in the all-poly tibial component and the tibial bone metaphysis. We used a standard tourniquet (Stille AB, Solna, Sweden) 110 mm wide, which was inflated to 275 mmHg during the entire operation if the patient was randomized to tourniquet use. Postoperative paracetamol and morphine analogs were given according to a standard protocol for pain management, with addition of extra morphine analogs on demand. BODY.EVALUATION: The radiostereometric X-ray examinations were performed 3 days after surgery, and after 6 months, 1 year, and 2 years. The blind analysis of the RSA data was done using the UmRSA computer program (RSA Biomedical AB, Umeå, Sweden). Pain was assessed by visual analog scale (VAS) at 8 time points during the hospital stay (at 0800 h, 1400 h, and 2000 h on the first 2 postoperative days and at 0800 h and 1400 h on the third postoperative day). The mean value of all these VAS values was used for analysis. ROM was measured before surgery and after 3 days, 4 days, 6 weeks, 3 months, 6 months, 1 year, and 2 years. These measurements were performed by a physiotherapist who did not know whether a tourniquet had been used. Blood loss was measured during surgery by weighing the surgical sponges and by subtracting the amount of irrigation fluid used from the content of the suction drain. Postoperative overt bleeding volume (in the drains) was estimated by measuring the hemoglobin content of the drains in relation to the blood hemoglobin concentration. Total blood loss was estimated by the hemoglobin dilution method (Lisander et al. 1998) based on blood volume according to (Nadler et al. 1962). Postoperative use of morphine-analog drugs was described as morphine equivalents for the 4 first days (including the day of surgery). BODY.STATISTICS: The primary effect variable was migration measured by RSA (MTPM) after 2 years. Secondary effect variables were other RSA outcomes, pain assessment during hospital stay, range of movement up to 2 years after surgery, and blood loss. Because the RSA MTPM values were far from normally distributed, we used the Mann-Whitney test and calculated a non-parametric confidence interval for the difference between group medians using the Hodges-Lehmann estimate (SPSS 19.0 software). Pain was analyzed as the sum of measurements as described above, using the Mann-Whitney test. Bleeding, morphine consumption, and range of motion appeared normally distributed and were analyzed with Student’s t-test. The 2-year result was regarded as final, and is given more emphasis. The gain in range of motion (from preoperatively to 2 years postoperatively) was dependent on range of motion before surgery, and was therefore analyzed by analysis of covariance (ANCOVA) using preoperative range of motion as covariate. BODY.RESULTS: BODY.PRIMARY VARIABLE: MTPM at 2 years showed a skewed distribution, with a few patients having considerable migration (Figure 2). There was no statistically significant group difference, and the non-parametric 95% confidence interval (CI) for the difference between group medians ranged from – 0.13 to 0.15 mm (Figure 3). The number of outliers, defined as having a 2-year MTPM of more than 1 mm, was 4 for the tourniquet group and 2 for the non-tourniquet group. Figure 2.The MTPM values (mm) at 2 years show a skewed distribution with outliers. Figure 3.MTPM (mm) at 1 year, 2 years, and 1–2 years (log scale). Note that migration for 1–2 years is not the same as migration for 2 years minus migration for 1 year, as MTPM has no defined direction. BODY.SECONDARY VARIABLES: None of the other RSA variables at 6 months, 1 year, and 2 years (x-, y-, and z-translation and rotation) showed a statistically significant difference (Table 2). Table 2.RSA variables at 6 months, 1 year, and 2 years (x–, y–, z–translation and rotation). No variables showed a significant difference Translation Rotation x y z x y z mean SD mean SD mean SD mean SD mean SD mean SD 6 months T –0.03 0.09 –0.09 0.22 0.06 0.10 0.05 0.38 0.01 0.23 0.01 0.22 NT –0.01 0.13 –0.11 0.31 0.10 0.15 0.05 0.64 0.01 0.21 0.08 0.79 1 year T –0.03 0.08 –0.10 0.25 0.07 0.09 0.02 0.39 0.01 0.23 0.06 0.25 NT –0.02 0.15 –0.12 0.27 0.11 0.15 –0.09 0.69 0.01 0.21 0.03 0.99 2 years T –0.14 0.46 –0.06 0.27 0.05 0.17 –0.21 1.51 –1.60 8.19 0.01 0.73 NT –0.07 0.22 –0.15 0.42 0.16 0.33 0.05 0.71 0.07 0.33 –0.56 1.33 T: tourniquet; NT: no tourniquet. VAS Pain values during the first 4 days were higher in the tourniquet group (median 49 vs. 41 mm; p = 0.01) (Figure 4). Non-parametric 95% CIs for the difference between group medians ranged from 2.5 to 15. Figure 4.Pain during the first 4 days, expressed as the mean of all consecutive measurements. Total overt bleeding was less in the tourniquet group (317 vs. 615 mL; p = 0.002) (Figure 5). 4 patients in the tourniquet group and 3 in the non-tourniquet group needed blood transfusions. As estimated by the hemoglobin dilution method, the total blood loss was 1,184 (SD 346) mL in the tourniquet group and 1,236 (SD 349) mL in the non-tourniquet group (95% CI for the difference between means: –256 to 152 mL). Figure 5.Peroperative bleeding. Figure 6.Blood loss (mL) according to the hemoglobin dilution method. Morphine consumption did not show any statistically significant group difference. The 95% CI for the difference between means (tourniquet minus no tourniquet) ranged from –31 to 18 mg morphine equivalents. This should be related to the average total use of 218 mg. 1 patient in the tourniquet group needed mobilization under anesthesia at 6 weeks, and range of motion was not measured thereafter. Complete data for range of motion were available for 21 patients in the tourniquet group and 22 patients in the non-tourniquet group. Range of motion, expressed as a sum of all measurements, was less in the tourniquet group (p = 0.01). The range of motion at 2 years was still less in the tourniquet group (tourniquet: mean 113°; non-tourniquet: mean 124°; 95% CI for difference between means: 5–17°; p = 0.001) (Figure 7). Patients with a small range of motion preoperatively had gained more in range at 2 years. The gain, after correction for preoperative status, was dependent on tourniquet treatment. This gain was statistically significant after correction for preoperative range of motion (p = 0.001, ANCOVA). Figure 7.Range of motion. BODY.CLINICAL RESULTS: Mean operating time was 85 (SD 30) min for the tourniquet group and 81 (SD 23) min for the non-tourniquet group. Mean hospital stay was 4.9 and 4.5 days, respectively. 2 patients in the non-tourniquet group were excluded from analysis, 1 patient because of deviation from the protocol during surgery and afterwards (femoral nerve block, no drain) and 1 patient because of a hematological malignancy. She was in remission at the time of inclusion, but had a relapse requiring cytostatic treatment. The RSA data were complete for all remaining patients except one at the 2-year follow-up (no tourniquet) (Figure 1). After the study period, 1 patient in the non-tourniquet group had his prosthesis revised because of loosening. This patient showed gross migration already at 1 year. 1 patient in the tourniquet group also showed gross migration, but at the time of writing there have been no clinical symptoms of loosening. BODY.DISCUSSION: Our RSA data do not support the view that tourniquet use will improve fixation. We found no statistically significant effect on prosthesis migration. The confidence interval excludes an increase in median value of 0.13 mm if a tourniquet is not used. Such a small difference is not likely to have any clinical importance. However, the distribution of the migration values was far from normal, and there were a number of outliers. These outliers are probably patients who may be at risk of future loosening (and one of these has already undergone revision). Indeed, RSA data may often show discontinuous distributions, reflecting distinct subcategories of the postoperative course (some are well fixed and others not) (Aspenberg et al. 2008). In this study, there were similar numbers of outliers in both groups. Apart from prosthesis fixation, bleeding is generally considered to be a good reason for tourniquet use. However, the possible advantage of less bleeding should be balanced against the disadvantage of increased postoperative pain and a smaller final range of motion. We found a reduction in overt bleeding in the tourniquet group, as has been described in a recent meta-analysis (Alcelik et al. 2011). However, there was no substantial difference in total bleeding as estimated by the hemoglobin dilution method, and any increase exceeding 22% from lack of tourniquet use could be excluded with 95% confidence. It is conceivable that the hidden, postsurgical blood loss is less in patients in whom the surgeons have been able to identify and cauterize bleeding vessels. The main reasons for refraining from tourniquet use, as judged by our results, are postoperative pain and range of motion. Before the study, we had the impression of a clear reduction in the patients’ complaints about thigh pain after we stopped using a tourniquet. Although patients reported more pain in the tourniquet group, there was no statistically significant difference in the use of morphine-analog analgesics. This may reflect that the difference between the groups was only minor, so that most patients’ pain was controlled by standard postoperative analgesics given to all patients. The range of motion may be more important: the difference between 113° and 124° may mean the difference between being able to ride a bicycle or not. There have been very few previous randomized trials on tourniquet use and range of motion. Short-term better flexion has been shown in 2 studies (Abdel-Salam and Eyres 1995, Wakankar et al. 1999), but neither publication reported a difference between treatment groups in the longer term. Total range of motion was not reported. The mechanism behind the reduced range of motion is unclear. However, the postoperative thigh pain might reflect muscle injury due to physical damage, as well as reperfusion injury, which might both cause a degree of muscle fibrosis. The increased pain might also reduce the patient’s ability to perform postoperative training, which has been shown to negatively affect the final range of motion (Newman 1984, Silver et al. 1986, Clarke et al. 2001). The main weaknesses of our study lie in the limitations of the RSA method to predict clinical outcomes, in spite of the fact that RSA studies are recommended before new joint prosthesis are commercially deployed (Derbyshire et al. 2009). The study size is sufficient for construction of relevant confidence intervals, but not for analysis of the number of outliers in the respective groups. This must be considered. The findings regarding range of motion and pain—although highly statistically significant—should be interpreted with caution, as they were not primary variables. Estimates of bleeding have a high degree of uncertainty, as the number of patients may be too small. In conclusion, our RSA data do not support the use of a tourniquet to improve fixation. On the contrary, tourniquets appear to cause more postoperative pain and less range of motion.

TITLE: A randomized controlled trial of a group-based gaze training intervention for children with Developmental Coordination DisorderGaze training for children with DCD The aim of this study was to integrate a gaze training intervention (i.e., quiet eye training; QET) that has been shown to improve the throwing and catching skill of children with Developmental Coordination Disorder (DCD), within an approach (i.e., group therapy) that might alleviate the negative psychosocial impact of these motor skill deficits. Twenty-one children with DCD were split into either QET (8 male 3 female, mean age of 8.6 years (SD = 1.04) or technical training (TT) groups (7 male 3 female, mean age of 8.6 years (SD = 1.84). The TT group were given movement-related instructions via video, relating to the throw and catch phases, while the QET group were also taught to fixate a target location on the wall prior to the throw (QE1) and to track the ball prior to the catch (QE2). Each group partook in a 4-week, group therapy intervention and measurements of QE duration and catching performance were taken before and after training, and at a 6-week delayed retention test. Parental feedback on psychosocial and motor skill outcomes was provided at delayed retention. Children improved their gaze control and catching coordination following QET, compared to TT. Mediation analysis showed that a longer QE aiming duration (QE1) predicted an earlier onset of tracking the ball prior to catching (QE2) which predicted catching success. Parents reported enhanced perceptions of their child’s catching ability and general coordination in the QET group compared to the TT group. All parents reported improvements in their child’s confidence, social skills and predilection for physical activity following the trial. The findings offer initial support for an intervention that practitioners could apply to address deficits in the motor and psychosocial skills of children with DCD. Trial registration: ClinicalTrials.gov NCT02904980 BODY.INTRODUCTION: Developmental coordination disorder (DCD) is a condition estimated to affect around 6% of children [1]. The condition is categorised as a marked impairment in the development of motor coordination that interferes with activities of daily living below the level expected for the child’s chronological age, which must not be attributable to neurological conditions, sensory problems or low intelligence [2]. While the aetiology of DCD is still poorly understood, children with DCD suffer motor deficits related to internal (forward) modelling, rhythmic coordination, executive function, gait and postural control, catching and interceptive action, and sensoriperceptual function [3]. In an effort to understand more about the mechanisms behind such deficits, researchers have explored the role and control of vision in children with DCD, compared to typically developing (TD) children. A body of evidence has linked DCD to significant impairments in general visuomotor control and the processing of task-relevant, visual information [3]; the ability to use predictive information to guide action [4]; the pursuit tracking of objects [5]; and the ability to maintain fixation on visual targets [6]. While some of this research is laboratory-based, these deficits in the control of vision have obvious implications for the production and control of coordinated movement in the ‘real-world’. For example, the ability to maintain a fixation on a visual target and track an object is fundamental for aiming and interception skills that are the building blocks for activities in sport and playground games. Throwing and catching is a perfect example of a task where these visual abilities are critical and it is unsurprising that children with DCD find this task difficult. In a recent study [7], we explored the visual control of children in a task that required them to throw a ball against a wall and subsequently catch it. In this study we measured–using a measure of optimal visual control derived from sport (the quiet eye (QE) [8])—the duration of time children spent fixating a target before throwing (QE1) and the duration of time spent pursuit tracking the ball prior to the catch attempt (QE2). These QE durations provide periods of pre-programming that assist with thrower’s predictions of the location of the bounce point on the wall and subsequent location and timing of the interception point of the catch [7,9]. Results suggested that highly proficient children demonstrated longer QE aiming fixations (QE1) before the release of the ball and longer QE pursuit tracking durations (QE2) on the ball prior to the catch. Mediation analyses revealed that the superior performance of the high motor proficient children was underpinned by an earlier and longer QE2 before the catch attempt [7]. Interestingly, in further studies we have demonstrated that teaching participants to adopt the QE strategy of skilled children through observing video footage of their eye-movements (QE training; QET), improved catching technique in children with average motor skill ability [9,10] and children with DCD [11]. In the latter study, DCD children who were given a brief QET intervention experienced significant improvements in their catching coordination and catching kinematics. Moreover, these benefits were maintained even after a 6-week detraining period. A control group, who received typical movement-focused instructions, revealed no improvement in catching technique at the 6-week delayed retention test [11]. The authors concluded that QET served to improve the attentional control of these children, providing more optimal aiming behaviour (QE1) and more time to track the early flight phase of the ball as it came towards them (QE2). As impairments in the ability to maintain target-focused fixations and pursuit tracking of objects has been shown to be characteristic of DCD [6], we believe that QET, a strategy that has been shown to teach this form of top-down attentional control [8], may be effective in the treatment of this condition. The motor deficiency associated with DCD also has severe consequences for the psychosocial development and wellbeing of the children who suffer from it. For example, children with DCD report being excluded (by either their peers or themselves) from partaking in physical activities or playground games [12]. This exclusion has an additive negative effect on their psychosocial development, resulting in children with DCD experiencing loneliness, victimisation and feelings of rejection by their peers [13] and lower levels of confidence, self-esteem and self-worth [14]. In an attempt to alleviate the tendency for social exclusion, studies have examined the effectiveness of group-based training interventions for children with DCD. Group-based therapy might not only be more effective than individual-based therapy in improving motor skills in children with DCD [15], but may also improve psychosocial variables [13]. Furthermore, not only is group therapy more time efficient and cost effective for the therapist, parents of children with DCD emphasise the importance of their children being able to participate in organised, physical activity groups and value therapy that improves their child’s perceptions of self-confidence and competence over those that just focus on improving motor abilities [16]. These views have led to suggestions that therapeutic interventions should therefore focus on enhancing the social, as well as the physical, skills of children with DCD [17]. The aims of this study were two-fold. First, we sought to build upon Miles et al.’s positive findings for QET for children with DCD [11] by adopting multiple sessions of training, in an attempt to increase the training effect and to more closely resemble typical therapy. We hypothesised that QET would significantly improve the catching performance of children with DCD compared to a group receiving typical, technical instructions by improving their visuomotor control. Specifically, we hypothesised that QET would facilitate more optimal aiming behaviour (longer aiming durations prior to the throw; QE1) and quicker and longer tracking durations of the ball prior to the catch (QE2). Furthermore, we hypothesised that these visuomotor improvements for the QET group would mediate the predicted performance advantage in catching for QET children. Specifically, mediation analysis should reveal that a longer QE aiming duration (QE1) predicts an earlier onset of tracking the ball prior to catching (QE2) which predicts catching performance [9]. A second aim was to explore the feasibility and effectiveness of using a group-based therapy approach as a framework for the delivery of this intervention. We hypothesised that regardless of the intervention received, parents would report that their children found the intervention to be more beneficial than typical, individual, therapy and would consequently report increases in their children’s confidence, enjoyment and predilection for sport or physical activity. BODY.METHODS: BODY.PARTICIPANTS: Twenty-one children, aged 7–11 years old, were recruited from local DCD support groups, social media, and local occupational therapy centres in the North West of England. Recruitment and follow-up took place between February 2015 and September 2015. All children scored below the 5th percentile on the Movement Assessment Battery for Children-2 [18] (MABC-2) carried out at the baseline testing phase. Using each child’s baseline score from the MABC-2 throwing and catching task, two randomised groups were formulated, by the second author. The QET group (8 males and 3 females) and the TT group (7 males and 3 females) were matched for age and MABC-2 score in a parallel group design (Table 1). 10.1371/journal.pone.0171782.t001Table 1BODY.DEMOGRAPHIC AND CLINICAL CHARACTERISTICS (MEANS AND SD) OF THE QUIET EYE TRAINING (QET) AND TECHNICAL TRAINING (TT) GROUPS.: QETTTpAge (yrs.) 8.55 (1.04) 8.60 (1.84) .993 MABC-2% 2.55 (2.09) 1.93 (2.19) .552 ADHD % 87.91 (17.10) 90.50 (14.70) .721 Children and their parents were blinded to their intervention allocation. Parents also completed the Attention Deficit/Hyperactivity Disorder (ADHD) Rating Scale-VI [19] prior to testing. No child scored above the 98th percentile for inattention or hyperactivity, which is recommended to be the minimum cut-off used as an indication of ADHD in research [19]. All children were classified as of ‘normal’ intelligence based on their teacher/parent reports. UK NHS ethical approval (15/NW/0279) was granted by the RES Committee North West—Greater Manchester South, before any testing was carried out, and parents and children provided written informed consent before taking part. Fig 1 shows a CONSORT flow diagram outlining participant recruitment and analysis (see also S1 Fig CONSORT checklist and the original study protocol in S1 File). Due to an oversight, the study protocol was not registered as a clinical trial before enrolment of participants but the authors confirm that all ongoing and related trials for this intervention are registered (ClinicalTrials.gov Identifier: NCT02904980). 10.1371/journal.pone.0171782.g001Fig 1BODY.A CONSORT FLOW DIAGRAM OUTLINING PARTICIPANT RECRUITMENT AND ANALYSIS.: BODY.THROWING AND CATCHING TASK: The throwing and catching task from the MABC-2 (8–10 year age bracket) was used to aid comparison with previous studies [8, 9, 10, 11]. It requires participants to stand behind a line situated 2 metres from a wall, throw a tennis ball against the wall using an under-arm action, and catch it before it bounces. In line with MABC-2 instructions, the task was first explained to the participant and demonstrated once, before the participant took five practice attempts. BODY.APPARATUS: Each participant was fitted with an Applied Science Laboratories’ Mobile Eye XG gaze registration system (ASL, Bedford, MA), measuring point of gaze at 30 Hz. A 30 Hz Digital SLR camera (Finepix S6500fd) was placed on a tripod 2 metres to the right of the throw line at shoulder height of the participant. This captured a side-on view (sagittal plane) of the participant’s movements during the throw and catch action. Additionally, children were fitted with four upper limb 3D inertial motion capture sensors (two on each arm) and 11 surface EMG electrodes to the upper and lower body muscles (Noraxon, USA). The analyses of detailed kinematic and muscle activity data will be submitted in a separate paper. BODY.PROCEDURE: Testing was divided into baseline, training, retention and delayed retention phases. During the baseline phase, children attended the laboratory individually, and following the completion of written consent, completed the MABC-2 protocol. Prior to completing the throwing and catching task, each participant was calibrated to the eye-tracker using nine locations on the wall to which he/she were required to throw the ball against. They then completed five blocks of 10 trials of the MABC-2 throw and catch task. Participants were then pseudo-randomly divided into two experimental groups based on this initial throwing and catching performance. The procedure for the retention and delayed retention phases replicated the baseline phase. The retention phase occurred 1 week after the end of the training phase (week 6) and the delayed retention phase took place 6-weeks after this point. During the delayed-retention phase, parents completed the parental feedback questionnaire while their child completed the throwing and catching task procedure. All parents and children were blind to their group allocation. BODY.TRAINING PROTOCOLS: The training phase consisted of each group attending the university sports hall on separate days during four weekly (1hr) group sessions (See Table 2). At the beginning of each session, each group watched a brief instructional video showing the same expert model completing the throwing and catching task. Both videos provided a split screen of the model showing a side-on view of their movement and a first person view taken from the eye tracker, showing the point of gaze while performing the task. The videos were edited to reinforce the different training instructions. The TT group videos highlighted the movement of the expert model whereas the QET group videos highlighted the gaze footage of the same expert model [11] (see S2 File). The individual in this video has given written informed consent (as outlined in PLOS consent form) to publish these case details. 10.1371/journal.pone.0171782.t002Table 2BODY.A WEEK-BY-WEEK BREAKDOWN OF THE TRAINING ACTIVITIES AND INSTRUCTIONS FOR QUIET EYE TRAINED (QET) AND TECHNICALLY TRAINED (TT) GROUPS.: WeekActivitiesQET InstructionsTT InstructionsWeek 1 –Accurate Throwing • 20 warm up MABC-2 throw and catches Focus your eyes on the target and count to two before you start a smooth throwing action Throw at a target using a smooth throwing action. • Watch instructional video for throwing • Target-related activities (throwing bean bags into buckets, throwing balls at cricket stumps, throwing at cut-out faces stuck on a wall). • Started at short distances and increase distance based on individual success • Competitive team games using the same tasks • De-brief and reinforced instructions Week 2 –Effective catching • 20 warm up MABC-2 throw and catches. Keep your eye on the ball until it comes back into your cupped hands Concentrate on the ball and cup your hands together. • Watch instructional video for catching • Catching-related activities (catching large sponge balls and beanbags, catching with a bucket instead of their hands, catching while moving around). • Varied distance and speed of the catch • Competitive team games using the same tasks. • De-brief and reinforced instruction Week 3 –Linking throwing and catching • 20 warm up MABC-2 throw and catches Questioning on previous instructions and instruction on combining coaching points together Questioning on previous instructions and instruction on combining coaching points together. • Watch instructional video linking the throw and catch • Throwing and catching tasks (throwing and catching between participants while walking around, throwing and catching a ball along a chain, rounders with a larger sponge ball where children hit the ball with their hands rather than a bat) Week 4 –Throwing and catching competitive games • 20 warm up MABC-2 throw and catches Questioning on previous instructions and reiteration of coaching points Questioning on previous instructions and reiteration of coaching points • Children chose their favourite games from the sessions and were prompted to remember the related coaching points After watching each video participants were questioned regarding its content to check their understanding. Participants then completed 20 ‘warm-up’ trials of the MABC-2 throwing and catching task in unison, while coaches reinforced the respective coaching instructions. Throughout the intervention both groups took part in exactly the same throwing, catching and related interception type activities but the instructions that were emphasised by the coaches were different for each group. In short, the TT group received technique-based instructions related to the mechanics of the skill, taken from a UK physical education resource [20]. The QET group received instructions that were related to controlling their eye movements so that they tracked the ball for longer [9,10,11]. BODY.MEASURES: BODY.QUIET EYE: QE measures were analysed using Quiet Eye Solutions software (www.quieteyesolutions.com). QE1 was defined as a targeting fixation located on the target on the wall (that remained within 1° of visual angle for more than 100ms) prior to and during the throw phase of the task. Onset of QE1 was defined as the final fixation duration prior to the initiation of foreswing of the throwing arm and offset occurred when gaze deviated off the target by more than 1° of visual angle for longer than 100ms [11]. QE2 was defined as the final tracking gaze on the tennis ball after it rebounded from the wall during the catch phase of the task. QE2 onset was defined as the start of the final tracking gaze on the ball (for more than 100 ms) before the grasping action was attempted, or the trial ended. QE2 offset occurred when the tracking gaze deviated off the ball for more than 100ms [11]. BODY.CATCHING PERFORMANCE: Catch success was scored using the sagittal motor video footage, and expressed as the total number of balls successfully caught out of 50 attempts. A measure of catching quality was also determined from the video footage providing a more sensitive measure of catching performance. Whereas catch success is binary (only a perfect catch can be considered successful), a technique score recognises differences in the quality of the attempt to catch, and awards more points to better attempts. For example, using the binary catching success measure, children who make no reaction to the ball as it comes back to them are awarded the same score (0) as a child who fumbles the ball or manages to catch it by trapping it against their arms. The catching performance scale recognises and quantifies these distinct differences in catch attempts. The catching performance scale (Table 3 [11]) consisted of an 11-point scale whereby catch attempts were given a score between ‘0’ (Makes no move towards the ball as it comes back) and ‘10’ (The catch is made exclusively with the palms and fingers). A second blinded researcher scored 10% of the trials to check for inter-rater reliability (92%). 10.1371/journal.pone.0171782.t003Table 3BODY.THE QUALITATIVE CATCHING PERFORMANCE SCALE.: OutcomeCodeDescriptionNo reaction 0 Makes no move towards the ball as it comes back Reaction, no contact 1 Makes some move towards ball, no contact, no attempt at a catch (delayed) Inaccurate/delayed reaction, no contact 2 Reacts to ball direction and makes effort to catch the ball. Delayed reaction, no contact before bounce 3 Reactions to ball direction and makes effort to catch the ball. Ball bounces/contacts some part of the body Delayed reaction, limited contact 4 Reacts to the ball, poor throw results in it bouncing/contacting another surface before catch can be made Ball contacts hands 5 The ball contacts one or both hands but there is no control Trap ball, no hands 6 Ball hits body and trapped with arms but not hands Fumble 7 Ball is fumbled and drops to the ground Trap 8 The ball is grasped by both hands, with the aid of the trunk or other body part Fumble but re-grasped 9 Clean catch completed after a fumble without ball hitting another surface Clean controlled catch 10 The catch is made exclusively with the palms and fingers BODY.PARENTAL QUESTIONNAIRE: The questionnaire consisted of two parts. Part I consisted of five 5-point Likert scale questions (1 = not at all; 3 = somewhat; 5 = very much so) relating to motor skill improvements, confidence and changes in their child’s predilection for physical activity. Part II consisted of three short answer open-ended questions that asked parents to document any changes they may have noticed post training; to list any aspects of the training that their child particularly enjoyed; and a final question that asked for any further comments about any aspect of the intervention. BODY.DATA ANALYSIS: All statistical tests were analysed using IBM SPSS version 22. Based on per-protocol analysis approach, QE and performance variables were subjected to mixed design 2 (Group: QET vs TT) x 3 (Phase: baseline, retention and delayed retention) ANOVA. Effect sizes were reported using partial eta squared statistics and sphericity assumptions were not violated for any variable. Based on our hypotheses, we focus our discussion on significant interactions (when found), which were followed up using repeated measures ANOVAs and Bonferroni corrected pairwise comparisons where appropriate. Mediation analyses were computed using the MEDIATE SPSS custom dialog [21]. For this, and in line with theorising by Miles et al [9], delayed retention catching success was entered as the dependant variable and QE1 duration was entered as the independent variable. QE2 onset was entered as the proposed mediator. This process allows inferences to be made about the indirect effects using percentile bootstrap confidence intervals. Parental questionnaire data (Part I) were analysed using Mann-Whitney U tests and 95% confidence intervals. Content analysis was used to analyse the free response statements (Part II) from the open-ended questions [22]. This involved two researchers reading each parental response and categorising key themes that occurred. Themes were then combined into five high-order themes through a process of discussion, before quantifying the frequency of these responses. BODY.RESULTS: Three participants (2 QET and 1 TT) dropped out of the study and failed to complete the delayed retention test. Consequently, three parents (2 QET and 1 TT) also failed to complete the parental questionnaire at delayed retention. BODY.QUIET EYE: A significant interaction, F(2,32) = 5.06, p = .012, ηp2 = .24, was found for QE1 duration. Post hoc t-tests, with an adjusted alpha value for multiple comparisons (0.05/3 = .0167), revealed no significant difference (p = .486) between QET (M = 241.09, SD = 134.48ms) and TT (M = 182.99, SD = 87.21ms) groups at baseline. However, the QET group had significantly longer (p < .001) QE1 aiming durations (M = 594.15, SD = 150.09ms) at retention compared to the TT group (M = 297.80, SD = 176.67ms). The significant difference between groups was maintained at delayed retention test where the QET group exhibited significantly longer (p < .001) QE1 aiming durations (M = 592.56, SD = 136.75ms) compared to the TT group (M = 269.61, SD = 149.21ms; see Fig 2 top). 10.1371/journal.pone.0171782.g002Fig 2BODY.MEAN (S.E.M.) QE1 DURATION (MS; TOP), QE2 ONSET (MS; MIDDLE) AND QE2 DURATION (MS; BOTTOM) DATA FOR QUIET EYE TRAINING (QET) AND TECHNICAL TRAINING (TT) GROUPS ACROSS BASELINE (QET :n = 11, TT n = 10), retention (QET n = 11, TT n = 10), and delayed retention tests (QET n = 9, TT n = 9). A significant interaction was also found for QE2 onset, F(2,32) = 4.97, p = .013, ηp2 = .24. Post hoc t-tests, with an adjusted alpha value for multiple comparisons (0.05/3 = .0167), revealed no significant difference (p = .878) between QET (M = 244.57, SD = 71.26ms) and TT groups (M = 244.84, SD = 48.81ms) at baseline. However, the QET group had significantly earlier (p = .005) QE2 onsets (M = 120.81, SD = 70.19ms) at retention compared to the TT group (M = 201.54, SD = 75.90ms). The significant difference between groups was maintained at delayed retention test where the QET group exhibited significantly earlier (p = .003) QE2 onsets (M = 102.36, SD = 44.59ms) compared to the TT group (M = 207.75, SD = 79.23ms; see Fig 2 middle). A significant interaction was also found for QE2 duration, F(2,32) = 3.44, p = .045, ηp2 = .18. Post hoc t-tests, with an adjusted alpha value for multiple comparisons (0.05/3 = .0167), revealed no significant difference (p = .484) between QET (M = 142.49, SD = 47.29ms) and TT (M = 128.39, SD = 32.71ms) groups at baseline. However, the QET group had significantly longer (p = .001) QE2 durations at retention (M = 236.85, SD = 43.30ms). compared to the TT group (M = 156.96, SD = 67.65ms). The difference between QET (M = 253.73, SD = 55.18ms) and TT (M = 162.14, SD = 92.67ms) groups at delayed retention tests failed to reach adjusted levels of significance (p = .022, see Fig 2 bottom). BODY.CATCHING PERFORMANCE: For catch success, a significant main effect was found for test, F(2,32) = 44.63, p < .001, ηp2 = .74, indicating that both groups caught significantly more balls from baseline (M = 8.17, SD = 8.42) to retention (M = 25.44, SD = 12.63) test (p < .001) and maintained this improvement (M = 28.44, SD = 913.45) at delayed retention (p = .351). The interaction effect was not significant, F(2,32) = 2.38, p = .108, ηp2 = .13, (Fig 3 top). 10.1371/journal.pone.0171782.g003Fig 3BODY.MEAN (S.E.M.) NUMBER OF CATCHES (0–50; TOP) AND CATCHING PERFORMANCE SCORE (0–10; BOTTOM) FOR QUIET EYE TRAINING (QET) AND TECHNICAL TRAINING (TT) GROUPS ACROSS BASELINE (QET :n = 11, TT n = 10), retention (QET n = 11, TT n = 10), and delayed retention tests (QET n = 9, TT n = 9). As the qualitative catching scores were not normally distributed, these data were subjected to natural log transformations prior to analysis. From this, a significant interaction, F(2,32) = 3.89, p = .031, ηp2 = .20, was found. A one way ANOVA revealed no significant between-group differences at baseline (p = .413; QET: M = 4.10, SD = 1.58 vs. TT: M = 3.73, SD = 2.02) or retention (p = .236; QET: M = 6.54, SD = 2.06 vs. TT: M = 5.45, SD = 2.30) but the QET group had significantly higher (p = .032) qualitative catching scores at delayed retention (QET: M = 8.07, SD = 1.61 vs. TT: M = 5.53, SD = 3.01). Within-group post hoc t-tests, with an adjusted alpha value for multiple comparisons (0.05/4 = .0125), revealed that the TT group did not significantly improve from baseline to retention (p = .028) or from retention to delayed retention (p = .529). The QET group significantly improved from baseline to retention (p < .001) and maintained this improvement (p = .014) at delayed retention (Fig 3 bottom). BODY.MEDIATION ANALYSIS: Results from mediation with bootstrapping (based on 10,000 sampling rate) indicated that there was a significant indirect effect for QE2 tracking onset (95% confidence interval = 0.01–0.06) in mediating the effect between QE1 aiming duration and catch success. BODY.PARENTAL QUESTIONNAIRE: BODY.LIKERT QUESTIONS (PART I): Significant group-based differences were found in the degree to which parents felt their child’s motor coordination had improved, supporting the objective performance data (see Table 4). 10.1371/journal.pone.0171782.t004Table 4BODY.PARENTAL RESPONSES GIVEN TO (PART I) LIKERT SCALE QUESTIONS (MEANS AND SD) AND (PART II) THE OPEN-ENDED QUESTIONS (% FREQUENCY OF PARENTS WHO MADE STATEMENTS) BETWEEN QUIET EYE TRAINING (QET) AND TECHNICAL TRAINING (TT) GROUPS.: QETTTCIPart I–Likert Questions Has your child’s throwing and catching improved? 4.38 (.52) 2.83 (.75) 0.91–2.20** Has your child’s overall coordination improved? 3.88 (.83) 2.67 (1.03) 0.28–2.15* Has your child’s confidence improved? 4.13 (.64) 3.83 (.75) -0.40–0.99 Has your child’s attitude towards physical activity improved? 3.88 (.99) 3.00 (1.67) -0.49–2.25 Has your child chosen to take part in physical activity more often since taking part in this study? 3.88 (.99) 4.00 (1.47) -1.37–1.13 Part II–Themes from Open-ended Questions Motor skill improvements 78% 56% -0.07–0.17 Improved confidence 89% 67% -0.07–0.15 Higher predilection for sport 78% 78% -0.10–0.10 Enjoyment 78% 89% -0.08–0.12 Social benefits 67% 67% -0.12–0.12 **p < .01 *p < .05 BODY.QUALITATIVE COMMENTS (PART II): The result of the content analysis was that we were able to group parents’ comments into five common themes (see Table 4). Frequently the themes of improved confidence, enjoyment, social benefits and predilection for continued participation in sport were reported together (as outlined below): Confidence / predilection / motor improvement. “She has definitely improved her confidence and will try more activities. Her throwing and catching skill have massively improved and we can see this when we watch her play sport.”(Parent of child 10 in QET group) Confidence / social benefits. “Increased confidence from knowing and seeing that he is not the only one who struggles–this has helped his confidence to keep persevering and practicing things that he finds difficult.” (Parent of child 5 in QET group) Confidence / social benefits. “Working with other children of the same ability was a unique experience and really beneficial to xxxxx’s confidence.” (Parent of child 6 in TT group) Enjoyment / social benefits. “Fun activities really helped to motivated her she also enjoyed talking to similar ability children.” (Parent of child 11 in QET group) Enjoyment / social benefits / predilection. “xxxxx has enjoyed meeting children of a similar ability and enjoyed the group session activities…….has taken more of an interest in different activities such as cricket and rounders.” (Parent of child 7 in QET group) Enjoyment / social benefits / confidence. “Enjoyed it all and really benefited from interacting with similar children. Increased her confidence” (Parent of child in 8 QET group) Another interesting theme was that parents favourably compared the environment from the study with what their children typically experienced. “There needs to be more things like this for these children. In normal PE/after school club activities these children feel like they aren’t good enough to play but here they are free to play and feel able to try different activities.” (Parent of child 8 in QET group) “This felt like more of a coaching club or after school club which I think really helped the children to relax and take part. Normal therapy sessions are totally the opposite of this (i.e boring!).” (Parent of child 12 in QET group) “He really dislikes his current physical therapy sessions and finds them boring, but these sessions are fun and engaging.” (Parent of child 9 in TT group) BODY.DISCUSSION: The aim of this study was to explore the efficacy of a group-based QET intervention for improving the catching coordination and success of children with DCD. By combining QET with group-based therapy, we hoped to integrate an intervention that improves skill acquisition in children with poor motor coordination, within an approach that may help ameliorate the psychosocial influence of these motor skill deficits. Our long-term aim was to work towards a holistic intervention for children with DCD that health professionals can use in their therapeutic practice. In line with our hypotheses and prior research [10,11], QET children learned to aim effectively which enabled them to predict the ball’s bounce point on the wall and track its trajectory for longer (Fig 2). Mediation analysis showed that longer aiming durations (QE1) predicted an earlier onset of tracking the ball prior to catching (QE2) which predicted catching success [9]. In short, this optimization of gaze provided children with earlier information on which to prepare the interceptive catch attempt [8, 9]. Interestingly, after completing the QET intervention the QE durations used by these DCD children when aiming (QE1 mean 592ms) and tracking the ball (QE2; mean 254ms) were almost identical to the durations that highly skilled children used to aim (QE1 mean 496ms) and track the ball (QE2 mean 255ms) in the same task [8]. These changes in visuomotor control were also reflected in children’s catching performance. While the improvement between groups in the dichotomous measure of catch success was not significantly different (despite the QET group improving by 51% compared to the TT improvement of 31%) the more sensitive catching score measure did show significant differences as we hypothesised. Specifically, QET individuals significantly improved their catching technique from baseline to retention and maintained this improvement at the delayed retention test. Conversely, the TT individuals did not significantly improve their catching technique throughout the experiment (see Fig 3). In effect, this meant that after training, the QET children were able to catch the ball cleanly with both hands before securing it against their body (mean score of 8), whereas the TT group were catching the ball by trapping it against their body with their arms, rather than grasping it with their hands (mean score of 6). Future analyses will attempt to quantify these changes in terms of patterns of motor coordination and muscular activity. Results from both the gaze and performance data highlight some important implications. First, regardless of the training intervention, all children were able to improve on this interceptive motor task; highlighting the important distinction that DCD should be considered as impaired motor proficiency rather than a fundamental inability to learn motor skills [23]. Second, DCD children can learn to make more functional eye movements that can directly compensate for the oculomotor atypicalities associated with this disorder [6]. Third, QET instructions appear to deliver more effective retention of interceptive skill than typical, explicit instructions focusing on movement control. These benefits are likely due to reduced demands on cognitive load supporting motor control [2425]: Rather than having to consciously think about the complex control of multiple limbs, these are guided implicitly once gaze control is optimised. Previous research has identified that implicit motor learning is an effective strategy for children with DCD [26], perhaps due to how it overcomes deficits in working memory resources [27]. Overall, while the specific mechanisms underpinning the QET benefits warrant further investigation [28], there appears to be clinical utility in exploring the application of QET to other tasks where children with DCD experience difficulties. Interestingly, the improvements in motor coordination displayed by the children were reflected in the perceptions of their parents. Parents of children in the QET group reported that their children had improved their throwing and catching ability and overall coordination to a greater extent than parents of TT children (see Table 4). We also found that parents of children in both groups reported that the intervention had improved their child’s confidence, social behaviours and predilection for sport as we hypothesised. Despite these promising findings, we must add a cautionary caveat here highlighting that we used an unvalidated parental feedback questionnaire and a relatively small sample size. Future research should use validated inventories (e.g., the Children Performance Skills Questionnaire [29]) with a larger sample of children in order to explore these findings further. Additionally, parental perceptions of their child’s predilection for sport or physical activity could also be more objectively measured (e.g., accelerometers) in future work in order to build on these initial results. Despite these limitations, there does appear to be considerable psychosocial benefits of group-based training irrespective of the specific content of the session and the extent of any motor improvements [14]. The positive results of the study lead us to suggest a number of cautionary applied recommendations to enhance the therapeutic experience of children with DCD. First, we wish to echo previous recommendations that interventions for children with DCD must aim to improve both the motor deficits and psychosocial consequences of the disorder [17]. Applied practitioners may benefit from developing innovative ways to incorporate group-based therapy sessions. While the clinical setting may not be the best place to achieve this aim, other organisations such as local councils, schools, national governing sporting bodies and professional sports clubs may have a part to play in supporting such initiatives. Either way, a group-therapy approach is something that children with DCD and their parents find beneficial and there is currently a lack of provision for these types of activities, particularly in the UK. Second, it seems that instructions that optimise the visual attention of children with DCD have real and durable benefits for motor skill attainment in this catching task. As children with DCD generally struggle to develop effective learning strategies on their own–they generally persist with ineffective ones [23]–equipping them with a strategy that has been proven to expedite the learning process in a number of tasks and populations seems a logical step for applied practitioners to take. Understandably, to achieve this will take a greater collaboration between the scientists who are familiar with these methods and the practitioners who will be at the forefront of their delivery. In conclusion, we aimed to develop a holistic intervention for children with DCD that would not only alleviate some of the visuomotor deficits that they experience but also improve some of the psychosocial consequences of the disorder. By combining an intervention (i.e., QET) that has been shown to improve motor learning in children with DCD within a group-based therapy framework, we believe that this aim has been achieved. Future research in now needed to establish the feasibility and practicality of incorporating these ideas into applied therapeutic practice. BODY.SUPPORTING INFORMATION: S1 FigBODY.THE CONSORT CHECKLIST.: (DOC) Click here for additional data file. S1 FileBODY.THE ORIGINAL STUDY PROTOCOL TAKEN FROM NHS ETHICS APPLICATION.: (DOCX) Click here for additional data file. S2 FileBODY.INSTRUCTIONAL VIDEOS.: Summarising the instructions given to the QET and TT groups. (ZIP) Click here for additional data file.

TITLE: Extended depth of focus contact lenses vs. two commercial multifocals: Part 2. Visual performance after 1 week of lens wearLentes de contacto de profundidad de foco extendido vs. dos lentes multifocales comerciales: Parte 2. Rendimiento visual tras una semana de uso ABSTRACT.PURPOSE: To compare the visual performance of prototype contact lenses designed via deliberate manipulation of higher-order spherical aberrations to extend-depth-of-focus with two commercial multifocals, after 1 week of lens wear. ABSTRACT.METHODS: In a prospective, participant-masked, cross-over, randomized, 1-week dispensing clinical-trial, 43 presbyopes [age: 42–63 years] each wore AIROPTIX Aqua multifocal (AOMF), ACUVUE OASYS for presbyopia (AOP) and extended-depth-of-focus prototypes (EDOF) appropriate to their add requirements. Measurements comprised high-contrast-visual-acuity (HCVA) at 6 m, 70 cm, 50 cm and 40 cm; low-contrast-visual-acuity (LCVA) and contrast-sensitivity (CS) at 6 m and stereopsis at 40 cm. A self-administered questionnaire on a numeric-rating-scale (1–10) assessed subjective visual performance comprising clarity-of-vision and lack-of-ghosting at various distances during day/night-viewing conditions and overall-vision-satisfaction. ABSTRACT.RESULTS: EDOF was significantly better than AOMF and AOP for HCVA averaged across distances (p ≤ 0.038); significantly worse than AOMF for LCVA (p = 0.021) and significantly worse than AOMF for CS in medium and high add-groups (p = 0.006). None of these differences were clinically significant (≤2 letters). EDOF was significantly better than AOMF and AOP for mean stereoacuity (36 and 13 seconds-of-arc, respectively: p ≤ 0.05). For clarity-of-vision, EDOF was significantly better than AOP at all distances and AOMF at intermediate and near (p ≤ 0.028). For lack-of-ghosting averaged across distances, EDOF was significantly better than AOP (p < 0.001) but not AOMF (p = 0.186). EDOF was significantly better than AOMF and AOP for overall-vision-satisfaction (p ≤ 0.024). ABSTRACT.CONCLUSIONS: EDOF provides better intermediate and near vision performance than either AOMF or AOP with no difference for distance vision after 1 week of lens wear. BODY.INTRODUCTION: Presbyopia is an age-dependent progressive reduction of the accommodative amplitude that results in an inability to focus accurately on near objects. Based on population projections, the global presbyopia prevalence is estimated to reach about 1.4 billion people by 2020.1 In developed countries alone, there are about 600 million people over the age of 45 who need some degree of vision correction for near. Although traditional spectacle lenses remain the most common correction method for presbyopes, recent prescribing trends2, 3 have shown that over the last decade multifocal contact lenses are gaining popularity.4 An increasingly more active lifestyle of presbyopes,5 demanding a more comfortable, less distracting correction method, could possibly be one of the reasons for this trend in the more developed countries. When compared to single vision lenses, visual performance with multifocal contact lenses is often compromised.6, 7 Simultaneous optical correction designed to correct appropriate viewing distances (i.e. far, intermediate and near) over a limited pupillary area leads to degradation in the visual performance. This imaging principle is often associated with an undesirable presence of ghosting,8 in addition to reduction in low-contrast visual acuity7, 9, 10 and contrast sensitivity.6 Depending on lens design (i.e. aspheric centre-near, centre-distance multifocal or concentric bifocal),7 near add power, pupil size,11 inherent spherical aberrations11, 12 and lens centration,7 the extent of visual performance reduction can range from mild to severe and can occur at one or more distances. In light of the visual compromises associated with current multifocal contact lens designs, a globally increasing presbyopic population and a greater demand in contact lens alternatives, the need to design and develop more satisfying presbyopic solutions is imminent. Via utilization of adaptive optics technology13, 14, 15, 16, 17 and Fourier optics simulations,12 it was demonstrated that interactions of several orders of higher-order spherical aberration terms with Zernike defocus can provide an extension in depth of focus of the corrected eye. Expanding on this concept,18 extended depth-of-focus prototype contact lenses for presbyopic refractive correction (EDOF) have been designed at the Brien Holden Vision Pty Ltd, Sydney, Australia and contract manufactured in poly-HEMA-based (58% water) material. In Part 1 of the study, we explored the performance of the proposed designs using through focus measures; we reported that lenses designed with deliberately manipulated higher order spherical aberration seem to be less susceptible to variations in pupil, inherent ocular aberrations and lens decentration; when compared to commercial alternatives. In this part of the paper, we focus on evaluating if and how these observed incremental improvements seen in ray-tracing experiments translate to clinical outcomes. Previously, we assessed the visual performance of prototype lenses against two commercial lenses in presbyopes after 1 h of lens wear, and found prototype lenses offered good vision at intermediate and near, without compromising distance vision.19, 20 Although, such initial on-eye results of prototype lenses are encouraging and supportive towards the theory of extension in the depth of focus achieved through manipulation of higher order spherical aberration, several authors have shown that the subjective visual performance of multifocal contact lenses differs from initial fitting and after several days of wear.21, 22, 23 The aim of the current study was therefore to assess the acuities, contrast sensitivity, stereopsis and subjective visual performance of the test and control lenses after one week of wear and to compare clinical findings with theoretical measures obtained through ray-tracing. BODY.METHODS: BODY.LENS DESIGNS: The power profiles of the prototype and controls lenses are shown in Fig. 1 of accompanying paper (Part I of the present study). As can be seen, the profile of the AIR OPTIX® Aqua multifocal (AOMF: lotrafilcon b, Alcon laboratories, TX USA) is designed with monotonic distribution of power decreasing from near zone to distance correction. On the other hand, the ACUVUE® OASYS® for presbyopia (AOP: senofilcon A: Johnson & Johnson Vision Care, FL, USA) is a concentric-ring, aspheric, zonal bifocal, where the distinct zones for distance and near correction are separated equally over the optic zone diameter. In contrast with commercial lenses, the prototype EDOF have a series of smooth, non-monotonic, aperiodic, power variation across the optic zone diameter that were designed using deliberate manipulation of multiple higher-order spherical aberration terms. Here, the objective was to achieve an extension in depth of focus that would facilitate a balanced visual performance across a range of predefined dioptric distances. The profiles are designed to satisfy presbyopes with low, medium and high-add requirements. BODY.PARTICIPANTS: This was a prospective, participant-masked cross-over, randomized, 1-week dispensing clinical trial conducted at the Clinical Research Trials Centre of the Brien Holden Vision Institute in Sydney, Australia, commencing in September 2014 and concluding in May 2015. A Human Research Ethics Committee (Bellberry, Adelaide, South Australia) approved the clinical trial. Written informed consent from every participant was obtained prior to commencing any study procedures. The clinical trial conformed to the principles of the Declaration of Helsinki and was registered on the Australian New Zealand Clinical Trials Registry (ACTRN12614000011684) and United States clinical trials registry (NCT02214797). The inclusion criteria for the study were: distance high-contrast visual acuity (HCVA) vision correctable to at least 6/12 in each eye with spherical contact lenses; astigmatism ≤1.00 DC; having a spherical equivalent refraction between −6.00 D and +3.00 D; requiring at least +0.50 D near addition over full distance subjective Rx and having no ocular conditions which would preclude safe contact lens wear. The enrolled participants were a mix of both experienced and non-contact lens wearers. The latter went through a 1-week period of lens adaptation with commercial single vision contact lenses for lens handling (i.e. insertion/removal) training. As they were presbyopes, an appropriate pair of ready-made readers were also given for their near vision needs, only to be used during the adaptation period. BODY.BASELINE PROCEDURES: All participants attended a baseline visit for subjective distance refraction measurement of near add power and determination of eye dominance. All clinical tests were performed under photopic conditions (∼400 lux). Subjective distance refraction was performed using standard optometric techniques. The near addition was the minimum plus power over full subjective distance Rx needed to read 0.0 logMAR print on a high-contrast, black on white Logarithmic Visual Acuity Chart – ETDRS 2000 Series Chart “1′ (Precision Vision, IL, USA) at 40 cm while wearing a trial frame with full aperture lenses. Participants were stratified as low, medium and high presbyopes based on the near add with low = +0.50 to +1.25 D add, medium = +1.50 to +1.75 D add and high = ≥+2.00 D add. Sensory dominance method was used for determining the dominant eye, as explained in detail elsewhere.19, 24 If the sensory dominance technique was unsuccessful then sighting dominance was used to identify the dominant eye, as described elsewhere.19, 24 BODY.ENROLMENT, CONTACT LENS PARAMETERS AND FITTING: Upon completion of the baseline visit, the participants were considered successfully enrolled and were allocated to three contact lens types (one test and two controls) based on a cross-over, balanced block, randomization scheme generated through http://randomization.com/. The randomization scheme determined the order in which lens types were worn by each participant. Lens identity was masked from participants, who wore all three lens types for approximately one week (minimum period of 5 days) each. A minimum two-night wash-out period with habitual correction between lens types was imposed. For more details, please see participant flow diagram in Fig. 1.Figure 1Participant flow diagram for the clinical trial. The EDOF prototypes constituted the test lenses. Three different designs, targeting participants with low, medium and high reading additions were used. The base curve and diameters of EDOF were 8.4 mm and 14 mm, respectively. AOMF and AOP constituted the control lenses. The base curves and diameters of AOMF and AOP 8.6 and 8.4 mm, and 14.2 and 14.3 mm, respectively. Both test and control lenses were available from −6.00 D to +3.50 D in 0.25 D steps. With the prototype lenses, presbyopes needing low and medium addition power wore low and medium add designs bilaterally, while those needing high reading addition wore medium-high combination, with the medium-add design in the non-dominant eye. For control lenses, the designs were dispensed based on the manufacturer's package insert and fitting guides.25, 26 BODY.METROLOGY EVALUATION OF PROTOTYPE LENSES: Lens metrology was performed on prototype EDOF lenses to verify that they conformed to the specifications. A quality control protocol was deployed which directed to assess five randomly chosen lenses in each available power for all prototype contact lenses. A custom-written software algorithm was deployed to post-process standard export raw outputs from NIMO TR1504 (Lambda-X, Belgium). A dedicated algorithm compared measured values with the nominal profile to determine conformity to design specifications. The criteria used for quality control was the sum of absolute differences between the measured and nominal profile across the half-chord diameter. The sum of absolute differences value divided by total number of points on the half-chord yielded an average sum of absolute differences for each profile. In addition to power, the same sample were subjected to checks on the sagittal height measurements with a Microscope MM-400 (Nikon, Japan); diameter with a Profile Projector V-12 (Nikon, Japan) and centre-thickness using a soft contact lens thickness gauge ET-3 (Rehder Development Co., CA, USA). The base curve for each lens was then calculated using these measurements. BODY.CONTACT LENSES DISPENSED: The initial lens power used for test and control contact lenses was based on each participant's spherical equivalent subjective distance refraction. Lenses were allowed to settle for about 10 min followed by over-refraction using a trial frame. Maximum plus to achieve best distance monocular high contrast visual acuity was used. The end point of distance refraction was at least 6/7.5 + 1 (0.08 LogMAR) binocular high contrast visual acuity and the inter-ocular high contrast acuity difference was kept to within 4 letters (0.08 logMAR) to avoid monovision or modified monovision effects. The final lens power was chosen based on the best visual outcome for each participant at both distance and near and within the above constraints. A Test Chart 2000 Pro (Thomson Software Solutions, Hertfordshire, UK) was used for all distance based measurements and high-contrast black on white near LogMAR charts (Precision Vision, USA) were used for all intermediate and near measurements. The control lenses were silicone hydrogels and were dispensed as per manufacturer's guidelines with a lens care system (ClearCare®, Alcon Laboratories, TX, USA), while the test lenses (hydrogels) were prescribed as daily disposables. EDOF, AOMF and AOP contact lenses have distinctly different tints, which made it impractical to mask the investigators. BODY.ACUITY, CONTRAST SENSITIVITY AND STEREOPSIS MEASUREMENTS: Distance high-contrast visual acuity at 6 m was taken monocularly and binocularly. All other acuity measurements were taken binocularly. Low-contrast visual acuity and contrast sensitivity were measured at 6 m. The HCVA and low-contrast visual acuity (LCVA) charts were set at 100% and 10% contrast levels, respectively and distance contrast sensitivity was measured at 6, 12 and 18 cycles/degree with randomized letter triplets whose contrast is varied logarithmically from top to bottom of the chart (Thomson Software Solutions, Hertfordshire, UK). HCVA was measured for intermediate (70 cm) and near (50 cm and 40 cm). The near LogMAR chart is designed for 40 cm, so HCVA measurements at 70 cm and 50 cm were converted to equivalent values in logMAR for these distances prior to statistical analyses (please see appendix). All acuity measurements were resolution acuity, i.e. participants were instructed to read the smallest line possible. Stereopsis was measured at 40 cm with the Stereo Fly Test Circles (Stereo Optical, IL, USA). The investigator entered all the acuity measurements directly into a central database. All acuity measurements performed at the assessment visit were included in the analysis. BODY.SUBJECTIVE QUESTIONNAIRE: The subjective questionnaire-instrument used was aimed to encompass a broad range of visual tasks/demands needed on a daily basis. The questionnaire-instrument was administered three times, two take-home (to be filled in different days between the dispensing and assessment visits) and the third one at the assessment visit. The average of responses for the three questionnaires was used for the analysis. All the subjective ratings were based on a numerical rating scale scored on 1–10 interval, in 1-unit steps. Similar questionnaires were used in our previous short-term assessments of contact lenses.19, 20 Unlike these short-term studies, the current study required participants to experience contact lenses under ‘real-world’ conditions, and so vision performance was separated under day and night-time conditions and additional questions were asked for vision when driving and night-time halos. Briefly, the questions comprised clarity of vision (1 = blurred, 10 = clear) and ghosting (1 = none, 10 = severe) for distance, intermediate and near and vision when driving (1 = blurred, 10 = clear) specifically under day and night-time conditions. Participants also rated vision stability (1 = very unstable, 10 = very stable), night-time haloes (1 = not bothersome, 10 = extremely bothersome), overall comfort (1 = uncomfortable and 10 = comfortable) and overall vision satisfaction (1 = not satisfied and 10 = satisfied). Participants recorded average daily contact lens wear-time and number of hours with acceptable vision via recall at each assessment visit. BODY.LENS FIT ASSESSMENT: Lens fit assessment was performed at dispensing and assessment visits. After vision and subjective ratings were complete, lenses were observed on eye with slit-lamp biomicroscopy and assessed for centration (horizontal and vertical, mm), primary lens movement (mm), primary lens lag (mm) and tightness (%). BODY.DATA TRANSFORMATIONS: To simplify interpretation, the scale for ghosting rating and the scale for night-time haloes have been reversed to be consistent with the other ratings, such that a higher rating refers to a better result (i.e. less ghosting and less bothersome haloes). Here and throughout the document, the ghosting variable will be referred to as ‘lack of ghosting’ and the halo variable will be referred to as ‘lack of bothersome haloes’. As the stereopsis data was positively-skewed, a log transform to the raw data was performed prior to statistical analyses. All the data are summarized as observed means ± SD for variables measured on an interval scale and as percentages for all categorical variables. BODY.STATISTICAL ANALYSIS: A minimum sample of 27 participants was needed to demonstrate a statistically significant paired difference of 1 ± 1.8 units between lenses types for subjective variables (primary outcome) with 80% power and 5% level of significance. This sample also had 90% power to detect 0.10 ± 0.15 logMAR difference in visual acuity (secondary outcome) between the lens types. All the analyses were performed using SPSS 21 (IBM, USA), and the level of significance was set at 5%. Results of sub groups are presented only if interactions were significant at 5% level. Bonferroni adjustments were made for post hoc analyses. A 3-way linear mixed model with subject random intercepts and repeated effects of lens types was deployed to test the hypothesis if the test lenses performed differently to the two controls. For the subjective variables (clarity of vision and lack of ghosting), the model included the following study design factors: lens-type (EDOF, AOMF or AOP), test-distance (far, intermediate and near), time-of-day (day/night). For vision-stability and driving-vision variables, the linear mixed model only had lens-type and time-of-day and their interaction as study design factors. For overall vision satisfaction and ocular comfort variables, lens-type was the only study design factor in the model. The add group (low/medium/high) was added as a confounding subject factor in all models. The interaction of lens type with all other factors were tested. The significance of lens type was assessed within a sub level of a factor only if the respective interaction term was significant. In all other instances the results are generalized for the whole group. This modelling approach was used instead of several repeated measures/paired t-tests to avoid inflating the type I error rate. Due to the unequal distribution of add-groups (Table 1), results for subjective variables are presented as ‘observed means’ (actual mean results obtained) and ‘estimated means’ (mean results calculated assuming an equal distribution between add-groups).Table 1Demographic factors for low, medium and high-add participants. Myopes were defined as participants requiring a negative spherical-equivalent distance subjective refraction and all other participants were defined as hyperopes. Table 1FactorTotalLow-addMed-addHigh-addn = 43n = 13n = 18n = 12Age (years) 53 ± 5a 48 ± 4a 54 ± 5a 56 ± 4a Age range (years) 42–63 42–57 48–63 49–63 Female:male (%) 53:47 62:38 44:56 58:42 Neophytes:experienced contact lens wearers (%) 30:70 15:85 44:56 25:75 Ethnicity (%)  Caucasian:Asian:Others 74:12:14 85:8:8 67:17:17 75:8:17 Myopesb  Spherical equivalent (D) −2.92 ± 1.50a −2.94 ± 1.27a −2.81 ± 1.96a −3.00 ± 1.34a  Range (D) −6.00 to −0.75 −5.25 to −0.75 −6.00 to −0.7 −6.00 to −1.25 Hyperopesb  Spherical equivalent (D) 1.62 ± 0.84a 1.31 ± 0.83a 1.56 ± 0.90a 1.85 ± 0.73a  Range (D) 0.25–3.25 0.50–2.25 0.25–3.25 0.75–2.75 Keratometryb  Flat power (D) 43.27 ± 1.38a 43.06 ± 1.53a 43.35 ± 1.50a 43.39 ± 1.02a  Steep power (D) 43.84 ± 1.45a 43.64 ± 1.57a 43.94 ± 1.59a 43.91 ± 1.12a aData expressed as mean ± SD. bResults presented for both eyes combined. For HCVA, lens-type, test-distance and their interactions were considered as study design factors. For distance, LCVA (both in high and low illumination conditions) and stereopsis measure, only the lens-type was considered as study design factors. For the distance contrast sensitivity variable, the lens-type, spatial-frequency (cyc/deg) were considered as study design factors. If the interactions of lens-type with test-distances and or spatial frequency were found to be significant, further post hoc tests were deployed to determine significance of lens type within sub levels of the interacting factor. Again, the add-group (low/medium/high) was added as a confounding subject factor in all models. The difference between the dispensed study lens power and vertex-corrected spherical equivalent refraction at baseline was also compared using the linear mixed model to see if they were different between test and control lenses. BODY.RESULTS: BODY.LENS METROLOGY: Five randomly picked samples from each power of the three prototype designs (Range: −6.00 D to +3.00 in 0.25 D steps, a total of 540 lenses) were requested for metrology evaluation. For power-profile measurements, the average range of sum of absolute differences measure for all lenses was 0.05–0.16 D, which is clinically insignificant. For sagittal height, diameter, centre thickness and base curve variables, all lenses were within tolerance specifications (ISO 18369 Section 2).27 BODY.DEMOGRAPHICS: A total of 43 participants completed the study (13 low-add participants, 18 medium-add participants and 12 high-add participants), of whom 53% were female, the mean ± SD age was 53 ± 5 years, the age range was 42–63 years, and 70% were experienced contact lens wearers. Participant details for each add-group are presented in Table 1. BODY.DATA TRANSFORMATIONS: For all acuity, contrast sensitivity and stereopsis measures, data from the assessment visit was used in the final analysis. However, with the subjective measures, data from the assessment visit and take-home questionnaires were averaged, as they were no different from each other (p > 0.05). BODY.ACUITIES, CONTRAST SENSITIVITY AND NEAR STEREOPSIS: The observed mean (±SD) of visual acuities at various distances, contrast and/or illumination levels, distance contrast sensitivity and near stereopsis for the three study lenses are presented in Table 2. For high-illumination HCVA, there were significant differences between lens-type (p < 0.001), test-distance (p < 0.001) and add-group (p = 0.006). As expected, the acuities were better for distance followed by intermediate and near; and the low adds achieved better acuity followed by medium and high groups. However, considering the interactions, neither lens-type and add-group, nor lens-type and test-distance achieved significance (p ≥ 0.080). Therefore, the post hoc model only included lens-type as a factor at each test-distance. The model indicated that participants wearing EDOF, on average, demonstrated better HCVA (average across all distances) than AOP (p < 0.001) and AOMF (p = 0.038).Table 2Observed mean ± SD of visual acuities, contrast sensitivity and near stereopsis variables for the three study lenses. Table 2Study variableEDOFAOMFAOPHigh illumination, HCVA  Distance (LogMAR) −0.07 ± 0.06 −0.07 ± 0.08 −0.06 ± 0.08  At 70 cm (LogMAR) −0.07 ± 0.08 −0.03 ± 0.09 0.00 ± 0.10  At 50 cm (LogMAR) 0.01 ± 0.10 0.03 ± 0.11 0.06 ± 0.12  At 40 cm (LogMAR) 0.10 ± 0.11 0.13 ± 0.13 0.12 ± 0.11 High illumination, LCVA (Distance) 0.27 ± 0.10 0.22 ± 0.10 0.27 ± 0.09 Low illumination, LCVA (Distance) 0.35 ± 0.12 0.30 ± 0.10 0.37 ± 0.12 Distance contrast sensitivity  (6 cyc/deg, Log units) 1.44 ± 0.11 1.47 ± 0.11 1.44 ± 0.09  (12 cyc/deg, Log units) 1.21 ± 0.16 1.26 ± 0.15 1.21 ± 0.16  (18 cyc/deg, Log units) 0.95 ± 0.23 1.01 ± 0.19 0.92 ± 0.20 Stereopsis at 40 cm (arc sec) 61 ± 37 97 ± 129 74 ± 63 HCVA = high contrast visual acuity. LCVA = low contrast visual acuity. EDOF = extended depth of focus. AOMF = AIR OPTIX Aqua multifocal. AOP = ACUVUE OASYS for Presbyopia. For high-illumination LCVA, there were significant differences between lens-type (p = 0.019) and add-group (p < 0.001), however the interaction between lens-type and add-group was not significant (p = 0.292). Post hoc paired analysis demonstrated that EDOF lens was inferior to AOMF by two letters (p = 0.021) and not different from AOP (p = 1.00). For the low-illumination, LCVA at 6 m, there were significant differences between lens-type (p = 0.001) and add-group (p = 0.001), however the interaction between lens-type and add-group was not significant (p = 0.079). Post hoc paired analysis demonstrated that EDOF lens was inferior to AOMF by two letters (p = 0.012) and not different from AOP (p = 1.00). For both high- and low-illumination, the low add participants achieved better LCVA, followed by medium and high add groups. For distance contrast sensitivity, significant differences were found for lens-type (p = 0.001), spatial-frequency (p < 0.001) and add-group (p = 0.021) factors, however the interaction between lens-type and spatial-frequency factors did not achieve significance (p = 0.529). Nevertheless, the interaction between lens-type and add-group was found to be significant (p = 0.023). Therefore, the data was split by add-group and the contrast sensitivity data was averaged across all spatial frequencies. The model suggested that EDOF lenses were inferior to AOMF by 1–2 letters in the medium and high add group (p < 0.007) but not different in the low-add group (p = 0.876). Distance contrast sensitivity with EDOF lenses were no different from AOP for all add-groups (p > 0.519). For the near stereopsis variable, there was a significant difference between lens types (p = 0.008). Participants wearing EDOF had significantly better stereopsis than those wearing AOMF (Δ = 36 seconds-of-arc, p = 0.028) and AOP (Δ = 13 seconds-of-arc, p = 0.05). This observation was independent of the add-group (p = 0.731). The difference between the prescribed study lens power and vertex-corrected spherical equivalent at baseline for test and control lenses were insignificant (−0.01 ± 0.08, 0.01 ± 0.05 and −0.01 ± 0.05 for EDOF, AOMF, AOP, respectively, p = 0.102). BODY.SUBJECTIVE VARIABLES: The observed subjective ratings (mean ± SD) obtained from participants wearing all three study lenses at various test distances, during day and night-time conditions, are detailed in Table 3.Table 3Observed mean ± SD of subjective variables for the three study lenses. Table 3Subjective variables (on a 1–10 scale, higher the better)EDOFAOMFAOPDistance vision clarity Day 7.8 ± 1.7 7.9 ± 1.8 7.1 ± 2.2 Night 7.5 ± 1.8 7.5 ± 1.7 7.0 ± 2.0 Intermediate vision clarity Day 7.9 ± 1.6 7.5 ± 1.7 6.9 ± 1.6 Night 7.6 ± 1.7 7.2 ± 1.8 6.7 ± 1.5 Near vision clarity Day 7.4 ± 1.8 6.6 ± 2.0 6.0 ± 1.9 Night 7.1 ± 1.9 6.3 ± 2.2 5.7 ± 1.9 Distance lack of ghosting Day 8.2 ± 2.0 8.3 ± 2.2 7.5 ± 2.6 Night 7.9 ± 2.2 7.9 ± 2.2 7.4 ± 2.6 Intermediate lack of ghosting Day 8.5 ± 1.7 8.1 ± 2.0 7.5 ± 2.0 Night 8.3 ± 1.6 7.9 ± 2.1 7.6 ± 1.9 Near lack of ghosting Day 8.2 ± 1.9 7.6 ± 2.2 7.1 ± 2.3 Night 8.1 ± 1.8 7.3 ± 2.3 7.1 ± 2.2 Vision stability Day 8.1 ± 1.4 7.9 ± 1.7 7.3 ± 2.0 Night 8.2 ± 1.2 7.8 ± 1.7 7.4 ± 1.7 Driving vision Day 8.0 ± 1.6 8.0 ± 1.5 7.2 ± 2.1 Night 7.5 ± 1.7 7.4 ± 1.4 6.8 ± 2.0 Lack of bothersome haloes at night 8.9 ± 1.5 8.3 ± 2.0 8.3 ± 2.2 Overall vision satisfaction 7.6 ± 1.8 6.7 ± 2.1 5.7 ± 2.6 Ocular comfort 8.5 ± 1.3 7.6 ± 1.9 8.0 ± 1.6 EDOF = extended depth of focus. AOMF = AIR OPTIX Aqua multifocal. AOP = ACUVUE OASYS for Presbyopia. For the clarity of vision variable, significant differences were observed between lens-type (p < 0.001) and test-distance (p < 0.001). The interaction of lens type with test-distance was significant at the 5% level of significance (p = 0.044). Time-of-day was a significant factor (p = 0.001). The ratings obtained during night-time were lower to those obtained during day-time, however the difference between lens types did not interact with time-of-day (p = 0.936) or add-group (p = 0.585). The model indicates that for day/night-time conditions, participants wearing prototype lenses rated their clarity of vision significantly clearer than AOP distance (Δ = 0.7 units, p = 0.021), intermediate (Δ = 1.0 unit, p < 0.001) and near (Δ = 1.3 units, p < 0.001); and significantly clearer than AOMF at intermediate (Δ = 0.4 units, p = 0.028) and near (Δ = 0.8 units, p < 0.001). The estimated means of clarity of vision ratings for all three study lenses determined from the linear mixed model, for each test distance (day and night combined) can be seen in Fig. 2.Figure 2Estimated means (means calculated assuming an equal distribution between add-groups) for ‘clarity of vision’ ratings at each test distance (day and night time measures combined) determined from the linear mixed model for all study lenses. The error bars indicate the upper bound of the 95% confidence interval. The symbol ‘¥’ represents statistically significant difference observed between extended depth-of-focus prototype (EDOF) and AIR OPTIX Aqua multifocal (AOMF) lenses and symbol ‘§’ represents statistically significant difference observed between EDOF and ACUVUE OASYS for presbyopia (AOP) lenses. For the lack of ghosting ratings variable, there were significant main effects with lens-type (p < 0.001) and test-distance (p < 0.001) as factors. The lack of ghosting ratings obtained at near were lowest, followed by intermediate and far test distances. Time-of-day was not significant (p = 0.077) nor were any of the interactions (p > 0.056). As the interaction between lens-type and test-distance were insignificant at 5%, the model was adjusted to look at effect of lens type for an average of lack of ghosting measure across test distances and day/night conditions. The results indicate that on average, participants wearing EDOF rated significantly less ghosting than AOP (Δ = 0.8 units, p < 0.001) but not AOMF (Δ = 0.3 units, p = 0.186). The estimated means of lack of ghosting ratings for all three study lenses determined from the linear mixed model, for all test distances combined (day and night combined) is presented in Fig. 3.Figure 3Estimated means (means calculated assuming an equal distribution between add-groups) for ‘lack of ghosting’ ratings averaged across all test distances (day and night time measures combined) determined from the linear mixed model for all study lenses. The error bars indicate the upper bound of the 95% confidence interval. The symbol ‘¥’ represents statistically significant difference observed between extended depth-of-focus prototype (EDOF) and AIR OPTIX Aqua multifocal (AOMF) lenses and symbol ‘§’ represents statistically significant difference observed between EDOF and ACUVUE OASYS for presbyopia (AOP) lenses. For the vision-stability variable, there was a significant difference between lens-types (p < 0.001) but not with time-of-day (p = 0.787), add-group (p = 0.476) or their interaction (p = 0.788). Participants rated their vision-stability with EDOF significantly better than with AOP (Δ = 0.9 units, p < 0.001) and no different from AOMF (Δ = 0.3 units, p = 0.177). For driving vision, significant differences were found between lens type (p < 0.001) and time of day (p = 0.009). however, their interaction was not significant (p = 0.849). The ratings obtained during night-time were lower to those obtained during day-time The model predicted that driving vision was significantly better with EDOF compared to AOP (Δ = 0.7 units, p = 0.003) but not different to AOMF (Δ = 0 units, p = 1.00). With the overall-vision satisfaction variable, there was a significant effect of lens type (p < 0.001), with participants wearing EDOF rating their overall-vision satisfaction significantly higher than wearing AOMF (Δ = 0.9 units, p = 0.024) and AOP (Δ = 1.9 units, p < 0.001). Fig. 4 presents the estimated means of overall vision satisfaction for all three study lenses determined from the linear mixed model.Figure 4Estimated means (means calculated assuming an equal distribution between add-groups) for ‘overall vision satisfaction’ ratings at each test distance (day and night time measures combined) determined from the linear mixed model for all study lenses. The error bars indicate the upper bound of the 95% confidence interval. The symbol ‘¥’ represents statistically significant difference observed between extended depth-of-focus prototype (EDOF) and AIR OPTIX Aqua multifocal (AOMF) lenses and symbol ‘§’ represents statistically significant difference observed between EDOF and ACUVUE OASYS for presbyopia (AOP) lenses. With the overall-comfort variable, there was a significant effect of lens type (p = 0.003). Participants rated their overall-comfort significantly higher with EDOF than with AOMF (Δ = 0.9 unit, p = 0.009) and AOP (Δ = 0.5 unit, p = 0.011). With the lack of bothersome haloes variable, there was no significant effect for the lens-type suggesting that the night-time halo experience with EDOF was no different from AOMF and AOP (p > 0.146). The average daily contact lens wear-time and number of hours with acceptable vision in participants wearing the three study lenses are detailed in Table 4. For the average daily contact lens wear-time variable, there was a significant effect of the lens-type (p = 0.022) and add group (p = 0.004) but not their interaction (p = 0.680). The low-add participants, on averaged, had a higher lens wear time, followed by medium and high add groups. The model suggests that participants reported significantly greater wearing hours with EDOF over AOP (Δ = 1.3 h, p = 0.012) but not AOMF (Δ = 0.5 h, p = 0.616). For the question on the number hours with acceptable vision with study lenses, again, there was a significant effect of the lens-type (p = 0.001) and add group (p = 0.006) but not their interaction (p = 0.869). The model suggests that participants reported significantly greater number of hours with acceptable vision when wearing EDOF compared to AOP (Δ = 2.9 h, p < 0.001) but not AOMF (Δ = 0.9 h, p = 0.224).Table 4Observed means ± SD of CL wear time for the three study lenses. Table 4CL wear timeEDOFAOMFAOPNumber of hours with acceptable vision 9.5 ± 3.3 8.4 ± 4.1 6.6 ± 4.3 Average daily wear time 10.8 ± 2.9 10.3 ± 3.1 9.5 ± 3.3 EDOF = extended depth of focus. AOMF = AIR OPTIX Aqua multifocal. AOP = ACUVUE OASYS for Presbyopia. BODY.LENS FITTING: Centration, primary gaze movement, primary gaze lag and tightness (mean ± SD) for all three study lenses at the Assessment Visit are detailed in Table 5. There were no significant differences between lens types for horizontal centration (p = 0.170) while a significant difference was found between lens types for all other lens fitting measures (p ≤ 0.007). All differences between lens types were clinically insignificant.Table 5Lens fitting parameters (mean ± SD) for the three lens types at the assessment visit. Table 5Lens fitting parameterEDOFAOMFAOPHorizontal centrationa (mm) −0.04 ± 012 −0.05 ± 0.09 −0.07 ± 0.11 Vertical centrationb (mm) 0.02 ± 010 −0.04 ± 0.08 0.00 ± 0.09 Primary gaze movement (mm) 0.16 ± 010 0.13 ± 0.08 Primary gaze lag (mm) 0.06 ± 0.07 0.09 ± 0.06 0.05 ± 0.06 Tightness (%) 50 ± 9 46 ± 7 55 ± 6 EDOF = extended depth of focus. AOMF = AIR OPTIX Aqua multifocal. AOP = ACUVUE OASYS for Presbyopia. aPositive values indicate nasal decentration. Negative values indicate temporal decentration. bPositive values indicate superior decentration. Negative values indicate inferior decentration. BODY.DISCUSSION: Overall, the EDOF prototypes demonstrated some improvements over the commercially available zonal-refractive and aspheric centre-near designs, AOP and AOMF, respectively. The improvements in visual performance with EDOF lenses were noticed in clarity of vision (at one or more visual distances), lack of ghosting average across all distances (only with AOP) and overall vision satisfaction variables. Most of these observations were in-line with the outcomes from our previous short-term (1-h) evaluations.19, 20 The insignificant interaction between ‘lens-type’ and ‘add-group’ suggests that the improvements in performance seen with prototypes were not driven by a particular participant pool. The current study's findings were also in-line with many of the predictions from the theoretical modelling, presented in Part 1 of the study. Improved performance for intermediate and/or near with the new prototype lenses over the controls remained unaffected by the time of day, indicated as performance invariance to pupil diameter changes as described in Part 1. The predicted poor performance with decentered AOMF lenses in Part 1 did not occur because AOMF lenses were well-centred on eye. The theoretical modelling from this profile was in-line with the outcomes the current study. The major discrepancy between theoretical modelling and clinical trial outcomes occurred for near visual performance with AOP lenses. Compared to EDOF, modelling indicated similar distance and near performance with AOP but worse intermediate due to the bimodal nature of AOP. The clinical trial found similar distance but worse intermediate and near. This unexpected near result was probably influenced by the intermediate result – participants experienced a poor intermediate and so expected to experience a poor near, despite the predicted result from theory. This result indicates that while theoretical modelling may be a useful indicator of lens performance, the final determinant of performance is a human clinical trial. When considering conventional, pupil-dependent multifocal optics, that are based on either centre-distance or centre-near principles, it can be drawn from theory that for any given pupil, the through-focus performance is expected to follow a ‘see–saw’ response. In other words, the better the lens performance is for distance, the worse the performance would be at near and vice versa. This can be observed in the theoretical modelling obtained with AOMF designs. As previously discussed, the EDOF prototypes presented here are optimized via deliberate manipulation of higher order spherical aberrations to yield an extension in depth of focus. This facilitates improvement in visual performance at intermediate and near, without incurring a significant compromise at distance. Comparing prototypes with controls, it is reasonable to state that the design achieved its purpose, at least as gauged by the clarity of vision variable. There were no significant differences for distance acuities between the prototype and control lenses. Although the observed visual acuity with the prototype lenses was better than with the control lenses at intermediate and near test distances, the differences were not clinically relevant as they were within 2 letters. Unlike our previous short-term studies,19, 20 the current trial used a conventional ‘resolution-based’ acuity test instead of a ‘comfort-based’ reading assessment to measure intermediate and near visual performance. As a result, the average near visual acuity measures with both prototype and control lenses were on average 0.2–0.3 logMAR better than our previous observations with similar lenses. In our previous studies, we reported that the ‘comfort-based’ acuity was able to demonstrate clinically significant differences between prototypes and controls, suggesting that a less sensitive variable like resolution-based acuity may not be an ideal choice when comparing presbyopic visual performance between multifocal contact lenses. This study is in agreement with several previous studies assessing AOMF and AOP for many acuity-based measurements including HCVA at 6 m9, 28, 29; LCVA at 6 m under low illumination29; distance contrast sensitivity9; HCVA at 70 cm29 and 40 cm90,29 and near stereopsis.29 A short-term study by Vasudevan et al.28 reported much better LCVA at 6 m and HCVA at 40 cm for participants wearing AOMF and AOP than the current study. However, these discrepancies may be due differences between Vasudevan et al. vs. this study for contrast setting when measuring LCVA (36%28 vs. 10%) and participant age (41 ± 1 years28 vs. 53 ± 5 years). Enhancements in intermediate and/or near vision performance, while prescribing multifocals can also be achieved by practicing modified monovision techniques,30, 31, 32, 33 where one eye is prescribed to correct a range of distances spanning distance to intermediate, while the other eye is prescribed to correct from intermediate to near. The binocular summation capabilities of the visual system are expected to provide clear images for the whole range.34 In the current clinical trial, efforts were made to refrain from such a correction philosophy. The initial lens dispensing protocol was set up to keep the inter-ocular visual acuity difference within 4 letters for both test and control lenses. The fitting guides for both AOMF and AOP suggest “trouble-shooting” strategies for improving visual performance following initial lens fitting.25, 26 While these may be routinely performed in clinical practice, these strategies were not performed in the current study due to logistical considerations and so are a study limitation. However, this did not bias results as the limitation was applied to test and control contact lenses. An independent effort to validate our subjective questionnaires was not deemed necessary due to the simplistic nature and short construct of the questionnaire instrument used. Indeed, similar questions were asked in our previous short-term assessment of prototype contact lenses.19, 20 The primary aim of the current study was to evaluate if visual performance gauged via subjective responses obtained with the prototypes were different from the commercial controls. Given that the study design was executed in a randomized, cross-over manner, the reliability, content and/or criterion bias (if any) remains constant throughout all repeated administrations of the questionnaire. Thus, the bias (if any) will be washed out when considering differences between the subjective ratings with different lens types. We acknowledge that a double-blind trial would have been more robust. However, due to the study logistics, difference in lens handling tints, slight differences in the prescribed fitting guides between the prototype and controls, we were not able to mask the investigators. However, given that subjective measures are more sensitive than acuity measures23 when assessing visual performance with multifocal contact lenses, we allowed participants to enter their subjective performance directly into the database without investigator's intervention. Thus, we believe that the investigators being unmasked did not bias the overall findings of the study. Some of the participants could have been habitual wearers of the control lenses used in this study. One may argue that this could potentially introduce bias, as they may have been able to identify their lenses. Upon analyzing our baseline data, we identified only 2/43 participants as habitual wearers of AOMF and none habitually wearing AOP. Therefore, it is unlikely that such a small proportion would affect the overall outcome of the study. One other shortcoming of this study was that the wearing modalities of prototype and controls were different. The former lens was used as a daily-disposable, while the latter were prescribed for multiple-use with peroxide-based lens-care regimen. It is possible that the significantly higher comfort experienced with the test lenses may have been influenced by the wearing modality and therefore it may be perceived that it influenced the visual performance results. However, it is observed that that AOMF performed 0.5–1.0 units better than AOP for various subjective variables despite AOMF 0.5 units worse for comfort compared to AOP. This observation is further supported by the data on the average number of wearing hours per day and the number of hours of acceptable vision per day between AOMF and AOP. Clearly, despite the fact that AOP are more comfortable than AOMF, AOP were on average worn for fewer hours and provided fewer hours of acceptable vision quality than AOMF. It demonstrates that this particular cohort seem to have the ability to distinguish between comfort and vision. Therefore, we do not think the disparity in wearing modality and/or comfort confounded this studies’ overall findings. One may argue that the prototype lenses could have been offered as a frequent replacement lenses (like controls) to further reduce bias, if any. As the optical undulations engraved onto the prototype lenses were sub-micron in dimension, we avoided any chance of affecting the profiles by prescribing them as re-usable lenses. On the other hand, the control lenses were intended by the manufacturer to be used as a monthly-replacement lens; furthermore, the cost involved with using control lenses as daily disposables were prohibitive. Nevertheless, we acknowledge that using the controls as multiple-use may have created some resentment in participants towards these lenses, potentially leading to lower subjective ratings, and so it is important to further investigate prototype lens performance with daily disposable control lenses to confirm this assumption. BODY.CONCLUSIONS: After 1 week of lens wear, EDOF prototype contact lenses offer significant improvements in subjective visual performance over AOMF and AOP for clarity of vision at intermediate and near without compromising distance vision. BODY.CONFLICTS OF INTEREST: This project is entirely funded by the Brien Holden Vision Institute and the Institute has proprietary interests in the intellectual property governing extended depth-of-focus contact lenses (Patent application number WO2014059465 A1) and receives royalties from the sale of centre-near contact lenses described in this manuscript.

TITLE: Prognostic and predictive importance of the estrogen receptor coactivator AIB1 in a randomized trial comparing adjuvant letrozole and tamoxifen therapy in postmenopausal breast cancer: the Danish cohort of BIG 1-98 ABSTRACT.PURPOSE: To evaluate the estrogen receptor coactivator amplified in breast cancer 1 (AIB1) as a prognostic marker, as well as a predictive marker for response to adjuvant tamoxifen and/or aromatase inhibitors, in early estrogen receptor-positive breast cancer. ABSTRACT.METHOD: AIB1 was analyzed with immunohistochemistry in tissue microarrays of the Danish subcohort (N = 1396) of the International Breast Cancer Study Group’s trial BIG 1-98 (randomization between adjuvant tamoxifen versus letrozole versus the sequence of the two drugs). ABSTRACT.RESULTS: Forty-six percent of the tumors had a high AIB1 expression. In line with previous studies, AIB1 correlated to a more aggressive tumor-phenotype (HER2 amplification and a high malignancy grade). High AIB1 also correlated to higher estrogen receptor expression (80–100 vs. 1–79%), and ductal histological type. High AIB1 expression was associated with a poor disease-free survival (univariable: hazard ratio 1.35, 95% confidence interval 1.12–1.63. Multivariable: hazard ratio 1.29, 95% confidence interval 1.06–1.58) and overall survival (univariable: hazard ratio 1.34, 95% confidence interval 1.07–1.68. Multivariable: hazard ratio 1.25, 95% confidence interval 0.99–1.60). HER2 did not seem to modify the prognostic effect of AIB1. No difference in treatment effect between tamoxifen and letrozole in relation to AIB1 was found. ABSTRACT.CONCLUSIONS: In a subset of the large international randomized trial BIG 1-98, we confirm AIB1 to be a strong prognostic factor in early breast cancer. Hence, although tumor AIB1 expression does not seem to be useful for the choice of tamoxifen versus an aromatase inhibitor in postmenopausal endocrine-responsive breast cancer, AIB1 is an interesting target for new anti-cancer therapies and further investigations of this biomarker is warranted. BODY.INTRODUCTION: One of the biggest challenges in today’s breast cancer care is to adjust adjuvant treatment, according to both tumor and patient characteristics, for optimal treatment aimed at improved prognosis. Although both tamoxifen and aromatase inhibitors have been shown to improve survival in estrogen receptor-positive breast cancer, the disease will recur in many patients despite adjuvant treatment (7–9% breast cancer recurrences five years after randomization in BIG-98 [1]). Moreover, in the metastatic setting most tumors eventually develop resistance to the given treatment. Hence, further studies of potential prognostic and predictive biomarkers are essential in order to optimize and individualize breast cancer treatment. An interesting biomarker in relation to endocrine treatment is AIB1 (amplified in breast cancer 1). AIB1 belongs to the p160 steroid receptor coactivator family and interacts with the estrogen receptor in a ligand-dependent manner to enhance transcription [2, 3]. However, it has also been shown to interact with other transcription factors and signaling pathways inducing hormone-independent proliferation [4–6]. In human breast cancer, AIB1 correlates with factors indicating a more aggressive phenotype (HER2 amplification, DNA non-diploidy, a high tumor grade, a high S-phase fraction, and high Ki67) [5, 7–10]. Several studies have also indicated AIB1 to be associated with endocrine treatment effect [5, 7, 8, 11–13], although results have not been unanimous. We have previously shown AIB1 to be both a prognostic marker and a predictive marker for adjuvant tamoxifen in a randomized trial of premenopausal women receiving adjuvant tamoxifen for 2 years versus control, and in independent cohorts [9, 10, 14]. These data were extended also to postmenopausal patients in an independent randomized trial of adjuvant tamoxifen versus control [15]. Women with estrogen receptor-positive breast cancer expressing high levels of AIB1 have a worse prognosis, but respond well to tamoxifen. The prognosis of women with low tumor expression of AIB1, on the other hand, is not further improved by tamoxifen, although early on they have a better prognosis. However, previous studies of AIB1 in relation to aromatase inhibitors are very sparse, and its predictive value for treatment with aromatase inhibitors has not been evaluated in any larger clinical trial. If patients with low tumor expression of AIB1 would still benefit from aromatase inhibitors, AIB1 might be a predictive marker for the choice between tamoxifen and aromatase inhibitors, which is something we lack in the clinic today. We use the Danish subcohort of the large randomized Breast International Group (BIG) 1-98 trial of adjuvant tamoxifen versus letrozole (as monotherapy or sequentially) with the aim to investigate AIB1 as a prognostic and predictive biomarker in relation to adjuvant endocrine treatment in estrogen receptor-positive postmenopausal breast cancer. BODY.PATIENTS AND METHODS: BODY.BIG 1-98: The design of the BIG 1-98 trial, as well as the Danish cohort, has been described in detail before [16–18]. Briefly, this is a randomized, phase 3, double-blinded trial of postmenopausal, estrogen receptor-positive, early breast cancer. Patients were randomized to either monotherapy with tamoxifen or letrozole for 5 years, or to sequential therapy with 2 years of tamoxifen or letrozole followed by an additional 3 years with the other drug (letrozole/tamoxifen). The trial enrolled 1396 Danish patients from 1998 to 2003 included in the intention-to-treat population. Primary tumor samples were available for 1323 of patients and tissue microarrays were constructed for 1281 of these [18–20]. In 1997, the Danish Medicines Agency and the Danish National Committee on Biomedical Research Ethics approved the double-blinded BIG 1-98 trial, and the Ethical Committee of the Capital Region approved the current biomarker study before its activation (KF 02-178/97, KF 12-142/04, RH-2015-166; I-suite 04070). The BIG 1-98 trial is registered on the clinical trial site of the USA National Cancer Institute’s website (http:www.clinicaltrials.gov/ct/show/NCT00004205). The remark criteria were considered for presentation of data below [21]. BODY.CENTRAL ASSESSMENT OF THE ESTROGEN RECEPTOR, PROGESTERONE RECEPTOR, AND HER2: The International Breast Cancer Study Group’s Central Pathology Laboratory carried out a central review on whole tissue sections for estrogen and progesterone receptors by immunohistochemistry, and for HER2 by immunohistochemistry and fluorescent in situ hybridization as previously described [1, 22]. Tumors expressing estrogen or progesterone receptors in ≥1% of tumor cells were considered hormone receptor positive, and those with a HER2-to-Centromere-17 ratio ≥2 considered HER2-amplified. The pathology central review was carried out without knowledge of other characteristics, treatment assignment, or outcomes. BODY.IMMUNOHISTOCHEMISTRY FOR AIB1: Tissue microarrays were constructed from formalin-fixed and paraffin-embedded tumor blocks by a tissue microarray builder using 2-mm tissue cores [18]. Each tumor was represented by two cores. Immunohistochemistry for AIB1 was performed in an Autostainer-Plus, Dako. As a primary antibody for AIB1 detection, a mouse-monoclonal IgG antibody was used at 1:100 dilution (Cat no #611105 BD Bioscience, CA, USA), as previously described [7, 10]. This antibody has been used in several previous clinical trials [3, 7, 8], and its specificity has been confirmed by both Western blot and Northern blot, and in situ hybridization [3, 8]. Immunohistochemical staining (nuclear) was examined by two independent viewers blinded for clinical/tumor characteristics (Sara Alkner and Kristina Lövgren). Each sample was semi-quantitatively scored from 0 to 3 for percentage of stained nuclei and staining intensity. Proportion score 0 represented no stained nuclei, 1:1–10%, 2:11–50%, and 3:51–100%. Staining intensity 0 represented negative staining, 1 weak, 2 moderate, and 3 intense staining. Proportion and intensity scores were added to a total score ranging from 0 to 6. As in several previous publications from our group, a total score of ≥5 was used to define high AIB1 [7, 9, 10, 14]. Cases classified differently (high vs. low AIB1) between viewers (8%) were reexamined to reach consensus. In case of discrepant staining between the two cores from the same patient, the highest score was used. BODY.STATISTICAL ANALYSIS: All clinical data were collected and monitored by the International Breast Cancer Study Group and subsequently transferred to the Danish Breast Cancer Cooperative Group, where the statistical analyses were conducted. Follow-up time was quantified in terms of a Kaplan–Meier estimate of potential follow-up. The primary end-point was disease-free survival, defined as the time from randomization to the first of the following events: recurrence at local, regional, or distant sites; a new invasive cancer in the contralateral breast; any second (non-breast) malignancy; or death without a prior cancer event. Secondary end-point was overall survival, defined as the time from randomization to death, irrespective of cause of death. Time-to-event outcomes were analyzed according to intention to treat. Follow-up was censored at last disease assessment, and in cross-over arms for predictive analysis at 2 years: time of scheduled cross-over. Baseline characteristics were compared using two-sided Fisher’s exact tests or Wilcoxon rank sum test (age and tumor size). AIB1 expression (low/high) was compared via a stratified log-rank test of disease-free and overall survival, with randomization option (two-arm or four-arm) and treatment arm as a stratification factor, and Kaplan–Meier plots were generated. Cox proportional hazards models were used to estimate hazard ratios and 95% confidence intervals stratified by randomization option; multivariable models were adjusted for age at surgery, tumor size, tumor type and grade, estrogen receptor status, HER2 status, and nodal status. Assumptions of proportional hazard were tested using Schoenfeld residuals and by including a time-dependent component. The interactions of treatment by subpopulation of AIB1 were tested by Cox proportional hazards models including treatment groups, an indicator of the subpopulation, and the interaction term, and likewise for interaction of HER2 and the estrogen receptor by subpopulation of AIB1. Level of statistical significance was set to 5%. Statistical analyses were performed with the SAS v9.4 program package. BODY.RESULTS: BODY.AIB1 EXPRESSION AND CORRELATION TO OTHER TUMOR MARKERS: Tissue microarray cores from the primary tumor were available from 1281 (92%) of the 1396 Danish participants in the BIG 1-98 trial. Of these, 1244 (97%) were assessable for AIB1. All had estrogen receptor-positive (≥1%) tumors as confirmed by the central assessment. Estimated median potential follow-up was 9.0 years for all patients with full follow-up, and in cross-over arms 7.9 years. There were 440 disease-free survival events and 310 overall survival events. Patient flow and tumor characteristics are described in Fig. 1 and Table 1, respectively. Excluded patients had a higher frequency of small, node-negative tumors and had an earlier year of surgery. A high AIB1 expression was found in 46% of tumors, similar to results from previous studies [9, 10].Fig. 1Flow chart of inclusion versus exclusion in the study cohort Table 1Demographic table, study population CharacteristicStudy populationExcludedTotal P a N (%) N (%) N (%)All 1244 (100.0) 152 (100.0) 1396 (100.0) Year of surgery  1998–99 285 (22.9) 52 (34.2) 337 (24.1) 0.004  2000–01 559 (44.9) 51 (33.6) 610 (43.7)  2002–03 400 (32.2) 49 (32.2) 449 (32.2) Age at randomization (years)  45–54 148 (11.9) 24 (15.8) 172 (12.3) 0.96  55–64 691 (55.5) 74 (48.7) 765 (54.8)  65–75 405 (32.6) 54 (35.5) 459 (32.9) Tumor size (mm)  0–20 596 (47.9) 88 (57.9) 684 (49.0) 0.002  21–50 606 (48.8) 62 (40.8) 668 (47.9)  51+ 41 (3.3) 2 (1.3) 43 (3.1) Histological type  Invasive Ductal 1036 (83.3) 132 (86.8) 1168 (83.7) 0.52  Invasive Lobular 173 (13.9) 16 (10.5) 189 (13.5)  Other 34 (2.7) 4 (2.6) 38 (2.7) Tumor grade  I 269 (25.0) 41 (30.3) 310 (25.6) 0.31  II 639 (59.5) 78 (57.8) 717 (59.3)  III 166 (15.5) 16 (11.9) 182 (15.1) Number of positive lymph nodes  0 436 (35.1) 69 (45.4) 505 (36.2) 0.04  1–3 531 (42.7) 48 (31.6) 579 (41.5)  4–9 177 (14.2) 21 (13.8) 198 (14.2)  ≥10 99 (8.0) 14 (9.2) 113 (8.1) Estrogen receptor  <1% 0 (0.0) 23 (44.2) 23 (1.8) 0.62  1–79% 217 (17.4) 6 (11.5) 223 (17.2)  80–100% 1027 (82.6) 23 (44.2) 1050 (81.0) AIB1  Low 678 (54.5) 14 (53.8) 692 (54.5) 1.00  High 566 (45.5) 12 (46.2) 578 (45.5)   Score 5 380 (30.6) 8 (30.8) 388 (30.6)   Score 6 186 (15.0) 4 (15.4) 190 (15.0) HER2  Normal 1118 (91.0) 42 (88.0) 1160 (90.9) 0.44  Amplified 110 (9.0) 6 (13.0) 116 (9.1) Unknown values and estrogen receptor values less than 1% were excluded from analysis aFishers exact test, except for age at randomization and tumor size where Wilcoxon rank sum test was used to compare the distribution of non-aggregated data AIB1 amplified in breast cancer 1 High AIB1 expression was associated with a higher tumor grade, HER2 amplification, high estrogen receptor expression (80–100% vs. 1–79%), a high Ki-67, and ductal histological type (Table 2).Table 2AIB1 association with demographic and prognostic variables CharacteristicAIB1LowHighTotal P a N (%) N (%) N (%)All 678 (100.0) 566 (100.0) 1244 (100.0) Year of surgery  1998–99 178 (26.3) 107 (18.9) 285 (22.9) 0.003  2000–01 280 (41.3) 279 (49.3) 559 (44.9)  2002–03 220 (32.4) 180 (31.8) 400 (32.2) Age at randomization (years)  45–54 72 (10.6) 76 (13.4) 148 (11.9) 0.51  55–64 381 (56.2) 310 (54.8) 691 (55.5)  65–75 225 (33.2) 180 (31.8) 405 (32.6) Tumor size (mm)  0–20 329 (48.5) 267 (47.3) 596 (47.9) 0.57  21–50 322 (47.5) 284 (50.3) 606 (48.8)  51+ 27 (4.0) 14 (2.5) 41 (3.3) Histological type  Invasive ductal 541 (79.9) 495 (87.5) 1036 (83.3) 0.001  Invasive lobular 111 (16.4) 62 (11.0) 173 (13.9)  Other 25 (3.7) 9 (1.6) 34 (2.7) Tumor grade  I 184 (32.6) 85 (16.7) 269 (25.0) <.0001  II 334 (59.2) 305 (59.8) 639 (59.5)  III 46 (8.2) 120 (23.5) 166 (15.5) Number of positive lymph nodes  0 237 (35.0) 199 (35.2) 436 (35.1) 0.98  1–3 288 (42.5) 243 (42.9) 531 (42.7)  4–9 99 (14.6) 78 (13.8) 177 (14.2)  ≥10 53 (7.8) 46 (8.1) 99 (8.0) Estrogen receptor 0.006  1–79% 137 (20.2) 80 (14.1) 217 (17.4)  80–100% 541 (79.8) 486 (85.9) 1027 (82.6) HER2  Normal 636 (95.5) 482 (85.8) 1118 (91.0) <.0001b  Amplified 30 (4.5) 80 (14.2) 110 (9.0) KI67  ≤14% 356 (54.9) 214 (38.2) 570 (47.1) <.0001  >14% 293 (45.1) 346 (61.8) 639 (52.9) Treatment armb  Tam 200 (29.5) 164 (29.0) 364 (29.3)  Let 205 (30.2) 175 (30.9) 380 (30.5)  T → L 140 (20.6) 116 (20.5) 256 (20.6)  L → T 133 (19.6) 111 (19.6) 244 (19.6) Unknown values and estrogen receptor values less than 1% were excluded from analysis aFishers exact test, except for age at randomization and tumor size where Wilcoxon rank sum test was used to compare the distribution of non-aggregated data bExclusion of HER2-amplified cases (as in analyses regarding predictive value of AIB1) only marginally changed the percentage of AIB1-high versus AIB1-low tumors in the respective treatment arm (data not shown) AIB1 amplified in breast cancer 1 BODY.AIB1 AS A PROGNOSTIC FACTOR FOR DISEASE-FREE AND OVERALL SURVIVAL: High AIB1 expression was significantly associated with a worse disease-free and overall survival (Table 3; Fig. 2), although for overall survival, significance did not remain in the multivariable analysis. Kaplan–Meier estimates 10 years after randomization showed a disease-free survival of 64% (95% confidence interval 59–68%) for patients with a low AIB1 versus 56% (95% confidence interval 51–61%) for high AIB1. The corresponding numbers for overall survival were 74% (95% confidence interval 69–77%) versus 68% (95% confidence interval 64–73%). An exploratory analysis with a further subdivision of AIB1 into AIB1 score 6 versus score 5 versus score <5 indicated the prognostic effect of AIB1 to be strongest in patients with the highest AIB1 tumor expression (hazard ratio AIB1 score 5: disease-free survival 1.30, overall survival 1.25. Hazard ratio AIB1 score 6: disease-free survival 1.47, overall survival 1.54. AIB1 score <5 as reference. Disease-free survival P = 0.005, overall survival P = 0.02). All analyses were repeated including only HER2-normal and HER2-unknown cases with similar results (data not shown). No difference in association between AIB1 and disease-free/overall survival was seen in relation to HER2 status (disease-free survival P = 0.51, overall survival P = 0.60).Table 3Association between AIB1 (high vs. low), disease-free survival (DFS), and overall survival (OS), respectively PopulationResponse subgroupUnadjusteda Adjustedb HR(95% CI) P HR(95% CI) P All patients DFS 1.35 (1.12–1.63) 0.002 1.29 (1.06–1.58) 0.01 (N = 1244) OS 1.34 (1.07–1.68) 0.01 1.25 (0.99–1.60) 0.07 Only HER2–negative or unknown DFS 1.27 (1.04–1.55) 0.02 1.28 (1.03–1.57) 0.02 (N = 1134) OS 1.24 (0.97–1.58) 0.08 1.23 (0.95–1.58) 0.11 aCox proportional hazards model stratified for two- or four-arm random assignment option and treatment arm bCox proportional hazards model stratified for two- or four-arm random assignment option, treatment arm and age at surgery (45–64 vs. 65–75 year). Model further adjusted for estrogen receptor status (1–79 vs. 80–100%), tumor histological grade (I vs. II, and III vs. II), lymph node status, histological type, and tumor diameter Fig. 2Disease-free survival and overall survival in relation to AIB1. a Disease-free survival in relation to AIB1. b Overall survival in relation to AIB1 BODY.AIB1 AS A PREDICTIVE MARKER FOR TREATMENT WITH TAMOXIFEN VERSUS LETROZOLE: Patients with HER2-amplified tumors were excluded, since these patients would not currently be treated with endocrine therapy alone. Hazard ratios for the treatment effect of letrozole versus tamoxifen were as follows—for disease-free survival: AIB1 low 0.85 (95% confidence interval 0.61–1.18) and AIB1 high 1.08 (95% confidence interval 0.76–1.54), and for overall survival: AIB1 low 0.91 (95% confidence interval 0.60–1.37) and AIB1 high 0.91 (95% confidence interval 0.60–1.41). No treatment effect heterogeneity was found between the AIB1-high and AIB1-low populations (Table 4; Fig. 3). Hence, these data indicate a similar benefit of letrozole versus tamoxifen regardless of AIB1 status.Table 4Treatment effect letrozole versus tamoxifen (N = 1134) ResponseSubgroupAdjusteda HR(95% CI) P P heterogeneityb Disease-free survival All 0.95 (0.75–1.21) 0.68 AIB1 Low 0.85 (0.61–1.18) 0.32 AIB1 High 1.08 (0.76–1.54) Overall survival All 0.91 (0.68–1.23) 0.54 AIB1 Low 0.91 (0.60–1.37) 0.99 AIB1 High 0.91 (0.59–1.41) Only HER2-negative or unknown tumors included. Analysis in monotherapy arms only (follow-up in cross-over arms truncated 2 year after randomization) aCox proportional hazard model stratified for two- or four-arm random assignment option and age at surgery (45–64 vs. 65–75 year). Model further adjusted for estrogen receptor status (1–79 vs. 80–100%), tumor histological grade (I vs. II, and III vs. II), lymph node status, histological type, and tumor diameter bTest for treatment effect heterogeneity Fig. 3Association between AIB1, treatment (tamoxifen/letrozole), and survival. a Disease-free survival. b Overall survival. Analyses made in monotherapy arms only (follow-up in cross-over arms truncated 2 years after randomization). HER2-amplified tumors excluded BODY.DISCUSSION: We have investigated the estrogen receptor coactivator AIB1 as a prognostic and predictive factor in relation to endocrine treatment in the Danish cohort of BIG 1-98 (tamoxifen vs. letrozole). We found 46% of tumors to express high levels of AIB1, which is similar to previous publications [9, 10]. In line with earlier studies, AIB1 correlated to a more aggressive tumor phenotype (HER2 amplification and a high malignancy grade) [5, 7, 8, 23, 24]. In relation to estrogen and progesterone receptor status, results from previous studies have varied. Some show a high AIB1 to be associated with hormone receptor-positive disease [8, 25], while others report an association with estrogen and progesterone receptor negativity [5, 23, 26], or no correlation to receptor status at all [2, 7, 24]. Differences may possibly be explained by different study designs, cut-offs, and study populations. In this cohort, a high AIB1 was associated with a higher estrogen receptor expression. AIB1 has been reported to be a negative prognostic factor, both in estrogen receptor-positive and estrogen receptor-negative breast cancers [9–11, 14, 24, 26–29]. As a result of studies that show an inferior prognosis for AIB1-high tumors in endocrine-treated estrogen receptor-positive breast cancer cohorts [5, 7, 8], AIB1 has previously been suggested to be of importance for endocrine treatment resistance. However, our earlier investigations of a randomized premenopausal estrogen receptor-positive breast cancer trial (2 years adjuvant tamoxifen vs. control) clearly indicate that although AIB1-high tumors had an inferior prognosis early on, these patients responded very well to tamoxifen [10]. AIB1-low tumors had a better prognosis early on, but this was not further improved by tamoxifen. Importantly, these results have been confirmed in a randomized tamoxifen trial including postmenopausal estrogen receptor-positive disease [15]. Hence, we hypothesize that the association between a high AIB1 expression and poor prognosis is related to its prognostic significance, which cannot be entirely eradicated by adjuvant endocrine treatment. The strong prognostic effect of AIB1 makes it a very interesting target for new anti-cancer therapies. Although steroid receptor coactivators are large unstructured proteins making production of drugs against them challenging, there are ongoing efforts to pharmacologically target them [30, 31]. Today there are no markers that predict which population of postmenopausal estrogen receptor-positive breast cancer patients that are likely to have superior benefit from tamoxifen versus aromatase inhibitors. Due to AIB1’s predictive effect in relation to tamoxifen, we postulated that AIB1 status might be a useful marker. Previous trials regarding AIB1’s relation to aromatase inhibitors are very sparse, include small cohorts, and show conflicting results [12, 13]. The data presented here are, to our knowledge, the first time AIB1 is investigated in relation to aromatase inhibitors in a large randomized clinical trial. However, we found no evidence of differences in treatment effect between tamoxifen and letrozole in relation to AIB1 status. Hence, our study indicates that tumor expression of AIB1 cannot be applied as a predictive marker for selection of tamoxifen versus letrozole as adjuvant therapy in postmenopausal endocrine-responsive breast cancer. A relationship between AIB1 and HER2 has previously been suggested, with a worse prognosis with co-expression of AIB1 and HER2 [5, 8, 32]. We found a correlation between a high AIB1 and HER2 amplification. However, in line with our previous studies, no significant interaction between AIB1 and HER2 in relation to prognosis was detected [9, 14]. As in all earlier studies though, AIB1-high, HER2-amplified tumors represented only a small subgroup of the cohort. Although we had the advantage of using a large international controlled randomized trial, this study still has some potential limitations. Most importantly, the BIG 1-98 trial did not include a control group of patients not receiving endocrine therapy. Access to such a group would probably have clarified the prognostic and predictive value of AIB1 even more, especially in relation to letrozole. In addition, after an interim analysis showed a superior effect for letrozole, patients randomized to tamoxifen were allowed a treatment switch, reducing the possibility to detect differences in treatment effect [17]. Furthermore, although a large patient cohort is included, as in all studies, numbers are strongly reduced in subgroup analyses, such as investigations of AIB1-high/HER2-amplified tumors. Finally, although we used a cut-off to define a high AIB1, which has been used in several previous studies, AIB1 is still an explorative biomarker and an optimal cut-off is yet to be definitely determined. In conclusion, in a subset of the BIG 1-98 study population, we confirm tumor expression of AIB1 to be a strong negative prognostic factor. As the association with a high AIB1 and poor prognosis has now been repeatedly shown in different patient cohorts, AIB1 is an interesting target for anti-cancer therapies. However, no difference in treatment effect between tamoxifen and letrozole in relation to AIB1 was found. Hence, AIB1 cannot be of assistance for the choice of type of endocrine treatment in postmenopausal endocrine-responsive disease.

TITLE: Effects on heart function of neoadjuvant chemotherapy and chemoradiotherapy in patients with cancer in the esophagus or gastroesophageal junction – a prospective cohort pilot study within a randomized clinical trial ABSTRACT.BACKGROUND: Neoadjuvant therapy for cancer of the esophagus or gastroesophageal (GE)-junction is well established. The pros and cons of chemoradiotherapy and chemotherapy are debated. Chemoradiotherapy might impair cardiac function eliciting postoperative morbidity. The aim of this pilot study was to describe acute changes in left ventricular function following chemoradiotherapy or chemotherapy. ABSTRACT.METHODS: Patients with esophageal and (GE)-junction cancer enrolled at our center into a multicenter trial comparing neoadjuvant chemoradiotherapy and chemotherapy were eligible. Patients were randomized to receive cisplatin and 5-fluorouracil with or without the addition of 40 Gy radiotherapy prior to surgery. Left ventricular function was evaluated using echocardiography and plasma N-Terminal Pro-B-Type Natriuretic Peptide (NT-proBNP) before and after neoadjuvant treatment. The primary outcome measure was left ventricular global strain (GS). Clinical effects were assessed using repeated exercise tests. Linear mixed models were used to analyze the effects of treatment group, and the interaction between groups. ABSTRACT.RESULTS: 40 patients participated (chemoradiotherapy, n = 17; chemotherapy, n = 23). In the chemoradiotherapy group there was no change in left ventricular global strain but mitral annular plane systolic excursion (MAPSE) of the ventricular septum, early diastolic filling velocity (E-velocity), and the ratio of early to late ventricular filling velocities (E/A ratio) decreased significantly (p = 0.02, p = 0.01, and p = 0.03, respectively). No changes were observed in the chemotherapy group. There was a trend towards an interaction effect for MAPSE sept and E (p = 0.09 and p = 0.09). NT-proBNP increased following chemoradiotherapy (p = 0.05) but not after chemotherapy (p > 0.99), and there was a trend towards an interaction effect (p = 0.07). Working capacity decreased following neoadjuvant treatment (chemoradiotherapy p = 0.001, chemotherapy p = 0.03) and was more pronounced after chemoradiotherapy with a trend towards an interaction effect (p = 0.10). ABSTRACT.CONCLUSIONS: Neoadjuvant chemoradiotherapy but not chemotherapy before surgery for cancer of the esophagus or GE-junction seems to induce an acute negative effect on both systolic and diastolic left ventricular function. Future studies on neoadjuvant treatment for esophageal cancer are suggested to add measurements of cardiac function. ABSTRACT.TRIAL REGISTRATION: Clinical Trials.gov NCT01362127. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13014-014-0310-7) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Neoadjuvant therapy has improved long-term survival after esophagectomy for cancer of the esophagus or gastroesophageal- (GE) junction even though long-term results are still poor [1]. Currently both chemotherapy and chemoradiotherapy are used. Two randomized trials have addressed the advantage of one regimen over the other [2,3]. Outcome results have, however, been criticized either due to small sample size, slow recruitment or lack of power to detect clinically relevant differences in outcomes. Neoadjuvant chemoradiotherapy has been suggested to increase postoperative mortality and possibly also morbidity [4,2,5], although these concerns were not confirmed according to other studies [6,7]. Currently it appears that chemotherapy alone does not increase postoperative morbidity or mortality while it is unclear whether chemoradiotherapy do. Radiation therapy directed towards a tumor in the esophagus or GE-junction will inevitably irradiate the heart. Long-term side effects of modern thoracic radiotherapy include coronary artery disease and heart failure [8], but little is known about acute cardiac effects. Recent studies have demonstrated an acute decrease in left ventricular systolic and diastolic function following radiotherapy, but results are far from consistent [9-12]. Also N-Terminal Pro-B-Type Natriuretic Peptide (NT-proBNP), widely used to diagnose and prognose heart failure [13,14] has been observed to increase following radiotherapy [15]. The incidence of postoperative cardiac complications after esophagectomy is reported to be in the range of 15-30% for both chemo-and chemoradiotherapy [3,16,17]. The aim of this pilot study was to describe possible acute effects of neoadjuvant therapy on left ventricular function using echocardiography and NT-proBNP in patients with cancer of the esophagus or GE-junction. The primary outcome variable was global systolic left ventricular function measured as global strain (GS). The secondary outcome variables were ejection fraction (EF), regional systolic left ventricular function, diastolic function and NT-proBNP. In addition, general physical capacity was measured using repeated exercise test. BODY.PATIENTS AND METHODS: BODY.PATIENT INCLUSION: A multicenter randomized trial of neoadjuvant chemoradiotherapy versus chemotherapy for cancer in the esophagus or GE-junction was conducted between 2006 and 2013 in Sweden and Norway (NeoRes; EudraCTnr 2006-001785-16). The primary endpoint was the rate of complete histological response in the surgical specimen, which is a surrogate marker of improved long-term survival. The study was designed to include 180 patients based on a power calculation suggesting a sample size of 172 patients to achieve the primary endpoint assuming an increase in rate of complete histological response from 20% to 35% with a power of 80%. Inclusion criteria were age 75 years or less, histologically verified squamous cell carcinoma or adenocarcinoma of the esophagus or GE-junction, tumor stage T1-3 and N0-1 (according to AJCC TNM staging system 6th edition) [18], WHO performance status 0–1 and no major illness making neoadjuvant treatment unsuitable. Patients were stratified by histology before randomization. Randomization was done by a computer based program operational at the Regional Oncological Center in Stockholm. The data presented here originate from a cohort of patients within the NeoRes trial scheduled for surgery at the Karolinska University Hospital. Patients underwent an extended protocol with studies of heart function which started in 2008. Added inclusion criteria were planned thoracoabdominal surgery. The NeoRes study protocol was approved by the regional ethics committee (EPN Stockholm 2006/738-32) and was registered at the registration site of the US National Institute of Health (ClinicalTrials.gov NCT01362127). For the extended protocol additional ethics approval was obtained (amendment 2008/1822-32). All patients were given written and oral information and were included after a signed informed consent had been obtained. BODY.NEOADJUVANT TREATMENT: Chemotherapy was given in three cycles of 21 days. Cisplatin 100 mg/m2 was given on day 1 and 5-fluorouracil 750 mg/m2/24 hrs was given on days 1–5. Cisplatin was switched to carboplatin or oxaliplatin in case of hearing impairment or renal dysfunction. Dose reduction was allowed for side effects. In the chemoradiotherapy group, concomitant radiotherapy was administered during cycles two and three with a total dose of 40 Gy; 2 Gy/day, 5 fractions per week. Radiotherapy was planned using a computer tomography-based three-dimensional treatment planning system (EclipseTM, Varian Medical systems, Palo Alto, USA). Treatment was administered using a multiple field technique with optimization of beam entry direction and beam weights in order to achieve a homogenous dose to planning target volume and minimize dose to organs at risk. Field shaping using a multi leaf collimator was used. For tumours located at or above the level of carina, the caudal border of the clinical target volume was 5 cm below diagnosed tumour, whereas the supraclavicular nodes defined the cranial border. For tumours located below the carina, the cranial border of the clinical target volume included 5 cm of radiographically uninvolved esophagus while the coeliac lymph nodes defined the caudal border, down to upper part of L1. In lateral, anterior and posterior directions clinical target volume encompassed gross tumour volume and the paraesophageal area with a margin of 1 cm but not including anatomical barriers as pleura, pericardium or bone. Dose–volume data of the heart including the pericardium with exclusion of the great vessels were extracted from individual dose distribution data in the treatment planning system. Planning target volume receiving >95% of planned radiation dose (PTV > 95%) all well as planning target volume receiving 95–105% of planned dose (PTV 95–105%) was measured. The percentage of the heart volume receiving 10 Gy or more (V10) and 30 Gy or more (V30) was measured. V30 was minimized as much as possible according to protocol. Surgery was scheduled 4–6 weeks after completion of neoadjuvant treatment. BODY.ECHOCARDIOGRAPHY: Strain is a validated, relatively new, echocardiographic parameter for assessment of left ventricular function [19-21]. Strain gives an average of longitudinal shortening in the distance between individual speckles (natural acoustic markers) in the selected view of the ventricle. Global strain (GS) is thus the average strain of segments obtained from one projection. GS was chosen as the primary outcome parameter in this study as it is considered more sensitive for measuring left ventricular function and not as user dependent as EF [22] [23,24]. Other echocardiographic parameters were chosen in accordance with European Association of Echocardiography guidelines [25]. For regional function we measured atrioventricular movement using tissue Doppler, a robust parameter sensitive to early impairment of left ventricular longitudinal systolic function [26]. Echocardiography was performed before start of neoadjuvant therapy and repeated 4–6 weeks after completion of neoadjuvant therapy. A Vivid 7 ultrasound scanner (GE Vingmed, Horten, Norway) and a standard 2.5 MHz transducer were used. All but three exams were performed by the same laboratory technician according to research protocol. Post processing analysis was performed by a single analyser, ML, using Echo PAC (GE Vingmed, Horten, Norway) according to the manufacturer’s instructions. Both the echocardiography examiner and interpreter were blinded to study group allocation. All exams were interpreted after inclusion was closed. A standard cardiac exam was performed on all subjects. One or more loops of three heartbeats were recorded online for each view and the best cardiac cycle selected for analysis during post processing. EF was calculated according to Simpson biplane. Mitral inflow was measured using pulsed wave Doppler. Peak velocity in the early rapid filling phase when the ventricle relaxes (E-Wave) and peak velocity of the late filling due to atrial contraction (A-Wave) were measured by pulsed Doppler across the mitral valve during diastole. Strain was measured using tissue tracing analysis in the apical four-chamber view centred on the left ventricle. For GS analysis a single cycle was used and all analyzed segments were approved by the program as well as by the analyzer. Tissue Doppler-based tissue tracing was used to measure atrio-ventricular plane movement. Mitral annular plane systolic excursion (MAPSE) was measured as the median value of three heart cycles using a region of interest (ROI) of 6×6 mm in the basal septum (sept) and basal anterolateral (lat) wall (Figure 1). Pictures were adjusted for optimal resolution and alignment of cardiac walls with the ultrasound beam.Figure 1 Echocardiographic measurements. Schematic illustration of echocardiographic measurements from apical four-chamber view. EF, ejection fraction measured according to Simpson Biplane; EDV, end diastolic volume; ESV end systolic volume; MAPSE, mitral annular plane systolic excursion; GS, global strain; E and A denotes blood velocities over the mitral valve during diastole. BODY.NT-PROBNP: Venous blood was collected in EDTA tubes before neoadjuvant treatment and on admission for surgery 4–6 weeks after neoadjuvant therapy. Electrochemiluminescence immunoassay with Modular Analytics E170 (Roche Diagnostics, Mannheim, Germany) was used for the analyses, which were all performed by the Karolinska University Hospital accredited laboratory for clinical chemistry. BODY.EXERCISE TEST: Exercise tests of maximum work capacity measured in Watts were completed on a bicycle ergometer under the supervision of a dedicated staff member of an accredited physiology department. Maximum capacity was determined when patients were unable to maintain 60 revolutions per minute due to either, fatigue, shortness of breath or chest pain. The test could also be stopped by the staff due to the occurrence of arrhythmias, electrocardiogram changes or pathological blood pressure reactions. Testing was performed either at Karolinska University Hospital or at the patient’s referring hospital. BODY.STATISTICS: Data were analyzed as intention to treat using a linear mixed model analysis for repeated measures data. The model was used to analyze the effects of within-group change and the interaction between groups. This was defined as the basic model. In addition to the basic model, the influence of different covariates and factors was analyzed. The group effect was the difference between radiotherapy and chemotherapy, and the trend effect was the change between the first measure and the second measure. The within-group change was the difference between the first and second measure in respective groups. The interaction effect was the difference in change between groups. The covariance structure used in the models was unstructured. Before performing analyses, tests of normal distribution were performed and log transformation was performed for non-parametric data. Due to the small data set, several mixed models analyses were performed and in each analysis one of a set of covariates was added to the basic model to test for any impact on the results. The set of covariates was gender, age, BMI, ASA classification, hypertension, ischemic heart disease, smoking, chronic obstructive pulmonary disease, anemia, diabetes, and alcohol abuse. The Bonferroni correction was used to account for multiple comparisons. Patient characteristics are presented as median (range) and data regarding heart function as mean (95% confidence interval). Subject characteristics were analyzed using 2-tailed Mann Whitney U test and 2-tailed Fisher’s exact test as appropriate. BODY.RESULTS: BODY.PATIENT CHARACTERISTICS: A total of 40 patients participated in the study (Figure 2). There were no statistically significant differences between the study groups regarding demographic and disease-specific characteristics as seen in Table 1. Six patients, (two in the chemoradiotherapy group) did not complete neoadjuvant treatment due to adverse events, persisting neutropenia in three cases, renal failure in one, peripheral neuropathy (foot drop) in one and circulatory instability in one. Five patients (two in the chemoradiotherapy group) had a reduced chemotherapy dose due to side effects. One patient had a reduced radiation dose due to thoracic pain. Target radiation dose was achieved in all other patients as shown by >95% and PTV 95–105%. Median PTV >95% was 99% (range 96–100%) and median PTV 95–105% was 97% (range 83–100%). Radiation dose to the heart was defined by V10 and V30. Median V10 to the heart was 74.9% (range 50.0–92.2%) and median V30 was 29.0% (range 0–80.1%).Figure 2 Patient flow chart. Flow chart of screened, enrolled and analyzed patients. Table 1 Patient characteristics Characteristics Chemotherapy (n = 23) Chemoradiotherapy (n = 17) P Age years median (range) 62 (46–71) 66 (56–75) 0.09 Male/Female 19/4 15/2 1.00 BMI median (range) 23 (18–33) 26 (21–35) 0.06 Weight change kg median (range) −10 (−24, +2) −10 (−21, +6) 0.62 Tumor type Adenocarcinoma, n (%) 16 (70) 13 (76) 0.73 Squamous-cell carcinoma, n (%) 7 (30) 4 (24) 0.73 Clinical T stage T1 0 0 _ T2 9 4 _ T3 14 13 _ Clinical N stage N0 5 3 >0.99 N1 18 14 >0.99 Cardiovascular disease, n (%) 7 (30) 10 (59) 0.11 Hypertension, n (%) 7 (30) 9 (53) 0.20 Ischemic heart disease, n (%) 1 (4) 2 (12) 0.56 Smoking ongoing or previous, n (%) 10 (43) 4 (24) 0.32 COPD, n (%) 5 (22) 1 (6) 0.22 Anemia, n (%) 6 (26) 4 (23) 1.00 Diabetes, n (%) 3 (13) 3 (18) 1.00 ASA class, n I/II/III/IV 0/15/8/0 0/10/7/0 0.75 BMI, Body mass index; COPD, chronic obstructive pulmonary disease; ASA, American Society of Anesthesiologists. BODY.ECHOCARDIOGRAPHY: Echocardiographic parameters were similar between groups before the start of neoadjuvant treatment. The neoadjuvant treatment induced no change in GS or EF in either of the groups (Table 2). MAPSE sept decreased significantly in the chemoradiotherapy group (mean change −1.4 mm, CI −2.4, −0.5, p = 0.02) and there was a trend towards an interaction effect (p = 0.09). E/A ratio in the chemoradiotherapy group also decreased significantly (mean change −0.22, CI −0.4, 0.1, p = 0.005) due to a significant decrease in E velocity (mean change −14.7 cm/s, CI −23.8, −5.8, p = 0.03) coupled with an unchanged A velocity (mean change +1.8 cm/s, CI −7.1, 10.7, p = 0.98). A trend towards an interaction effect was found for E velocity (p = 0.09) but not for E/A (p = 0.39). Adding the covariates gender, age, BMI, ASA classification, hypertension, ischemic heart disease, smoking, chronic obstructive pulmonary disease, anemia, diabetes and alcohol abuse to the model did not change the results significantly. See also Figure 2a-b in the additional file for scatterplots [Additional file 1]. No dose–response relationship was found between V30 and the echocardiographic changes (data not shown).Table 2 Results Chemotherapy Chemoradiotherapy Variable Pre neoadjuvant Post neoadjuvant P a Pre neoadjuvant Post neoadjuvant p a p b EF (%) 59 (56–62) 57 (53–60) >0.99 60 (57–64) 59 (55–63) >0.99 0.80 GS (%) −17.6 (−16, −19) −15.7 (−14, −17) 0.26 −17.3 (−16, −19) −16.1 (−14, −18) >0.99 0.59 MAPSE sept (cm/s) 12.5 (11.5–13.5) 12.1 (11.2–13.1) >0.99 12.6 (11.4–13.8) 11.1 (10.1–12.2) 0.02 0.09 MAPSE lat (cm/s) 11.5 (10.4–12.6) 11.2 (10.2–12.3) >0.99 11.2 (10.0–12.4) 11.0 (9.8–12.1) >0.99 0.96 E (cm/s) 72.0 (62.6–81.4) 68.1 (62.2–74.1) >0.99 78.8 (68.4–89.3) 64.1 (57.2–70.9) 0.01 0.09 A (cm/s) 67.8 (58.2–77.5) 74.6 (63.9–85.3) 0.37 82.0 (71.1–92.7) 83.7 (71.6–95.9) 0.98 0.41 E/A 1.08 (0.93–1.25) 0.95 (0.81–1.10) 0.43 0.97 (0.82–1.14) 0.77 (0.65–0.92) 0.03 0.39 NT–ProBNP (ηg/l) 93 (58–149) 108 (70–167) >0.99 65 (32–130) 154 (92–260) 0.05 0.07 Exercise test (W) 150 (135–165) 133 (115–151) 0.03 151 (133–151) 118 (96–140) 0.001 0.10 Data are presented as mean (95% confidence interval). aMixed models test of within-group changes; bMixed models test of interaction effect; EF, ejection fraction; GS, global strain; MAPSE, mitral annular plane systolic excursion; NT-proBNP, N-terminal pro-brain natriuretic peptide. BODY.NT-PROBNP: After neoadjuvant treatment we observed a significant increase of NT-proBNP in the chemoradiotherapy group (mean 140%, CI 27–357%, p = 0.05), but no significant change after chemotherapy (mean 14%, CI 1–82%, p > 0.99) and a trend towards an interaction effect (p = 0.07) (Table 2). The addition of the covariates listed above did not change results significantly. See also Figure 2c in the additional file for scatterplot [Additional file 1]. No dose–response relationship was found between V30 and the NT-proBNP changes (data not shown). BODY.EXERCISE TEST: All tests were stopped by the patient due to fatigue or shortness of breath without indications of cardiac ischemia. Both neoadjuvant regimens were followed by a significant decrease in the patients working capacity (p = 0.03 and p = 2). The addition of the covariates listed above did not change results significantly. See also Figure 2d in the additional file for scatterplot [Additional file 1]. BODY.DISCUSSION: Cardiac effects of neoadjuvant chemoradiotherapy in the treatment of cancer of the esophagus or GE-junction are important as they could have direct implications on anesthetic management and postoperative management as well as imply a need to individualize neoadjuvant treatment. This study provides results indicating that neoadjuvant chemoradiotherapy for cancer of the esophagus or GE-junction induces an acute impairment of heart function whereas neoadjuvant chemotherapy does not. Although we did not find any effect on our primary outcome variable global strain, a small but statistically significant decrease in septal function, was observed after chemoradiotherapy. The septum receives the highest radiation doses during chemoradiotherapy for esophageal cancer as shown by Hatakenaka et al. using magnetic resonance imaging [10]. In that study, regional wall motion was decreased for the mid anteroseptal, mid inferoseptal and mid inferior segments, which is in accordance with our findings. Moreover, in the study by Hatakenaka the largest decrease in wall movements was observed in the palliative patients who received the highest radiation doses indicating a dose-dependent response. Two retrospective studies have shown a decrease of EF after chemoradiotherapy using different combinations of chemotherapeutic agents and again using higher radiation doses (45–50 Gy) [11,27]. We were unable to show an effect on global systolic function, which may either be related to our small sample size or to the use of lower radiation doses. Chemoradiotherapy also decreased the blood flow velocities over the mitral valve during the fast, passive filling phase of the left ventricle (E), coupled with an unchanged blood flow during atrial contraction (A) and accordingly a decreased E/A. These data suggest an impaired diastolic function as a consequence of impaired relaxation of the left ventricle. Hatakenaka and coworkers also reported an impairment of left ventricular relaxation after radiotherapy [10]. Neoadjuvant chemoradiotherapy also increased NT-proBNP. This biomarker has been studied as a predictor for the risk of perioperative cardiac complications with cutoff values between 201–791 ηg/ml being suggested [28,29]. Pre and perioperative levels of NT-proBNP are also strong predictors for atrial fibrillation even if cutoff levels are under debate [30]. We were unable to demonstrate a dose–response relationship between V30 and the echocardiographic changes or NT-proBNP levels. This may be due to the small number of patients enrolled but also dependent on the fact that V30 reflects the radiation dose targeting the whole heart rather than different segments. We refrained from doing a segmental analysis since this would require a larger dataset. The clinical relevance of our findings is unclear. The level of systolic impairment detected was small and probably not clinically significant in a patient with an otherwise well-functioning left ventricle. Diastolic changes were larger with mean E/A levels after neoadjuvant therapy reaching grade I diastolic dysfunction which could have clinical implications. NT-proBNP levels increased following chemoradiotherapy and might indicate an increased risk for postoperative cardiac events and atrial fibrillation. This also shows that repeated NT-proBNP measurements could be a simple method to describe impairment from neoadjuvant treatment in future studies. Preoperative working capacity, as assessed during an exercise test, trajects into perioperative risks after esophagectomy [31,32]. We also observed that currently practiced neoadjuvant therapies, decreased the work capacity significantly in both groups. The effect was more pronounced in the chemoradiotherapy group but there was no significant interaction effect. Cisplatin and 5-fluorouracil are commonly used drugs in chemotherapy regimens for esophageal cancer. These drugs are known to be associated with cardiotoxic side effects [33]. Therefore it was interesting to note that we were unable to detect echocardiographic or biochemical signs of decreased left ventricular function from chemotherapy alone. The question whether neoadjuvant chemoradiotherapy is associated with an increased risk of postoperative morbidity compared with chemotherapy remains unresolved [5,7]. Only two previous randomized trials (n = 74 and n = 119) have directly compared neoadjuvant chemotherapy versus chemoradiotherapy [2,3]. However, functional studies of the heart were not a part of any of these prospective protocols. Impaired left ventricular systolic and diastolic function following neoadjuvant chemoradiotherapy could well have a bearing on the incidence and grade of the innately high postoperative cardiovascular and pulmonary morbidity after esophagectomy. Taken together our data emphasize the relevance of dedicated studies aimed at further clarifying the details and consequences of the cardiotoxicity of current chemoradiotherapy regimens. One pathway that needs to be explored is cardiovascular function during the perioperative period. One important limitation of our study is the small sample size, where the cases represented a cohort of patients consecutively enrolled from a larger, multicenter, randomized study. Two issues emerge as a consequence of this: the power of the observations and the possibility of selection bias. It should however, be emphasized that Karolinska University Hospital was the largest including center and the patients in our cohort represent 23% of the total study population. The randomization was not stratified to each center which may explain the small difference in the size of the treatment groups. There were no statistical differences in comorbidities between the study groups and the subsequent analysis of the effects of comorbidities in the linear mixed models did not display any significant impact on the results. Work capacity was also similar between the groups before neoadjuvant treatment suggesting comparable patients groups. The second limitation, which we tried to confront, was the missing values within our cohort. The design of the present clinical study was very complex in a logistic perspective, in that we were faced with a short timeframe during which we had to plan and complete the echocardiographic examinations, i.e. from the randomization to the start of neoadjuvant treatment. During this limited time frame several other investigations and procedures (central lines for chemotherapy, PET-CT, endoscopic ultrasonography, respiratory and exercise test) had to be performed, usually at other hospitals. In addition, we chose to concentrate the echocardiography investigations at the tertiary referral center to increase the validity of the echocardiography. In order to partially compensate for the impact of missing data we adopted a linear mixed model statistics where the patients were analyzed as intention to treat. Linear mixed models give the opportunity to analyze all available data and not having to exclude cases where the dataset is incomplete. It also gives the option to correct for co-variables and is not restricted to a spherical or compound symmetry where we rely on the assumption that data are missing at random. By doing this we were able to mitigate some of the impact of missing data and further explore how patient comorbidity affected the results. No effect was found. BODY.CONCLUSIONS: Neoadjuvant chemoradiotherapy seems to induce a slight acute impairment of both systolic and diastolic left ventricle function, whereas chemotherapy does not. The systolic impairment was small and probably not clinically significant in a patient with an otherwise well-functioning left ventricle. The change in diastolic function was larger and might have clinical implications. These effects of chemoradiotherapy may enhance the risk of postoperative morbidity and should be taken into clinical consideration in patients with cardiac comorbidity. Corresponding and related effects on the heart need to be further explored in future studies of neoadjuvant chemoradiotherapy for cancer of the esophagus and GE-junction.

TITLE: Efficacy and safety of canagliflozin in subjects with type 2 diabetes and chronic kidney disease ABSTRACT.AIMS: Canagliflozin is a sodium glucose co-transporter 2 inhibitor in development for treatment of type 2 diabetes mellitus (T2DM). This study evaluated the efficacy and safety of canagliflozin in subjects with T2DM and stage 3 chronic kidney disease [CKD; estimated glomerular filtration rate (eGFR) ≥30 and <50 ml/min/1.73 m2]. ABSTRACT.METHODS: In this randomized, double-blind, placebo-controlled, phase 3 trial, subjects (N = 269) received canagliflozin 100 or 300 mg or placebo daily. The primary efficacy endpoint was change from baseline in HbA1c at week 26. Prespecified secondary endpoints were change in fasting plasma glucose (FPG) and proportion of subjects reaching HbA1c <7.0%. Safety was assessed based on adverse event (AE) reports; renal safety parameters (e.g. eGFR, blood urea nitrogen and albumin/creatinine ratio) were also evaluated. ABSTRACT.RESULTS: Both canagliflozin 100 and 300 mg reduced HbA1c from baseline compared with placebo at week 26 (–0.33, –0.44 and –0.03%; p < 0.05). Numerical reductions in FPG and higher proportions of subjects reaching HbA1c < 7.0% were observed with canagliflozin 100 and 300 mg versus placebo (27.3, 32.6 and 17.2%). Overall AE rates were similar for canagliflozin 100 and 300 mg and placebo (78.9, 74.2 and 74.4%). Slightly higher rates of urinary tract infections and AEs related to osmotic diuresis and reduced intravascular volume were observed with canagliflozin 300 mg compared with other groups. Transient changes in renal function parameters that trended towards baseline over 26 weeks were observed with canagliflozin. ABSTRACT.CONCLUSION: Canagliflozin improved glycaemic control and was generally well tolerated in subjects with T2DM and Stage 3 CKD. BODY.INTRODUCTION: Progression of chronic kidney disease (CKD), leading to end-stage renal failure, is a common complication in patients with type 2 diabetes mellitus (T2DM) 1. Antihyperglycaemic agent (AHA) treatment options are limited as a number of classes of agents may have decreased efficacy and be associated with increased risk of adverse effects in patients with CKD 2. For example, there are labelled restrictions on the use of thiazolidinediones, metformin, sulphonylureas, and, more recently, glucagon-like peptide-1 (GLP-1) agonists in this cohort 3,4. Moreover, AHAs commonly used in this population, such as sulphonylureas, have been associated with an increased risk of hypoglycaemia and weight gain 3–5. Thus, new treatment options are needed for this growing population of patients with co-existing T2DM and renal insufficiency 1. Canagliflozin is an inhibitor of the sodium glucose co-transporter 2 (SGLT2) in development for the treatment of patients with T2DM. Canagliflozin lowers the renal threshold for glucose (RTG) and increases urinary glucose excretion (UGE), resulting in decreased plasma glucose in patients with hyperglycaemia, as well as a mild osmotic diuresis and a net caloric loss (by loss of glucose) promoting weight loss 6–10. Because the rate of UGE is proportional to the glomerular filtration rate (GFR) (as well as to the blood glucose concentration) 7,8,11, the effect of canagliflozin to augment UGE would be anticipated to be diminished in subjects with CKD. Therefore, the efficacy of canagliflozin in improving glycaemic control and reducing body weight may be affected in this subject population. In addition to assessing the efficacy response to canagliflozin in this population, it is also important to assess the safety and tolerability profile of SGLT2 inhibition in subjects with CKD. This phase 3 study evaluated the efficacy and safety of canagliflozin compared with placebo in subjects with inadequately controlled T2DM and stage 3 CKD. This study included subjects with a lower, more restricted estimated glomerular filtration rate (eGFR) range of ≥30 and <50 ml/min/1.73 m2 based upon the modification of diet in renal disease (MDRD) equation 12, compared with the typical eGFR range for stage 3 CKD of ≥30 and <60 ml/min/1.73 m2 . BODY.MATERIALS AND METHODS: BODY.STUDY DESIGN AND SUBJECTS: This 52-week, randomized, double-blind, placebo-controlled, phase 3 study was conducted at 89 centres in 19 countries and consisted of an AHA adjustment period (if required; consisting of a dose titration period of up to 4 weeks and an 8-week dose stable period); a 2-week, single-blind, placebo run-in period; a 26-week, double-blind, core treatment period; and a 26-week, double-blind, extension period (data to be reported in a separate publication). Eligible subjects were men and women aged ≥25 years with T2DM who had inadequate glycaemic control (HbA1c ≥7.0 and ≤10.5%) and stage 3 CKD (eGFR ≥30 and <50 ml/min/1.73 m2), and were either not on AHA therapy or were on a stable AHA regimen (monotherapy or combination therapy with any approved agent including metformin, sulphonylurea, dipeptidyl peptidase-4 (DPP-4) inhibitor, α-glucosidase inhibitor, GLP-1 analogue, pioglitazone or insulin) for ≥8 weeks (≥12 weeks with pioglitazone) prior to the week –2 visit. Subjects were required to have generally stable renal function, as determined by a ≤25% decrease in eGFR from the screening to the week –2 visits. Subjects on AHA regimens not consistent with local prescribing guidelines (e.g. metformin therapy) underwent an AHA adjustment period of up to 12 weeks before the placebo run-in period. Subjects were to remain on their stable AHA regimens through the completion of the 52-week treatment period (unless glycaemic rescue criteria were met, as discussed below). Subjects were excluded if they had repeated fasting plasma glucose (FPG) >15.0 mmol/l (270 mg/dl) during the pretreatment phase; a history of T1DM; renal disease that required immunosuppressive therapy, dialysis or transplant; nephrotic syndrome or inflammatory renal disease; New York Heart Association Class III-IV cardiovascular disease; myocardial infarction, unstable angina, revascularization procedure or cerebrovascular accident within 3 months prior to screening; or haemoglobin concentration <100 g/l (10 g/dl) at screening. The study protocol and amendments were approved by the institutional review boards at participating institutions and the study was conducted under the guidelines of Good Clinical Practices and the Declaration of Helsinki. All subjects provided written informed consent prior to participation. BODY.RANDOMIZATION AND STUDY TREATMENTS: Eligible subjects were randomly assigned to receive once-daily oral doses of canagliflozin 100 or 300 mg or placebo in a 1 : 1 : 1 ratio using an Interactive Voice Response System/Interactive Web Response System. Randomization was balanced by using permuted blocks of six subjects per block and stratified based on (i) the presence or absence of atherosclerotic cardiovascular disease (e.g. history of myocardial infarction, documented angina, transient ischemic attack or stroke or peripheral vascular disease) and (ii) whether a subject required an AHA adjustment period prior to randomization. During the double-blind, core treatment period, glycaemic rescue therapy (up-titration of current AHAs or step-wise addition of oral or non-oral AHAs) was initiated if FPG >15.0 mmol/l (270 mg/dl) after day 1 to week 6, >13.3 mmol/l (240 mg/dl) after week 6 to week 12, and >11.1 mmol/l (200 mg/dl) after week 12 to week 26. After randomization, HbA1c and FPG values were masked to the study centres unless these values met the prespecified glycaemic criteria for the initiation of rescue medication or after glycaemic rescue medication was started. BODY.STUDY ENDPOINTS AND ASSESSMENTS: The prespecified primary efficacy endpoint was the change from baseline in HbA1c at week 26. Prespecified secondary efficacy endpoints evaluated at week 26 were the proportion of subjects reaching HbA1c <7.0% and change from baseline in FPG. Other efficacy endpoints included change from baseline in blood pressure (BP) and percent change from baseline in body weight and fasting plasma lipids. Overall safety and tolerability were assessed by adverse event (AE) reports, safety laboratory tests, vital sign measurements, physical examinations and 12-lead electrocardiograms. Selected AEs of interest, including genital mycotic infections and urinary tract infections (UTIs), were prespecified for additional data collection. Events of hypoglycaemia were collected using a separate case report form that collected concurrent fingerstick glucose values and the presence of symptoms indicating a severe event (i.e. requiring the assistance of another individual or resulting in seizure or loss of consciousness). Measures of renal function, including eGFR, serum creatinine, blood urea nitrogen (BUN) and urine albumin/creatinine ratio (ACR) were also assessed. BODY.STATISTICAL ANALYSES: Sample size calculation was based on demonstrating the superiority of canagliflozin to placebo, as measured by the change in HbA1c from baseline to week 26. An estimated 61 randomized subjects per group were needed to achieve ≥90% power, assuming a group difference of 0.5% and a common standard deviation (SD) of 0.85% (based on relevant information from patients with T2DM and renal impairment), and using a two-sample, two-sided t-test with a type I error rate of 0.05. In order to provide additional safety information for canagliflozin, this study planned a modestly greater sample size of 80 randomized subjects per treatment group (∼240 total subjects) for enrolment. Efficacy analyses were conducted using the modified intent-to-treat (mITT) population, which consisted of all randomized subjects who received ≥1 dose of study drug, according to the randomized treatment assignment. The last observation carried forward (LOCF) approach was used to impute missing data. If subjects received rescue therapy, all postrescue data were censored and the last postbaseline value prior to the initiation of rescue therapy was used for analyses. Safety analyses were performed in randomized subjects who received ≥1 dose of study drug according to the predominant treatment received (the allocation of treatment assignment in the efficacy and safety analyses were the same as no subject took incorrect double-blind study drug for a predominant part of the double-blind treatment period). Primary and continuous secondary efficacy endpoints were assessed using an analysis of covariance (ancova) model with treatment and stratification factors as fixed effects and corresponding baseline values and baseline eGFR as covariates. Least squares (LS) mean differences and two-sided 95% confidence intervals (CIs) were estimated based on this model for the comparison of each canagliflozin group versus placebo. The categorical secondary endpoint (proportion of subjects reaching HbA1c < 7.0%) was analyzed using a logistic model with treatment and stratification factors as fixed effects and baseline HbA1c and eGFR values as covariates. Renal safety parameters, including change in eGFR and ACR, were analyzed using an ancova model with treatment and stratification factors as fixed effects and adjusting for the baseline covariate. Differences in LS means between groups (each canagliflozin dose vs. placebo) and two-sided 95% CIs were estimated. All statistical tests were interpreted at a two-sided significance level of 5% and all CIs at a two-sided confidence level of 95%. A closed testing of prespecified primary and secondary endpoints based on the treatment difference was implemented in order to preserve the overall type I error rate at 5%. The p values for the treatment comparisons were calculated and are reported for prespecified comparisons only. If a prespecified comparison was not found to be statistically significant, subsequent prespecified tests were not to be conducted; descriptive statistics (95% CI for between-group differences) are provided. BODY.RESULTS: BODY.SUBJECT DISPOSITION AND BASELINE CHARACTERISTICS: Of the 272 randomized subjects, 269 received ≥1 dose of study drug and were included in the mITT analysis population (figure 1). A total of 35 (12.9%) subjects discontinued before the week 26 visit, with fewer discontinuations in the canagliflozin 300 mg group compared with the canagliflozin 100 mg and placebo groups. A smaller proportion of subjects treated with canagliflozin 100 or 300 mg received glycaemic rescue therapy before the week 26 visit compared with those treated with placebo (4.4, 3.3 and 14.3%, respectively). Baseline demographic and disease characteristics were similar across treatment groups (Table 1). Mean baseline HbA1c was 8.0%, mean age was 68.5 years and mean body mass index was 33.0 kg/m2; the mean duration of T2DM for subjects was 16.3 years. Mean baseline eGFR was 39.4 ml/min/1.73 m2 and median baseline ACR was 30.0 µg/mg. Approximately 80% of subjects had a history of ≥1 diabetic microvascular complication, with nephropathy being the most common complication. A total of 98% of subjects were on background AHA therapy at baseline, with insulin (74%) and sulphonylureas (31%) being the most common background therapies (Table 2). Most subjects were on antihypertensive therapy (Table 1), including agents acting on the renin-angiotensin system (87%), diuretics (73%), β-blocking agents (56%) and calcium channel blockers (42%). Figure 1Study flow diagram. PBO, placebo; CANA, canagliflozin; mITT, modified intent-to-treat. *mITT analysis set. Table 1Baseline demographic and disease characteristics* CharacteristicPBO (n = 90)CANA 100 mg (n = 90)CANA 300 mg (n = 89)Total (N = 269)Sex, n (%)  Male 57 (63.3) 58 (64.4) 48 (53.9) 163 (60.6)  Female 33 (36.7) 32 (35.6) 41 (46.1) 106 (39.4) Age, years 68.2 ± 8.4 69.5 ± 8.2 67.9 ± 8.2 68.5 ± 8.3 Race, n (%)†  White 78 (86.7) 71 (78.9) 66 (74.2) 215 (79.9)  Black or African American 0 3 (3.3) 2 (2.2) 5 (1.9)  Asian 7 (7.8) 9 (10.0) 11 (12.4) 27 (10.0)  Other‡ 5 (5.6) 7 (7.8) 10 (11.2) 22 (8.2) HbA1c, % 8.0 ± 0.9 7.9 ± 0.9 8.0 ± 0.8 8.0 ± 0.9 FPG, mmol/l (mg/dl) 8.9 ± 2.4 (160.4 ± 43.2) 9.4 ± 2.6 (169.4 ± 46.3) 8.8 ± 3.2 (158.6 ± 58.0) 9.1 ± 2.8 (164.0 ± 49.6) Body weight, kg 92.8 ± 17.4 90.5 ± 18.4 90.2 ± 18.1 91.2 ± 18.0 BMI, kg/m2 33.1 ± 6.5 32.4 ± 5.5 33.4 ± 6.5 33.0 ± 6.2 Duration of T2DM, years 16.4 ± 10.1 15.6 ± 7.4 17.0 ± 7.8 16.3 ± 8.5 eGFR, ml/min/1.73 m2 40.1 ± 6.8 39.7 ± 6.9 38.5 ± 6.9 39.4 ± 6.9 Median ACR, µg/mg 31.3 23.7 30.1 30.0 Microvascular complications, n (%) 74 (82.2) 73 (81.1) 69 (77.5) 216 (80.3)  Neuropathy 45 (50.0) 36 (40.0) 38 (42.7) 119 (44.2)  Retinopathy 25 (27.8) 27 (30.0) 36 (40.4) 88 (32.7)  Nephropathy 61 (67.8) 69 (76.7) 65 (73.0) 195 (72.5) History of ASCVD, n (%) 51 (56.7) 50 (55.6) 46 (51.7) 147 (54.6) Antihypertensive therapy at baseline, n (%)  Agents acting on the renin-angiotensin system 77 (85.6) 79 (87.8) 79 (88.8) 235 (87.4)  Diuretics 62 (68.9) 65 (72.2) 70 (78.7) 197 (73.2)  β-blocking agents 50 (55.6) 51 (56.7) 50 (56.2) 151 (56.1)  Calcium channel blockers 33 (36.7) 40 (44.4) 39 (43.8) 112 (41.6) Antihyperlipidemic therapy at baseline, n (%) 70 (77.8) 74 (82.2) 68 (76.4) 212 (78.8) PBO, placebo; CANA, canagliflozin; FPG, fasting plasma glucose; BMI, body mass index; T2DM, type 2 diabetes mellitus; eGFR, estimated glomerular filtration rate; ACR, albumin/creatinine ratio; ASCVD, atherosclerotic cardiovascular disease; SD, standard deviation. *Data are mean ± SD unless otherwise indicated. †Percentages may not total 100.0% due to rounding. ‡Includes American Indian or Alaska Native, Native Hawaiian or other Pacific Islander, other and unknown. Table 2AHA therapies at baseline (mITT) Subjects, n (%)PBO (n = 90)CANA 100 mg (n = 90)CANA 300 mg (n = 89)Total (N = 269)Total subjects with AHA therapy 88 (97.8) 87 (96.7) 88 (98.9) 263 (97.8) AHAs (alone or in combination)  Sulphonylureas 33 (36.7) 24 (26.7) 27 (30.3) 84 (31.2)  Thiazolidinediones* 7 (7.8) 3 (3.3) 7 (7.9) 17 (6.3)  DPP-4 inhibitors 5 (5.6) 7 (7.8) 8 (9.0) 20 (7.4)  Biguanide 1 (1.1) 1 (1.1) 2 (2.2) 4 (1.5)  Other AHAs† 7 (7.8) 6 (6.7) 10 (11.2) 23 (8.6)  Insulin‡ 66 (73.3) 67 (74.4) 66 (74.2) 199 (74.0) Combinations§  Sulphonylurea + insulin 11 (12.2) 7 (7.8) 10 (11.2) 28 (10.4)  Other AHA¶ + insulin 12 (13.3) 8 (8.9) 10 (11.2) 30 (11.2)  Biguanide + insulin 0 1 (1.1) 2 (2.2) 3 (1.1)  Biguanide + sulphonylurea 1 (1.1) 0 0 1 (0.4) AHA, antihyperglycaemic agent; mITT, modified intent-to-treat; PBO, placebo; CANA, canagliflozin; DPP-4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1. *All subjects were on pioglitazone. †Including α-glucosidase inhibitors, GLP-1 agonists, glinides and other AHAs. ‡Including basal + bolus insulin, basal insulin alone and bolus insulin alone. §Subset of subjects on combinations of the AHAs listed above. ¶Including α-glucosidase inhibitors, thiazolidinediones, DPP-4 inhibitors, GLP-1 agonists, glinides and other AHAs. BODY.EFFICACY: BODY.GLYCAEMIC EFFICACY ENDPOINTS: HbA1c was significantly reduced from baseline with canagliflozin 100 and 300 mg compared with placebo at week 26 (figure 2A). Differences in LS mean changes relative to placebo were –0.30% for canagliflozin 100 mg (p < 0.05) and –0.40% for canagliflozin 300 mg (p < 0.001). A numerically higher proportion of subjects treated with canagliflozin 100 or 300 mg than with placebo achieved HbA1c < 7.0% at week 26 (27.3, 32.6 and 17.2%, respectively; figure 2B). Canagliflozin 100 and 300 mg provided numerically greater reductions from baseline in FPG at week 26 compared with placebo (figure 2C), with differences in LS mean changes (95% CI) of –0.85 (–1.6, –0.1) and –0.67 (–1.4, 0.1) mmol/l [–15.4 (–28.5, –2.3) and –12.2 (–25.4, 1.0) mg/dl], respectively. The comparison of canagliflozin 300 mg versus placebo in change in FPG was not statistically significant and, therefore, the statistical comparison of canagliflozin 100 mg versus placebo did not proceed based on the closed testing procedure. Figure 2Effects on efficacy parameters (LOCF). Change in HbA1c (A), proportion of subjects reaching HbA1c <7.0% (B), change in FPG (C), and percent change in body weight (D). LOCF, last observation carried forward; FPG, fasting plasma glucose; PBO, placebo; CANA, canagliflozin; LS, least squares; SE, standard error; CI, confidence interval; NS, not significant. *Statistical comparison for CANA versus PBO not performed owing to multiplicity control. †p = NS for CANA versus PBO. ‡Statistical comparison for CANA versus PBO not performed (not prespecified). BODY.OTHER EFFICACY ENDPOINTS: Canagliflozin 100 and 300 mg provided reductions from baseline in body weight over 26 weeks, whereas placebo was associated with a slight increase in body weight (figure 2D). Differences in LS mean percent changes (95% CI) relative to placebo at week 26 were –1.6% (–2.3, –0.8) and –1.8% (–2.6, –1.0) for canagliflozin 100 and 300 mg, respectively, corresponding to absolute changes of –1.4 and –1.6 kg, respectively. Both canagliflozin 100 and 300 mg were associated with greater decreases from baseline in systolic and diastolic BP compared with placebo at week 26 (LS mean changes of –6.1, –6.4 and –0.3 mmHg, respectively, in systolic BP and –2.6, –3.5 and –1.4 mmHg, respectively, in diastolic BP; Table 3). No notable changes in pulse rate were observed with canagliflozin 100 or 300 mg compared with placebo (mean change of –1.9, –1.1 and –2.5 beats/min, respectively). Canagliflozin 100 and 300 mg increased high-density lipoprotein cholesterol (HDL-C) compared with placebo (LS mean percent changes of 4.0, 3.0 and 1.5%, respectively; Table 3). An increase in triglycerides (LS mean percent changes of 11.9, 6.2 and 7.9%, respectively) and a decrease in low-density lipoprotein cholesterol (LDL-C; LS mean percent changes of –1.0, 6.4 and 6.3%, respectively) were seen with canagliflozin 300 mg compared with canagliflozin 100 mg and placebo. There was no notable difference in non–HDL-C between the canagliflozin 300 mg and placebo groups (LS mean percent changes of 2.8 and 3.8%, respectively). Table 3Summary of blood pressure and fasting plasma lipid findings at week 26 (LOCF)* ParameterPBO (n = 90)CANA 100 mg (n = 90)CANA 300 mg (n = 89)Systolic BP, n 89 90 89  Mean ± SD baseline, mmHg 132.1 ± 13.6 135.9 ± 13.1 136.7 ± 15.0  LS mean ± SE change –0.3 ± 1.5 –6.1 ± 1.5 –6.4 ± 1.5  Difference versus PBO (95% CI) –5.7 (–9.5, –1.9) –6.1 (–10.0, –2.3) Diastolic BP, n 89 90 89  Mean ± SD baseline, mmHg 73.9 ± 9.0 73.5 ± 8.8 75.7 ± 7.8  LS mean ± SE change –1.4 ± 0.9 –2.6 ± 0.9 –3.5 ± 0.9  Difference versus PBO (95% CI) –1.2 (–3.4, 1.0) –2.1 (–4.3, 0.2) Triglycerides, n 75 82 85  Mean ± SD baseline, mmol/l (mg/dl) 2.0 ± 1.1 (179.4 ± 96.2) 1.9 ± 0.9 (164.9 ± 81.1) 2.1 ± 1.2 (189.3 ±103.5)  LS mean ± SE change, mmol/l (mg/dl) –0.01 ± 0.11 (–0.7 ± 10.0) 0.02 ± 0.11 (1.8 ± 9.7) 0.22 ± 0.11 (19.5 ± 9.5)  Median (IQR) percent change 2.8 (–18.4, 22.8) –1.0 (–20.3, 17.3) 2.5 (–16.1, 25.8)  LS mean ± SE percent change 7.9 ± 4.8 6.2 ± 4.6 11.9 ± 4.6  Difference versus PBO (95% CI) –1.7 (–13.8, 10.5) 3.9 (–8.1, 15.9) LDL-C, n 75 82 84  Mean ± SD baseline, mmol/l (mg/dl) 2.5 ± 1.0 (96.3 ± 39.0) 2.4 ± 0.9 (91.3 ± 33.4) 2.3 ± 0.9 (87.2 ± 33.9)  LS mean ± SE change, mmol/l (mg/dl) 0.06 ± 0.08 (2.4 ± 3.0) 0.09 ± 0.08 (3.5 ± 2.9) –0.08 ± 0.08 (–3.1 ± 2.9)  Median (IQR) percent change 0.0 (–14.2, 22.3) 1.3 (–9.0, 15.5) 0.2 (–16.7, 16.1)  LS mean ± SE percent change 6.3 ± 3.6 6.4 ± 3.5 –1.0 ± 3.4  Difference versus PBO (95% CI) 0.1 (–8.9, 9.2) –7.2 (–16.3, 1.8) HDL-C, n 75 82 85  Mean ± SD baseline, mmol/l (mg/dl) 1.1 ± 0.3 (42.6 ± 10.0) 1.1 ± 0.2 (43.1 ± 8.6) 1.2 ± 0.3 (44.3 ± 12.2)  LS mean ± SE change, mmol/l (mg/dl) 0.00 ± 0.02 (0.1 ± 0.7) 0.03 ± 0.02 (1.3 ± 0.7) 0.02 ± 0.02 (0.8 ± 0.7)  Median (IQR) percent change 1.6 (–7.3, 8.2) 2.3 (–6.6, 10.9) 2.1 (–7.3, 11.8)  LS mean ± SE percent change 1.5 ± 1.8 4.0 ± 1.7 3.0 ± 1.7  Difference versus PBO (95% CI) 2.5 (–1.9, 7.0) 1.5 (–3.0, 5.9) LDL-C/HDL-C, n 75 82 84  Mean ± SD baseline, mol/mol 2.3 ± 1.0 2.2 ± 0.9 2.1 ± 0.8  LS mean ± SE change 0.04 ± 0.08 0.02 ± 0.07 –0.15 ± 0.07  Median (IQR) percent change –0.5 (–12.9, 16.6) –2.2 (–13.0, 13.8) 3.3 (–19.5, 12.4)  LS mean ± SE percent change 4.7 ± 3.8 4.7 ± 3.7 –4.3 ± 3.7  Difference versus PBO (95% CI) 0.0 (–9.7, 9.7) –8.9 (–18.6, 0.8) Non–HDL-C, n 75 81 85  Mean ± SD baseline, mmol/l (mg/dl) 3.4 ± 1.1 (131.9 ± 41.9) 3.2 ± 0.9 (123.9 ± 36.2) 3.3 ± 1.0 (125.7 ± 39.4)  LS mean ± SE change, mmol/l (mg/dl) 0.06 ± 0.09 (2.2 ± 3.5) 0.10 ± 0.09 (4.0 ± 3.4) 0.02 ± 0.09 (0.9 ± 3.4)  Median (IQR) percent change –1.0 (–13.7, 15.0) 0.8 (–9.3, 12.0) 1.9 (–11.5, 12.0)  LS mean ± SE percent change 3.8 ± 2.9 5.1 ± 2.8 2.8 ± 2.8  Difference versus PBO (95% CI) 1.2 (–6.1, 8.6) –1.1 (–8.3, 6.2) LOCF, last observation carried forward; PBO, placebo; CANA, canagliflozin; BP, blood pressure; SD, standard deviation; LS, least squares; SE, standard error; CI, confidence interval; IQR, interquartile range; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol. *Statistical comparison for CANA 100 and 300 mg versus PBO not performed (not prespecified). BODY.SAFETY: BODY.OVERALL SAFETY AND TOLERABILITY: The overall incidence of AEs, serious AEs and study discontinuations due to AEs was similar for canagliflozin 100 and 300 mg and placebo (Table 4). The incidence of drug-related AEs was higher in both canagliflozin groups compared with placebo, largely because of a higher incidence of several specific AEs discussed below. Table 4Summary of overall safety and selected AEs* Subjects, n (%)PBO (n = 90)CANA 100 mg (n = 90)CANA 300 mg (n = 89)Any AE 67 (74.4) 71 (78.9) 66 (74.2) AEs leading to discontinuation 5 (5.6) 4 (4.4) 2 (2.2) AEs related to study drug† 20 (22.2) 23 (25.6) 29 (32.6) Serious AEs 16 (17.8) 10 (11.1) 10 (11.2) Deaths 1 (1.1) 1 (1.1) 0 Selected AEs  UTI 5 (5.6) 5 (5.6) 7 (7.9)  Genital mycotic infection   Male†,§ 0 1 (1.7) 1 (2.1)   Female¶,‖ 0 1 (3.1) 1 (2.4)  Osmotic diuresis-related AEs   Pollakiuria** 1 (1.1) 2 (2.2) 4 (4.5)   Polyuria†† 0 0 0  Volume-related AEs   Postural dizziness 0 1 (1.1) 2 (2.2)   Orthostatic hypotension 0 0 1 (1.1) AE, adverse event; PBO, placebo; CANA, canagliflozin; UTI, urinary tract infection. *All AEs are reported for regardless of rescue medication, except for osmotic diuresis- and volume-related AEs, which are reported for prior to initiation of rescue therapy. †Possibly, probably or very likely related to study drug, as assessed by investigators. ‡PBO, n = 57; CANA 100 mg, n = 58; CANA 300 mg, n = 48. §Including balanitis and posthitis. ¶PBO, n = 33; CANA 100 mg, n = 32; CANA 300 mg, n = 41. ‖Including vulvovaginal mycotic infection. **Increased urine frequency. ††Increased urine volume. Canagliflozin was associated with slightly higher rates of genital mycotic infections in males and females compared with placebo (Table 4), but incidences were low across groups and none led to study discontinuation. The incidence of UTIs was higher with canagliflozin 300 mg compared with canagliflozin 100 mg and placebo, with no upper UTI AEs reported. All events were considered by investigators to be mild or moderate in severity, with none leading to study discontinuation. Incidences of pollakiuria (increased urine frequency) and AEs related to reduced intravascular volume (i.e. postural dizziness and orthostatic hypotension) were increased with canagliflozin 300 mg relative to canagliflozin 100 mg and placebo; these were low across groups, generally mild or moderate in intensity and infrequently led to discontinuation. There was no report of polyuria (increased urine volume) in any group. Most subjects (96.3%) were on background AHA therapy associated with an increased risk of hypoglycaemia (i.e. insulin or sulphonylurea agents). Among these subjects, the proportion with documented hypoglycaemia episodes was higher with canagliflozin 100 and 300 mg (52.9 and 51.2%, respectively) compared with placebo (36.4%). Six subjects experienced severe hypoglycaemia episodes [4 (4.7%), 1 (1.2%) and 1 (1.1%) with canagliflozin 100 and 300 mg and placebo, respectively]. There were no documented hypoglycaemia episodes reported among subjects who were not on insulin or a sulphonylurea agent. Overall, only small differences in safety laboratory parameters were observed with canagliflozin 100 and 300 mg relative to placebo (Table 5). At week 26, similar increases in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were observed with canagliflozin 100 mg (mean percent changes of 10.1 and 5.5%, respectively) and placebo (8.2 and 4.3%, respectively), whereas decreases were seen with canagliflozin 300 mg (–4.4 and –4.3%, respectively). Increases in serum magnesium were seen with canagliflozin 100 and 300 mg, whereas no change was observed with placebo (mean percent changes of 9.1, 14.6 and 0.0%, respectively). Dose-related increases in serum phosphate were seen with canagliflozin 100 and 300 mg compared with placebo (mean percent changes of 4.9, 9.5 and 1.0%, respectively). Canagliflozin 100 and 300 mg were associated with non–dose-related increases in haemoglobin compared with a minimal change with placebo (mean percent changes of 5.3, 3.1 and –0.5%, respectively); corresponding changes in haematocrit were observed (mean percent changes of 6.0, 4.8 and –0.1%, respectively; Table 5). Table 5Mean percent changes in clinical laboratory parameters from baseline to week 26* PBOCANA 100 mgCANA 300 mgALT, n 63 70 78  Mean baseline, U/l 23.7 20.8 22.9  Mean ± SD percent change 8.2 ± 48.5 10.1 ± 40.4 –4.4 ± 34.8 Alkaline phosphatase, n 63 70 78  Mean baseline, U/l 79.3 77.8 80.2  Mean ± SD percent change 5.3 ± 17.7 7.0 ± 19.6 –2.1 ± 15.5 AST, n 62 67 78  Mean baseline, U/l 23.6 21.9 23.7  Mean ± SD percent change 4.3 ± 30.9 5.5 ± 31.3 –4.3 ± 20.7 Bilirubin, n 63 70 78  Mean baseline, µmol/l 7.7 8.2 8.1  Mean ± SD percent change 4.1 ± 31.6 4.5 ± 31.9 7.4 ± 41.8 Magnesium, n 63 70 78  Mean baseline, mmol/l 0.8 0.8 0.8  Mean ± SD percent change 0.0 ± 9.3 9.1 ± 10.4 14.6 ± 12.9 Phosphate, n 63 70 77  Mean baseline, mmol/l 1.2 1.2 1.2  Mean ± SD percent change 1.0 ± 16.5 4.9 ± 16.0 9.5 ± 20.5 Urate, n 63 70 78  Mean baseline, µmol/l 433.7 434.4 442.5  Mean ± SD percent change 2.5 ± 18.6 –0.3 ± 16.9 –2.0 ± 20.0 Haemoglobin, n 62 69 76  Mean baseline, g/l 136.2 133.8 130.9  Mean ± SD percent change –0.5 ± 8.1 5.3 ± 7.4 3.1 ± 5.9 Haematocrit, n 62 69 76  Mean baseline, % 40.8 40.1 39.2  Mean ± SD percent change –0.1 ± 9.1 6.0 ± 7.6 4.8 ± 6.9 PBO, placebo; CANA, canagliflozin; ALT, alanine aminotransferase; SD, standard deviation; AST, aspartate aminotransferase. *Statistical comparison for CANA 100 and 300 mg versus PBO not performed (not prespecified). BODY.MEASURES OF RENAL FUNCTION: Changes in renal function parameters were observed with both canagliflozin doses compared with placebo. Decreases in eGFR from baseline were observed in all treatment groups and were larger in the canagliflozin 100 and 300 mg groups relative to the placebo group: LS mean percent changes of –9.1, –10.1 and –4.5%, respectively. The reductions in eGFR with canagliflozin were largest at week 3 (the first postbaseline measurement) and then trended back towards baseline over the 26-week treatment period (figure 3A). Increases in BUN were observed with canagliflozin 100 and 300 mg compared with placebo (LS mean percent changes of 12.1, 12.5 and 4.9%, respectively); these increases also occurred early and then trended towards baseline over the remaining treatment period. Canagliflozin 100 and 300 mg were associated with greater decreases in urine ACR compared with placebo, with median percent reductions of –29.9, –20.9 and –7.5%, respectively (figure 3B). Progression of albuminuria from baseline to week 26 was examined (i.e. from normoalbuminuria to micro- or macroalbuminuria, or from micro- to macroalbuminuria), with a lower proportion of subjects in the canagliflozin 100 and 300 mg groups progressing relative to those in the placebo group [5.1, 8.3 and 11.8%, respectively; odds ratio (95% CI) of 0.33 (0.08, 1.48) and 0.51 (0.14, 1.91) for the pairwise comparisons of canagliflozin 100 and 300 mg to placebo, respectively]. Figure 3Change in eGFR (A) and ACR (B) over time. #eGFR, estimated glomerular filtration rate; ACR, albumin/creatinine ratio; PBO, placebo; CANA, canagliflozin; LS, least squares; SE, standard error. *Statistical comparison for CANA versus PBO not performed (not prespecified). BODY.DISCUSSION: This study examined the efficacy and safety of canagliflozin in subjects with T2DM and stage 3 CKD, but in a lower, more restricted eGFR range of this classification (i.e. 30 to <50 ml/min/1.73 m2 rather than to <60 ml/min/1.73 m2). In this study population, both canagliflozin doses significantly lowered HbA1c compared with placebo over 26 weeks of therapy. Reductions in HbA1c with canagliflozin 100 and 300 mg (differences in LS mean changes vs. placebo of –0.30 and –0.40%, respectively) were clinically useful, particularly in the setting of stage 3 CKD in which available oral AHA therapy options are limited. In addition to providing reductions in HbA1c, a numerically greater proportion of subjects treated with canagliflozin (including about a third of subjects in the canagliflozin 300 mg group) achieved HbA1c <7.0% than those treated with placebo, indicating that canagliflozin provides meaningful clinical value in this patient population. Both canagliflozin doses provided numerically greater reductions in FPG compared with placebo, although these differences did not achieve statistical significance. A smaller proportion of subjects treated with canagliflozin required glycaemic rescue therapy compared with placebo-treated subjects. Both canagliflozin doses were also associated with reductions in body weight relative to placebo. The mechanism by which canagliflozin reduces body weight is thought to be related to the loss of calories associated with induction of UGE, although through the osmotic diuretic effect, reduced fluid volume may also contribute to the reduction in body weight – this may be particularly relevant in patients with renal impairment who tend to have sodium and fluid retention, and therefore have excessive fluid volume. Also likely related to the osmotic diuretic effect, canagliflozin provided numerically greater reductions in systolic and diastolic BP compared with placebo, an important observation given the often difficult-to-treat hypertension in patients with stage 3 CKD. The efficacy on glycaemic parameters and body weight reduction observed with canagliflozin in this population of subjects was less than that seen in subjects with T2DM who have normal or only mildly impaired renal function 10,14–17. This is not unexpected because the rate of UGE is related to both plasma glucose concentration and eGFR; with lower eGFR, the ability of canagliflozin to augment UGE is attenuated 7,8,11. With lesser increases in UGE, the glucose-lowering efficacy of canagliflozin is also reduced. Results from the current study are consistent with those from a phase 1 canagliflozin study showing reduced UGE and decreased RTG lowering in subjects with stage 3 CKD compared with subjects with normal renal function 18. Owing to the limited amount of UGE observed with canagliflozin treatment in patients with more severe renal insufficiency 18, SGLT2 inhibitors are not expected to be efficacious for patients with an eGFR <30 ml/min/1.73 m2 (i.e. stages 4 or 5 CKD) or for dialysis patients. Both canagliflozin doses were well tolerated, and incidences of AEs, serious AEs and study discontinuations due to AEs were similar across treatment groups. The AEs associated with SGLT2 inhibition seen in other canagliflozin phase 3 studies 14–17, including genital mycotic infections, a small increase in UTIs (with no reports of upper UTI AEs), and AEs related to osmotic diuresis (i.e. pollakiuria and polyuria), were also seen in this study, although at lower rates, which may reflect the attenuation of UGE in this study population. Canagliflozin acts by lowering RTG; this value is typically 10.0 mmol/l (180 mg/dl) in normal individuals, raised in patients with T2DM to approximately 13.3 mmol/l (240 mg/dl), and reduced to levels of approximately 4.4 to 5.0 mmol/l (80-90 mg/dl) in patients treated with canagliflozin 9,10. Because the usual threshold for hypoglycaemia is approximately 3.9 mmol/l (70 mg/dl), this would suggest a low risk for hypoglycaemia with canagliflozin – as has been observed in studies of healthy volunteers and patients with T2DM 7,9,10,16. When an agent not associated with hypoglycaemia is added to the regimen of a medication that is associated with hypoglycaemia, like insulin or a sulphonylurea agent, an increase in hypoglycaemia is usually observed 19–23. This was seen in this study for subjects on background therapy with insulin or a sulphonylurea agent, in whom both canagliflozin doses were associated with the expected higher incidences of hypoglycaemia relative to placebo. Importantly, the rate of severe hypoglycaemia in subjects on such agents was low and there were no documented hypoglycaemia episodes among subjects not on insulin or a sulphonylurea agent. Because the kidney is a target organ with canagliflozin treatment, the effects of canagliflozin on renal function were carefully assessed in this study. Canagliflozin 100 and 300 mg were associated with some changes in renal function (assessed by eGFR, serum creatinine, BUN and ACR) early on, with subsequently stable or improving eGFR values over the 26-week core treatment period (compared with a gradual, small decline in the placebo group). These transient changes in renal function with canagliflozin may be related to a mild osmotic diuretic effect of this agent, with small reductions in plasma volume, leading to a mild prerenal pattern. The proportion of subjects with progression of albuminuria with canagliflozin was slightly less than with placebo; the decrease in the ACR along with the stable renal function after the small initial decline is reassuring with regard to the lack of renal injury with this agent. The urinary ACR has been used as a biomarker, with reduction suggesting prevention of progression of renal injury 24,25, as seen with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs); whether the reduction in ACR seen with canagliflozin, along with stable renal function (after the small initial decrease), indicates the potential for renal protection with canagliflozin can only be assessed with longer-term and larger studies. As noted, the efficacy observed with canagliflozin in this renal-impaired population with T2DM was less than that observed in subjects with normal or only mildly impaired renal function. Nonetheless, this agent still provides important clinical value in this setting. It is important to note that physicians managing such patients have limited options, with several agents restricted (e.g. metformin or thiazolidinediones), and other agents that must be used carefully owing to safety concerns, including sulphonylurea agents and insulin that can lead to sodium retention, weight gain and hypoglycaemia. Treatment with canagliflozin added on to subjects' stable diabetes treatment regimens lowered HbA1c and resulted in more patients reaching HbA1c goal compared with placebo, indicating clinical utility. Additional studies are needed to assess the efficacy and safety of canagliflozin monotherapy in patients with renal impairment. It is interesting to note that another SGLT2 inhibitor, dapagliflozin, has not demonstrated HbA1c-lowering efficacy in this patient population 26; whether this indicates differences between agents in this class or differences in study design remains to be determined. In conclusion, canagliflozin 100 and 300 mg significantly reduced HbA1c and were associated with numerical reductions in FPG, body weight and BP compared with placebo after 26 weeks of therapy in subjects with T2DM and stage 3 CKD (eGFR ≥30 and <50 ml/min/1.73 m2). Canagliflozin was generally well tolerated, with an expected increase in hypoglycaemia among the >95% of subjects on insulin or a sulphonylurea agent. Canagliflozin was associated with transient changes in renal function parameters that recovered towards baseline over the study period. These findings suggest that canagliflozin may be an appropriate treatment option for patients with T2DM and stage 3 CKD.

TITLE: Maternal positioning to correct occiput posterior fetal position during the first stage of labour: a randomised controlled trialGuittier et al. ABSTRACT.OBJECTIVE: To evaluate the efficacy of the hands and knees position during the first stage of labour to facilitate the rotation of the fetal head to the occiput anterior position. ABSTRACT.DESIGN: Randomised controlled trial. ABSTRACT.SETTING: Geneva University Hospitals, Switzerland. ABSTRACT.POPULATION: A total of 439 women with a fetus in the occiput posterior position during the first stage of labour. ABSTRACT.METHODS: The women in the intervention group were invited to take a hands and knees position for at least for 10 minutes. Women allocated to the control group received the usual care. For both groups, 15 minutes after randomisation, women completed a short questionnaire to report their perceived pain and the comfort of their position. ABSTRACT.MAIN OUTCOME MEASURES: The rotation of the fetal head in occiput anterior position confirmed by ultrasonography 1 hour after randomisation. ABSTRACT.RESULTS: One hour after the randomisation, 35 of 203 (17%) fetuses were diagnosed as being in the occiput anterior position in the intervention group compared with 24 of 209 (12%) in the control group. This difference was not statistically significant (relative risk 1.50; 95% CI 0.93–2.43; P = 0.13). The change in the evaluation of comfort between the randomisation and 15 minutes after showed an improvement in 70 and 39 women, no change in 82 and 78 women and a decrease in 56 and 86 women in the intervention and control groups, respectively (P = 0.02). ABSTRACT.CONCLUSIONS: This study could not demonstrate a benefit of the hands and knees position to correct the occiput posterior position of the fetus during the first stage of labour, but the women reported an increase in their comfort level. ABSTRACT.TWEETABLE ABSTRACT: Hands and knees position does not facilitate rotation into occiput anterior but increases the comfort level of women. ABSTRACT.TWEETABLE ABSTRACT: Hands and knees position does not facilitate rotation into occiput anterior but increases the comfort level of women. ABSTRACT.INTRODUCTION: At the onset of labour, an occiput posterior (OP) position occurs in approximately 25% of fetuses in the cephalic position.1, 2, 3, 4 Persistent OP at delivery occurs in approximately 10% of fetuses.5, 6 Previous studies have shown an increase in short‐term and long‐term maternal and fetal complications that are associated with the OP position, such as prolonged labour, maternal exhaustion, fetal distress, instrumental delivery, caesarean delivery and severe perineal tears.7, 8, 9, 10, 11 The aetiology of persistent fetal OP presentation is poorly known. The shape of the pelvis, epidural analgesia, or parity may increase the risk of persistent OP for the delivery.12 Currently, medical and midwife teams have limited interventions to correct fetal head malposition and believe that the hands and knees position of women in labour facilitates the rotation of the fetus.13 Diagnosis of the OP position during vaginal examination is difficult because it is often associated with a deflection of the fetal head and fetal head oedema.14, 15 Several studies recommend verifying the position by ultrasonography to improve the reliability of the diagnosis of the OP position.16, 17 A Cochrane review on the effects of the hands and knees posture in late pregnancy or labour concluded that this intervention does not improve delivery outcomes.18 However, only one randomised controlled trial, including 147 participants during the first stage of labour, was included in the review. It reported a significant decrease of back pain in the hands and knees group (P = 0.008). Fetal head rotation to the occiput anterior (OA) position after the 1‐hour study period was observed in 11 of the 70 women (16%) allocated to the hands and knees group compared with five of 77 (7%) in the control group [relative risk (RR) 2.42, 95% confidence interval (95% CI) 0.88–6.62]. The authors concluded that the sample size of the study had an insufficient power to demonstrate the efficacy of the intervention.19 de Gasquet described several variations of the hands and knees position to facilitate the rotation of the fetal head to the OA position (Figure 1 ).20, 21 According to her hypotheses, maternal positions that are described as resting on the knees, with the chest leaning forward and back stretching, increased maternal comfort and encouraged the rotation of the fetal head almost immediately. Figure 1Two examples of hands and knees positions. Given the complications that are associated with a persistent OP position, it is important to evaluate interventions that may help fetuses to rotate to the OA position. According to previous studies, the hands and knees posture appears to be easy to implement, safe for the mother and fetus, and acceptable to women,18, 22 but their effectiveness on rotation and delivery outcomes remains to be evaluated. The aim of our study was to evaluate the efficacy of the hands and knees position to correct fetal head position from the OP position during the first stage of labour. ABSTRACT.METHODS: ABSTRACT.TRIAL DESIGN AND PARTICIPANTS: We conducted a randomised clinical trial comparing the hands and knees position with expectant management (no intervention). The study took place in the maternity unit of Geneva University Hospitals (4000 births/year). Before and during the trial, all midwives working in the delivery room were trained to help women to assume the evaluated positions during labour and delivery during a 4‐day workshop conducted by Dr de Gasquet. The recruitment of women took place in the delivery room. In the trial, we included nulliparous and multiparous women during the first stage of labour with a cervical dilatation between 2 and 9 cm and with a singleton pregnancy at term (≥37 weeks of gestation). We performed a transabdominal ultrasonography to reliably diagnose the fetal head position during the first stage of labour. The operator could be a doctor, a midwife or a research assistant. Each had personal skills in ultrasonography. In case of doubt, there was a double check. The position of the head was determined according to the position of the usual features of the face or the orientation of the midline and the position of the intracranial landmarks. After confirming the fetal head position, all women presenting a fetus in the OP position (including both left and right OP) were invited to participate in the trial. Women <18 years old, with a limited understanding of French or who had attempted the hands and knees position previously during the first stage of labour were not enrolled in the study. After obtaining written consent for participation and before randomisation, women completed a questionnaire including: sociodemographic data, perceived pain measured by a visual analogue scale (0 cm = no pain to 10 cm = the worst possible pain), and comfort level of their position using a Likert scale (very comfortable, comfortable, neutral, uncomfortable, very uncomfortable). ABSTRACT.RANDOMISATION: Randomisation was performed using randomly permuted blocks of varying sizes (4, 6 and 8), stratified by parity (nulliparous/multiparous) and epidural analgesia (yes/no). The ratio for hands and knees versus expectant management was 1:1. A research midwife or the attending midwife opened a web‐based system developed by the informatics department of the Geneva University Hospitals. After inclusion of the woman, the system returned the allocation. ABSTRACT.PROCEDURES: Immediately after obtaining the group allocation, the women in the intervention group were invited to choose one of the six positions described by Dr de Gasquet.20 The midwife presented pictures of six variants of hands and knees positions, and the woman decided which position would be the best for her.21 These positions have three common features: (1) resting on the knees and, if necessary, on the hands; (2) the abdomen is thrust forward; and (3) the back is stretched (Figure 1). A pillow should be placed between the legs of the woman in labour to limit discomfort. The woman decided if she wanted to place her abdomen on a cushion or leave it unsupported. We recommended that the participants maintained the position as long as they felt comfortable, but for at least 10 minutes. After this period of time, they could remain in the hands and knees position or change positions if they preferred. The time spent in the evaluated position was recorded. Women allocated to the control group received the usual care, i.e. they stayed in the position that they chose before inclusion in the trial. It could be standing, sitting, semi‐recumbent, lying on the back or on the side, but not a hands and knees position. After 1 hour and following ultrasonography verification of the fetal head position, they could adopt a hands and knee position if they chose to do so. For both groups, 15 minutes after randomisation, women completed a short questionnaire to report their perceived pain, as measured by a visual analogue scale, and the comfort of their position, as evaluated by the Likert scale. One hour after randomisation, we performed a second ultrasound scan to diagnose the fetal head position. The fetal head position was also recorded at the full dilatation of the cervix (before starting pushing efforts) and at delivery. Obstetric and neonatal outcomes were collected from the medical record. ABSTRACT.OUTCOMES: Our primary outcome measure was fetal head in the OA position 1 hour after randomisation or at delivery if delivery happened first. Secondary outcomes included an evaluation of the comfort of maternal positions, impact of the maternal position on perceived pain measured before the randomisation and 15 minutes after, duration of the first and second stages of labour, mode of delivery, perineal status and markers of neonatal asphyxia. ABSTRACT.STATISTICAL METHODS: Data analysis and reporting were performed according to the CONSORT guidelines for randomised controlled trials. A descriptive table of the baseline characteristics is reported for the participants for both groups. Primary and secondary outcomes were analysed on an intention‐to‐treat basis. Sub‐group analyses for the variables used to stratify the randomisation (parity and epidural) were performed. The means and their standard deviations were calculated for continuous variables, and the statistical significance of differences between groups was tested using Student's t‐test or the Mann–Whitney U‐test. The mean duration of the first and second stages of labour was calculated, excluding women who had a caesarean section. Proportions were compared between groups, and differences were tested using the Fisher test. The effects of the intervention were estimated by relative risks and their 95% confidence interval. P values were calculated with Fisher's exact test to test the significance of the differences. We calculated that a sample size of 438 women (219 per group) would be needed, with a risk of type I error of 5% and a power of 80%, to show a statistically significant difference in the incidence of the main outcome measure. We hypothesised that the clinically significant difference between groups in the proportion of fetuses rotating to the OA position 1 hour after randomisation would be 10% (10% in the control group versus 20% in the intervention group). The women received written information, and they signed informed consent forms. The data were treated confidentially, and participants were identified in the computerised database by a number. Data monitoring and quality assurance for this study was performed by a research assistant independent of the research team. The complete protocol of the study has been previously published.21 ABSTRACT.RESULTS: ABSTRACT.PARTICIPANTS: Between March 2011 and December 2013, 1418 women with a fetus in cephalic presentation were approached during labour and provided consent for ultrasonography. Among them, 766 (44%) had a fetus that was diagnosed in the OP. Among the 484 women meeting the inclusion criteria, 439 consented to participate in the trial and were randomised into the intervention group or the control group, 220 (50%) and 219 (50%), respectively. Five women in the intervention group did not assume an evaluated position, and three women in the control group adopted one of these positions during the first hour. All of the women were included in the analysis of their randomised group (Figure 2, flow chart). Overall, 289 of 439 participants (65%) were primiparous, and 412 of 439 participants (93%) had epidural analgesia at randomisation. Figure 2 CONSORT 2010 flow diagram. The baseline characteristics were comparable between groups, with the exception of the educational level, which was higher in the control group (Table 1). Table 1Comparison of the general and obstetric characteristics between women in both groups Postures group (n = 220)Control group (n = 219) General characteristics at randomisation Educational level, n (%) Education ≥15 years 149/198 (75.3) 132/202 (65.3) Education <15 years 49/198 (24.7) 70/202 (34.7) Ethnic origin, n/total (%) Caucasian 145 (65.9) 141 (64.7) Other 75 (34.1) 77 (35.3) Maternal age in years, mean (SD) 30.5 (4.8) 30.0 (4.8) Height in cm, mean (SD) 164.8 (6.6) 164.6 (6.3) Maternal weight gain during pregnancy in kg, mean (SD) 15.7 (4.9) 15.8 (5.2) Physical activity last trimester of pregnancy, n (%)a 136/211 (64.5) 142/214 (66.4) Obstetric characteristics at randomisation Nulliparity, n (%) 148 (67.3) 141 (64.4) Gestational age at randomisation, mean (SD) 39.6 (1.0) 39.4 (1.3) Pain (VAS, in cm), median (range) 0.3 (0–10) 0.4 (0–10) Comfort of position at randomisation, n (%) From comfortable to very comfortable 167/213 (78.4) 183/212 (86.3) Neither comfortable nor uncomfortable to very uncomfortable 46/213 (21.6) 29/212 (13.7) Analgesia local/regional, n (%) 208 (94.5) 204 (93.1) Spontaneous labour, n (%) 81 (36.8) 78 (35.8) Cervical dilatationb in mm, mean (SD) 45.4 (21.1) 44.9 (18.6) Intact amniotic sac, n (%) 25 (11.4) 32 (14.6) Station of the presenting part, n (%) Above ischial spines 216 (98.6) 218 (99.5) At ischial spines or below 3 (1.4) 1 (0.5) Fetal back position, n (%) Left 119 (54.6) 121 (55.3) Right 13 (6.0) 11 (5.0) Posterior 86 (39.4) 87 (39.7) Placental location, n (%) Anterior 101/215 (47.0) 100/215 (46.5) Posterior 75/215 (34.9) 84/215 (39.1) Fundal 16/215 (7.4) 16/215 (7.4) Lateral 23/215 (10.7) 15/215 (7.0) VAS, visual analogue scale. Denominators are displayed when there are missing values. aHave you practiced physical activity corresponding to approximately 30 minutes of walking per day or more during the last trimester of pregnancy? bCervical dilatation was assessed digitally. John Wiley & Sons, Ltd ABSTRACT.PRIMARY AND SECONDARY OUTCOMES: One hour after the randomisation, 35 of 203 (17.2%) fetuses were diagnosed as being in the OA position by ultrasonography in the intervention group compared with 24 of 209 (11.5%) in the control group (Table 2). This difference was not statistically significant (RR 1.50, 95% CI 0.93–2.43, P = 0.13, risk difference 5.7%). In the intervention group, 60 of 199 (30%) of the women maintained their chosen position for ≤15 minutes, 103 of 199 (52%) for 16–30 minutes and 36 of 199 (18%) for >30 minutes. Rotation after 1 hour occurred in 10 of 60 (17%), 18 of 103 (18%) and six of 36 (18%) fetuses when the evaluated posture was maintained for 0–15, 16–30 and >30 minutes, respectively (P = 0.99). In the control group, 59 of 190 (31%) mothers were in vertical positions (sitting, semi‐recumbent, standing) and 131 of 190 (69%) were in the lateral decubitus position. Rotation occurred in four of 59 (7%) and 17 of 131 (13%) fetuses when the women were in vertical and horizontal positions, respectively (P = 0.16). After the 1‐hour period of evaluation, the women were free to adopt a position of their choice. In the intervention group, 20 of 220 women (9%) repeated the hands and knees postures compared with 116 of 219 (56%) in the control group. Table 2Comparison of maternal, obstetrical and fetal outcomes Postures group (n = 220)Control group (n = 219)RR (95% CI) P value 15 minutes after randomisation Comfort of position, n (%)a Improvement 70/208 (33.6) 39/203 (19.2) 0.02 No change 82/208 (39.4) 78/203 (38.4) Decrease 56/208 (27.0) 86/203 (42.3) Pain (VAS, in cm), median (range) 0.8 (0–10) 0.7 (0–10) 0.72 One hour after randomisation (Ultrasonography diagnosis) Occiput anterior position 35/203 (17.2) 24/209 (11.5) 1.50 (0.93–2.43) 0.13 Occiput posterior or lateral position 168/203 (82.8) 185/209 (88.5) Fetal back position, n (%) Left 79/198 (39.9) 83/204 (40.7) 0.79 Right 105/198 (53.0) 110/204 (54.4) Anterior 3/198 (1.5) 3/204 (1.5) Posterior 11/198 (5.6) 7/204 (3.4) Fetal position at complete dilatation (Ultrasonography or clinical diagnosis) Occiput anterior position 82/182 (45.1) 99/192 (51.6) 0.87 (0.71–1.08) 0.25 Occiput posterior or lateral position 100/182 (54.9) 93/192 (48.4) Outcomes at delivery Duration of first stage labour in minutes, mean (SD) 354 (195) 369 (158) 0.39 Duration of second stage labour in minutes, mean (SD) 48 (31) 43 (31) 0.17 Analgesia local/regional, n (%) 211 (96.3) 211 (96.8) 1 (0.96–1.03) >0.99 Mode of delivery, n (%) Caesarean 54 (24.5) 35 (16.0) 0.08 Normal vaginal delivery 118 (53.6) 134 (61.2) Instrumental delivery 48 (21.8) 50 (22.8) Perineal status, n (%) Intact or first‐degree perineal tear 151 (68.6) 130 (59.4) 0.05 Second‐degree perineal tear or episiotomy 68 (30.9) 84 (38.4) Third‐degree perineal tear 1 (0.5) 5 (2.3) Blood loss in ml, mean (±SD) 409.6 (239.0) 378.4 (218.3) 0.16 Maternal complication,b n (%) 42 (19.1) 31 (14.2) 1.35 (0.88–2.06) 0.21 Duration of hospital stay, mean (SD) 3.5 (1.4) 3.3 (1.2) 0.03 Neonatal outcomes Weight in grams, mean (SD) 3422 (401) 3411 (406) 0.77 APGAR score <7 at 5 minutes, n (%) 4 (1.8) 4 (1.8) 0.96 (0.24–3.78) >0.99 Umbilical artery pH, mean (SD) 7.22 (0.07) 7.22 (0.06) 0.32 Neonatal resuscitation, n (%) 10 (4.5) 10 (4.6) 1.00 (0.42–2.34) >0.99 VAS, visual analogue scale. Denominators are displayed when there are missing or non applicable values. aChange in the evaluation of comfort between the randomisation and after 15 minutes. bHaemorrhage, fever, retained placenta. John Wiley & Sons, LtdA caesarean section was performed during the first stage of labour in 38 and 27 women in the intervention and control groups, respectively. At full dilatation, 82 of 182 (45%) fetuses were in the OA position in the intervention group and 99 of 192 (52%) in the control group (P = 0.25). The duration of the first and second stages of labour was not significantly different between groups. There were more caesarean sections in the intervention group [54 of 220 (25%) and 35 of 219 (16%) in the intervention and control groups, respectively]. Globally, the mode of delivery did not differ significantly between the intervention and control groups (P = 0.08). The median perception of pain at randomisation was 0.3 cm in the intervention group and 0.4 cm in the control group. After 15 minutes, the medians were 0.8 cm and 0.7 cm, respectively. The median difference in the perception of pain between randomisation and after 15 minutes was similar between groups (0 cm in the two groups, P = 0.76). The proportions of women reporting that they were comfortable or very comfortable at randomisation were 78% and 86% in the intervention and control groups, respectively. After 15 minutes, these proportions were 82% in the intervention group and 75% in the control group. The change in the evaluation of comfort between the randomisation and after 15 minutes showed an improvement in 70 and 39 women, no change in 82 and 78 women and a decrease in 56 and 86 women in the intervention and control groups, respectively (P = 0.02). ABSTRACT.SUBGROUP ANALYSIS: Among the 59 fetuses in OA position after 1 hour, 45 of 336 (13.4%) were randomised when the women were at a dilatation of the cervix between two and 6 cm, compared with 14 of 76 (18.4%) when dilatation was >6 cm (P = 0.28). The effect of the intervention was similar in the two subgroups (RR 1.30, 95% CI 0.75–2.24, in women randomised between two and 6 cm; RR 2.50, 95% CI 0.86–7.28, in women randomised after 6 cm; P value for interaction 0.28; adjusted RR 1.50, 95% CI 0.92–2.43). In the intervention group, there were 10 of 60 (16.7%) fetuses in OA position when women were kept in the hands and knees position for 0–15 minutes, 18 of 103 (17.5%) if 16–30 minutes and 6 of 36 (16.7%) if >30 minutes (P = 0.99). We did not observe a significant difference in rotation 1 hour after the randomisation between the six positions (P = 0.28). ABSTRACT.DISCUSSION: ABSTRACT.MAIN FINDINGS: In cases of the fetal head being in an OP position during the first stage of labour, we observed no efficacy of the hands and knees position for women to promote the rotation to an OA position. The hands and knees postures were, however, associated with an increase in maternal comfort. ABSTRACT.STRENGTHS AND LIMITATIONS: There was a small but not statistically significant increase in the number of fetuses in the OA position after 1 hour in our study. Stremler et al. conducted a similar trial, including 147 women with a fetus in the OP position.19 They found a nonsignificant difference in rotation of 11% between the hands and knees group and the control group after 1 hour. Hence, we based our sample size calculation on a difference of 10% between the groups, giving us limited power to show smaller differences. This suggests that there may be a benefit from this intervention, but the benefit is much smaller than was expected. To explore the reasons for a reduced benefit, we hypothesise that the duration of the intervention was too short. The hypothesis of de Gasquet was that the fetus should rotate within 10 minutes after women adopt a hands and knees position. We observed in a sub‐groups analysis that an increased duration in the position was not associated with an increase in success. To maximise the opportunity to show a benefit of the evaluated positions, all of the research assistants and most of the midwives in the delivery unit were trained by Dr de Gasquet to correctly position the women who were allocated to the intervention group. Among women screened with ultrasonography during labour, we identified that 44% of the fetuses were OP. This prevalence may be overestimated because of a preferential selection for screening women with a clinical suspicion of OP. Because labour is prolonged in the OP position, the probability of being screened may also be higher in this condition. However, this important prevalence was found in other studies.23 The randomisation was stratified by parity and epidural analgesia because these variables are important predictors of the outcome; hence, we wanted to insure a good balance between the groups. We observed that a large majority of the participants had epidural analgesia before the randomisation. This could be explained by the fact that the staff members were more motivated to propose the study and the women were more likely to participate when pain was controlled. It is possible that the effect of hands and knees positions is different in women without epidural analgesia. ABSTRACT.INTERPRETATION: We observed a good compliance of the women to the interventions related to their randomised group. This is in contrast with the results of the study by Hodnett et al.24 However, only a small proportion of women in the intervention group wished to adopt the hands and knees position again later in labour, and only half of the women who were included in the control group decided to take the hands and knees position after the evaluation 1 hour after randomisation. This suggests that, in our context, these positions are not very attractive for women in labour. Women in the intervention group were significantly more comfortable compared with the control group, but we did not observe a benefit in the perception of pain. This can be explained by the high proportion of epidural analgesia in the included women. We observed an increase in the risk of caesarean sections in the intervention group. This was not our primary outcome, and this difference may be a chance finding. The intervention was provided for a limited duration during the first stage of labour, and women in both groups were free to adopt any posture they chose after 1 hour. Half of the women in the control group assumed the evaluated posture after the evaluation period. This reduced the contrast between groups for the interpretation of the data at full dilatation and for the delivery. In addition, the percentage of fetuses in the OA position was similar between groups during the second stage of labour. These factors, and potentially other factors, should be taken into account when interpreting the increased risk for caesarean sections in the intervention group. We did not observe an influence on the results of the stage of the dilatation at randomisation, of the duration of the hands and knees position, or the hands and knees position chosen by women. These results must be interpreted with caution as they result from unplanned sub‐groups analysis. During our trial, a randomised trial including 220 women in labour with a fetus documented to be in an OP position reported no benefit from a sequence of three maternal positions, including hands and knees, during labour depending on the height of the fetal head. They also reported that the OP position in the second stage of labour is strongly associated with operative delivery.23 ABSTRACT.CONCLUSIONS: Our study could not demonstrate a benefit of the hands and knees position to correct the OP position of the fetus during the first stage of labour. However, women reported that they were more comfortable in these positions. Given the consequences of the persistent OP position during labour, further evaluation of interventions to promote the rotation of the fetal head is needed. ABSTRACT.DISCLOSURE OF INTERESTS: Full disclosure of interests available to view online as supporting information. ABSTRACT.CONTRIBUTION TO AUTHORSHIP: MJG and BdG conceptualised the trial. MJG, BdG, MB and OI drafted the protocol; the protocol was revised by all of the authors. MJG, VOG and MB performed the statistical analysis. BdG trained the delivery room midwives to correctly position women in cases with an OP presentation. All of the authors read and approved the final manuscript. ABSTRACT.DETAILS OF ETHICS APPROVAL: The study protocol was approved by the institutional ethics committee of the University of Geneva Hospitals (n° CER10‐182) the 1 September 2010. ABSTRACT.FUNDING: The study was supported by grants from the Swiss National Science Foundation (SNFS, DORE research grants), decision 13DPD6_134760/1. The funding sources had no role in the trial design, patient recruitment, data collection, analysis or interpretation, writing of the report or the decision to submit the paper for publication. ABSTRACT.SUPPORTING INFORMATION:   Click here for additional data file.   Click here for additional data file.   Click here for additional data file.   Click here for additional data file.   Click here for additional data file.

TITLE: Strengthening Exercises Improve Symptoms and Quality of Life but Do Not Change Autonomic Modulation in Fibromyalgia: A Randomized Clinical TrialEffects of Strengthening Exercises on Fibromyalgia ABSTRACT.OBJECTIVE: Autonomic dysfunction is an important mechanism that could explain many symptoms observed in fibromyalgia (FM). Exercise is an effective treatment, with benefits potentially mediated through changes in autonomic modulation. Strengthening is one of the less studied exercises in FM, and the acute and chronic effects of strengthening on the autonomic system remain unknown. The objective of this study was to assess the chronic effects of strengthening exercises (STRE) on autonomic modulation, pain perception and the quality of life (QOL) of FM patients. ABSTRACT.METHODS: Eighty sedentary women with FM (ACR 1990) were randomly selected to participate in STRE or flexibility (FLEX) exercises in a blinded controlled trial. The intensity of STRE was set at 45% of the estimated load of 1 Repetition Maximum (RM) in 12 different exercises. Outcomes were Visual Analog Scale (VAS) for pain, Heart Rate Variability (HRV) analysis, treadmill test, the sit and reach test (Wells and Dillon’s Bench), maximal repetitions test and handgrip dynamometry; and quality of life by the Fibromyalgia Impact Questionnaire (FIQ), the Beck and Idate Trait-State Inventory (IDATE), a short-form health survey (SF-36). ABSTRACT.RESULTS: The STRE group was more effective to strength gain for all muscles and pain control after 4 and 16 weeks (p<0.05). The FLEX group showed higher improvements in anxiety (p<0.05). Both groups showed improvements in the QOL, and there was no significant difference observed between the groups. There was no change in the HRV of the STRE and FLEX groups. ABSTRACT.CONCLUSIONS: Strengthening exercises show greater and more rapid improvements in pain and strength than flexibility exercises. Despite the benefits in fitness, pain, depression, anxiety and quality of life, no effect was observed on the autonomic modulation in both groups. This observation suggests that changes in autonomic modulation are not a target tobe clinically achieved in fibromyalgia. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT02004405 BODY.INTRODUCTION: Fibromyalgia (FM) is reported in 2–5% of the generalpopulation [1]. This syndrome, which impairs the quality of life (QOL) and increases financial expenses for the patient and society [2], is difficult to treat. The hallmarks of FM include widespread pain, fatigue, sleep dysfunction, muscular strength loss and cardiovascular fitness impairment [3]. FM patients show sympathetic hyperactivity and abnormal vagal balance. High sympathetic tonus increases the resting heart rate and reduces the heart rate variability (HRV) [4], [5]. Autonomic dysfunction might be an important mechanism associated with FM and could explain the major FM symptoms, such as fatigue, morning stiffness, sleep disorders, paresthesias, vestibular syndrome, palpitations, irritable bowel syndrome and Reynaud’s phenomenon [6]. Interestingly, a correlation between autonomic dysfunction and symptoms severity or quality of life has been previously described [7]. Undoubtedly, exercise is useful for the treatment of FM [2], [8], [9]. Most clinical trials have focused on aerobic fitness. Few studies have shown strengthening (STRE) as safe and effective for FM [8], [9]. However, until recently, the physiological mechanisms through which exercise induces clinical improvements remain unclear, and it is likely that better autonomic control might be involved. It has been shown that exercise improves autonomic modulation in healthy subjects and diabetic and heart disease patients [10]. Little is known about the acute and chronic physiological effects of STRE on autonomic modulation in FM [11]–[13]. Previous studies with small sample sizes have demonstrated that STRE improves total power and cardiac parasympathetic modulation in women with FM [11], [12]. Hence, we hypothesize that strengthening exercises are beneficial and might improve autonomic modulation. The objective of this study was to investigate the chronic effects of strengthening exercises on the heart rate variability and disease symptoms in FM patients. BODY.METHODS: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. This study was a randomized, evaluator-blinded, controlled parallel clinical trial comparing the STRE to flexibility exercises (FLEX) in women with FM from outpatient clinic of rheumatology of Federal University of Espírito Santo. The following inclusion and exclusion criteria were used. BODY.INCLUSION CRITERIA: Women, between 18 and 65 years old, who met the criteria according to the American College of Rheumatology, 1990 [14]. BODY.EXCLUSION CRITERIA: Cardiovascular, respiratory, metabolic, and rheumatic diseases that could limit exercise; Diseases associated with autonomic dysfunction, such as arterial hypertension, diabetes and coronary insufficiency; The use of medication, such as beta blockers, calcium channel blockers, and any other anti-hypertensive, anticonvulsants, non-tricyclic antidepressants, and opioid analgesics, including tramadol, cyclobenzaprine >10 mg/day, andamitriptyline >25 mg/day, which could interfere with cardiovascular or autonomic responses; Exercise within the last 3 months; Inability to understand the questionnaires; Positive treadmill test for myocardial ischemia; and Receipt of the social security benefits. Patients who were not taking medications during the first appointment were requested to use only paracetamol at a maximal dose of 2 g/day during the four months of the treatment. No other medications were allowed. BODY.RANDOMIZATION: The patients were sequentially randomized to STRE or FLEX groups according to the order of inclusion in the study. The first included patient was allocated to intervention and the second to control group and so on. The physician responsible for initial evaluation and inclusion also randomized patients to groups and obtained the consent form. The assessor was blinded to the group membership and. Only the study coordinator and 2 professionals (physical educator and physical therapist) were responsible for the training knew the group membership information. Primary outcomes were visual analogic scale for pain and Heart Rate Variability. Secondary endpoints were fitness outcomes (treadmill test, the sit and reach test, maximal repetitions test and handgrip dynamometry) and quality of life by the Fibromyalgia Impact Questionnaire (FIQ), the Beck and Idate Trait-State Inventory (IDATE), a short-form health survey (SF-36). BODY.EXERCISE PROTOCOL: All the patients underwent clinical and cardiological evaluations using the treadmill test (TT), before starting the exercise program. The exercise prescription in both groups was consistent with the recommendations of the ACMS [15], with 45 min bouts at 2 times a week for 16 weeks. The STRE group received supervised progressive training in the standing and sitting positions using weight machines. The intensity was moderate, with an overload of 45% of the estimated 1 RM, calculated based on maximal repetitions [15]. Eight major muscle groups were trained (quadriceps femoris, hamstrings, biceps brachii, triceps brachii, pectoral, calf, deltoid, and latissimusdorsi) in 12 different exercises, with 3 sets of 12 repetitions (leg Press, leg extension, hip flexion, pectoral fly, triceps extension, shoulder flexion, leg curl, calf, pulldown, shoulder abduction, biceps flexion and shoulder extension). The exercise program for the FLEX group included the major muscles [16]. BODY.EVALUATION TOOLS: BODY.THE EVALUATION OF FITNESS IMPROVEMENT: The participants underwent periodic muscle strength and flexibility evaluations every 30 days (before and 30, 60, 90 and 120 days after training) to adjust the training intensity and monitor the clinical evolution. Two expert professionals, a physiotherapist and a physical educator, both trained for the application of the analysis tools, performed the evaluations. The participants were subjected to the treadmill test (Ergo PC 13, Micromed version 2.3), the maximal repetitions test [15], handgrip dynamometry [17], and the sit and reach test (Wells and Dillon’s Bench) [18]. The treadmill test was performed to assess the cardiovascular risk and measure indirect oxygen consumption (VO2). A treadmill (Centurion 200, Micromed) was employed using a previously described ramp protocol tested under similar conditions [16]. BODY.PAIN PERCEPTION, FUNCTION, QUALITY OF LIFE AND MOOD EVALUATION: The pain perception was evaluated using the visual analog scale (VAS) at the beginning of the assessment and every 4 weeks thereafter. The QOL and mood were evaluated at the beginning of the assessment, and at 8 and 16 weeks thereafter. The trained examiners were blinded to the groups. The evaluation of the symptoms was performed using the Fibromyalgia Impact Questionnaire [19], a short-form health survey (SF-36) [20], and the Beck and Idate Trait-State Inventory (IDATE) questionnaire [21]. BODY.HEART RATE VARIABILITY (HRV) ANALYSES: HRV analyses were performed in the time and frequency domains, before and at the end (16 weeks) of the study. The electrodes were positioned in the distal region of the superior and inferior limbs. A continuous electrocardiographic recording of 10 minutes was performed with the participants in a supine body position in a quiet environment under a controlled temperature (22–24°C) using a digital electrocardiograph (Micromed, sample rate: 250 Hz) and specific software (Wincardio 4. 4a), which generated a beat-to-beat R-R interval series from the selected lead, with higher amplitude of the R wave (typically D2). The HRV analyses were performed using software developed in Matlab. The series were automatically pre-processed for the removal of ectopic beats and artifacts. The analyses in the time domain included the proportion of R-R intervals that differed in more than 50 ms of the adjacent intervals (pNN50) and the square root of mean of the sum of the squares of difference among the adjacent intervals (RMSSD). The power spectral analysis was performed through autoregressive modeling, using the Yule-Walker method with a Levison-Durbin recursive algorithm. The model order was adjusted to 16 in all analyses, consistent with Dantas et al (2012) [22]. The oscillatory components present in the time series were classified based on very low (VLF: 0–0.04 Hz), low (LF: 0.04–0,15 Hz), and high (0.15–0.40 Hz) frequencies. The values for the spectral indices were presented as normalized units. Normalization was achieved by dividing the spectral power of each oscillatory component by the total power of the spectrum minus the VLF power component. The LF/HF ratio was achieved by dividing the LF components by the HF components in normalized units [22]. BODY.STATISTICAL ANALYSES AND ETHICAL PROCEDURES: Considering both alternative hypothesis (H0) and main hypothesis (H1), sample size calculation was performed taking in account comparisons between media of VAS for pain from two fibromyalgia populations with same variance and same number of observations. We have used MedCalc Program to determine sample size, considering type 1 error of 5%, type 2 error of 20%. The used standard deviation of VAS for pain was previously published (16). These led to at least 58 participants to be randomized to two groups. The normality of the results was tested using the Shapiro-Wilk test. Student’s “t”-test for paired samples was used to perform intra-group comparisons at different times, when the data were normally distributed, and the nonparametric equivalent of Student’s t-test (Wilcoxon test) was used when the data showed an asymmetrical distribution. To compare the data between the STRE and FLEX groups, ANOVA for repeated measures was used, followed by Bonferroni’s post-hoc test. Bilateral tests were carried out adopting a 5% level of significance. The statistical software used for the analyses were SPSS version 20.0 and Biostat 5.0. Other outcomes were fitness measure by treadmill test, the sit and reach test (Wells and Dillon’s Bench), handgrip dynamometry; and quality of life by the Fibromyalgia Impact Questionnaire (FIQ), the Beck and Idate Trait-State Inventory (IDATE), a short-form health survey (SF-36). This project was approved through the Ethics Committee of the Health Science Center of the Federal University of Espírito Santo, protocol n° 119/10. Written informed consent was obtained from all patients. This study was not registered before enrolment of participants started because it is not necessary according national law. The authors confirm that all ongoing and related for this intervention are registered. BODY.RESULTS: Eighty sedentary women between 18 and 65 years old, who met the criteria according to the American College of Rheumatology (1990) were included from July 2010 to September 2012. Training ended September 2012. Fourteen patients were excluded (9 patients in the FLEX and 5 patients in the STRE group) did not complete the study (Figure 1). 10.1371/journal.pone.0090767.g001Figure 1BODY.STUDY DESIGN AND FLOW CHART.: The mean of the analyzed sample (n = 66) had 46±8.5 years, and 56.6% of the participants were married, 62.8% were unemployed and 45.2% had 8 or more years of education. The more frequent symptoms included fatigue (97%), sleep disturbance (86.4%), paresthesias (80.3%), irritability (75.8%), cognitive dysfunction (74.2%), oral dryness (72.7%) and morning stiffness (71.2%). The groups were similar in body weight, height, body mass index (BMI), maximal oxygen uptake (VO2), flexibility, pain perception, function (FIQ), life quality (SF-36), anxiety and depression. However, the STRE group was younger and stronger at the beginning of the study (Table 1). Approximately 7% of the patients (n = 9) in the STRE group and 8% (n = 8) of the patients in the FLEX group used low doses of cyclobenzaprine or amitriptyline. 10.1371/journal.pone.0090767.t001Table 1BODY.DEMOGRAPHICS AND CLINICAL CHARACTERISTICS OF TREATMENT.: Variables STRE (n = 35) FLEX (n = 31) p value Age (years) 44.34±7.94 48.65±7.60 0.028* Weight (kg) 65.75±9.30 67.70±13.63 0.504 Height (m) 1.59±0.07 1.56±0.06 0.061 CMI (kg/m2) 26.12±4.08 27.82±4.81 0.128 Pain (VAS) cm 7.81±1.59 8.38±1.46 0.184 FIQ 6.785 6.678 0.790 Depression (Beck) 25.83 22.78 0.225 Anxiety (IDATE TRAIT) 57.2 52.13 0.057 Anxiety (IDATE STATE) 46.69 44.45 0.407 SF-36 Physical Component 27.01 24.37 0.164 SF-36 Mental Component 33.47 36.98 0.181 VO2 max (ml/min) 31.20±10.87 28.90±9.82 0.372 Handgrip (kgf) 26.33±7.03 21.50±6.71 0.037* Wells’ Bench (cm) 20.44±7.91 20.29±8.58 0.955 The values are presented as the means ± SD. STRE: strengthening exercise, FLEX: Flexibility exercise. FIQ: Fibromyalgia Impact Questionnaire. SF-36: Short-Form healthy Survey. VO2 max: maximal oxygen uptake. Wilcoxon Test: intra-group comparison. ANOVA: inter-group comparison, analysis of variance for repeated measures, p value: inter-group difference, *p≤0.05. Pos hoc Bonferroni’s test. Handgrip and Wells’ Bench data refer to N = 18 in the STRE group and N = 20 in the FLEX group. The strengthening and flexibility programs were effective, asimprove strength and flexibility were observed after treatment. The STRE group showed earlier and more gradual and progressive strength gain than the FLEX group for all muscle groups (results not shown) and in the global strength measured using handgrip dynamometry (Figure 2, Table 2). 10.1371/journal.pone.0090767.g002Figure 2BODY.ISOMETRIC STRENGTH, FLEXIBILITY AND PAIN EVALUATION.: Mean and standard error of measures of strength (handgrip) (A), flexibility (Wells’ Bench) (B), Visual-Analogical Scale (VAS) (C), STRE: Resistance Exercise, FLEX: Flexibility Exercise. Wilcoxon Test: intra-group comparison (*), ANOVA: inter-group comparison (#), *p≤0.05. ANOVA: analysis of variance for repeated measures. Post-hoc Bonferroni’s test. 10.1371/journal.pone.0090767.t002Table 2BODY.PHYSICAL FITNESS ASSESSMENT AT BASELINE AND AFTER 16 WEEKS.: STRE before STRE after FLEX before FLEX after p value N = 35 N = 35 N = 31 N = 31 STRE × FLEX SF 36 - Functional capacity 39±22.81 47.86±19.83* 29.39±16.64 43.39±19.85* 0.364 HF (beat/min) 90.97±11.87 88.49±12.30 90.13±16.54 87.87±11.20 0.833 VO2 max 31.20±10.87 37.71±6.24* 28.90±9.82 32.54±7.50 0.002* Handgrip (kg) 26.33±7.03 29.75±7.04* 21.50±6.71 0.004* Wells’ Bench 20.44±7.91 25.34±7.48* 20.29±8.58 25.80±7.56* 0.85 Shoulder flexion (kg) 7.29±2.00 11.09±3.08* 6.68±2.24 8.13±2.84 <0.001* Leg (kg) 113.79±36.84 163.21±48.87* 100.43±35.96 132.24±50.55 0.013* The values are represented as the means ± SD. STRE: strengthening exercise, FLEX: Flexibility exercise. Handgrip and Well’s Bench data refer to a subsample of 38 patients. Wilcoxon Test: intra-group comparison. ANOVA: inter-group comparison, *p≤0.05. HF: heart frequency at rest. ANOVA: analysis of variance for repeated measures. p value: inter-group difference. Pos hoc Bonferroni’s test. A progressive improvement in pain perception (monthly evaluation) was observed in the two groups, but the effect in the STRE group was higher than that the FLEX group after 30 days and at 4 months, indicating earlier improvement and better pain control in the STRE group (Figure 2). The STRE group showed a significant improvement of fitness, not only in strength, but also in the maximal oxygen uptake. Neither of the groups showed any changes in the resting heart rate (Table 2). Both groups had good tolerance to exercise program and neither showed injury. A significant improvement of the function, depression and QOL was observed in both groups. However, there was no difference between the groups, except for anxiety, in which the FLEX group showed better control (Table 3). 10.1371/journal.pone.0090767.t003Table 3BODY.EFFECTS OF EXERCISE ON FUNCTION, SYMPTOMS, LIFE QUALITY, ANXIETY AND DEPRESSION.: STRE before STRE after FLEX before FLEX after p value N = 35 N = 35 N = 31 N = 31 STRE × FLEX FIQ 67.85±15.37 51.15±18.38* 66.78±17.24 51.15±18.38* 0.95 BECK 25.83±17.36 18.49±12.35* 22.77±18.56 16.39±9.46 0.452 IDATE-TRAIT 57.20±10.57 51.40±11.44* 52.13±10.67 45.19±11.74* 0.033* IDATE-STATE 46.69±10.18 45.11±10.01 44.45±11.58 39.06±10.92* 0.022* SF36- functional capacity 39.00±22.81 47.86±19.83* 29.39±16.64 38.39±19.85* 0.418 SF36- physical aspects 12.14±24.53 33.57±35.84* 9.68±26.36 28.23±38.59 0.414 SF36-Pain 27.68±13.49 42.68±14.82* 29.54±13.83 42.49±16.53* 0.994 SF36- General S. of Health 35.40±16.62 47.17±18.18* 45.58±20.43 56.65±20.93* 0.28 SF36-Vitality 25.14±17.38 41.14±20.15* 22.58±19.79 38.71±21.13* 0.533 SF36-Social aspects 41.76±21.40 55.66±17.92* 40.32±23.87 61.45±26.31* 0.306 SF36-Emotional aspects 27.62±40.81 40.00±41.07 35.48±41.22 51.61±43.75* 0.27 SF36-Mental Health 38.97±21.46 50.06±25.01 45.94±24.87 60.97±24.55 0.079 SF36- Physical Component 27.01±7.61 35.65±7.80* 24.37±7.58 34.15±9.20* 0.477 SF36- Mental Component 33.47±12.33 39.16±12.64 36.98±12.73 44.55±13.60 0.099 The values are represented as the means and ± SD. STRE: strengthening exercise, FLEX: flexibility exercise. Wilcoxon Test: p intra-group, ANOVA: inter-group comparison, analysis of variance for repeated measures, *p≤0.05. p value: inter-group difference. Post Hoc Bonferroni’s test. Although improvements in the symptoms were observed, no change in the autonomic modulation was detected after 16 weeks in both groups, as shown in table 4. 10.1371/journal.pone.0090767.t004Table 4BODY.LINEAR ANALYSIS OF THE HEART RATE VARIABILITY BEFORE AND AFTER 16 WEEKS OF TRAINING.: STRE (n = 35) FLEX (n = 31) p value Before 16 weeks Before 16 weeks STRE x FLEX Total power (ms2) 4095.81±1723.52 2884.52±1326.38 2414.78±785.46 1429.41±333.53 0.84 pNN50 (ms) 12.59±2.79 6.66±1.60* 5.99±1.52 5.51±1.46 0.75 RMSSD (ms) 48.40±10.31 39.16±8.62 36.86±7.28 30.37±4.50 0.96 LF (ms2) 1007.37±495.27 788.54±424.70 710.12±310.64 266.81±47.07 0.54 HF (ms2) 1680.60±797.25 1090.25±574.71 773.26±336.09 555.92±255.24 0.82 LFnu 42.80±3.03 43.42±3.53 43.68±3.72 46.7±4.48 0.73 Hfnu 47.24±2.92 47.83±3.40 43.85±3.83 44.83±4.16 0.73 LF/HF 1.36±0.23 1.88±0.56 1.88±0.51 2.73±1.00 0.64 The values are presented as the means ± SD. RMSSD: square root of the mean squared differences between adjacent normal RR intervals, in a time interval; PNN50: percentage of adjacent RR intervals differing longer than 50 ms; LF: low frequency; HF: analysis of variance for repeated measures, *p≤0.05, p value: entre-group difference. Pos Hoc Bonferroni’s test. High-frequency, Wilcoxon Test: intra-group comparison, ANOVA: inter-group comparison. BODY.DISCUSSION: The results showed that both treatments, strengthening and flexibility, improved the symptoms and QOL of patients with FM. However, in both groups, there was no interference in the autonomic modulation, as evaluated through heart rate variability. Strengthening exercises were more effective and faster for pain control, whereas flexibility exercises were better for anxiety control. These results are useful to prescribe exercises for FM, as the effects of both treatments can be complementary. In addition, these differences can facilitate the selection of the most suitable exercises, according to the clinical profile of each patient. Only one previous study compared strengthening to flexibility, and the results showed that the magnitude of the effects was wider in the STRE group. Moreover, there was no difference between the groups for all variables studied [23]. Previous reports have compared strengthening with aerobic training [24]–[27], and similar results between the groups in the clinical improvement of the patients were observed. These studies are consistent with the results obtained in the present study, showing that STRE is beneficial, safe and comparable with other exercises [24]–[27]. In our study, the STRE group underwent training on machines and using free weights (barbells and dumbbells), and the load was adjusted monthly, similar to the regiment used for healthy sedentary subjects. Despite the low physical fitness, FM patientsexhibit muscle trainability and adaptation similar to healthy individuals, as previously reported [11]–[13], [28]–[32]. The STRE group was younger and stronger. But these factors did not affect the conclusion that STRE was more effective for strength gain and pain control, as demonstrated through intra-group and inter-group analyses. The STRE generated global fitness improvements, not only for strength but also for flexibility and oxygen consumption, likely reflecting the fact that strengthis essential in daily tasks. Although effective for controlling symptoms, STRE did not affect the ANS, indicating that the involvement of other mechanisms, such as serotonin, endorphins, angiotensin II, cytokines, oxide nitric and GH increase and effects on the cerebral cortex [33]–[36]. The physiological effects varied according to the type of exercise. Thus, the production of serotonin, endorphins and increased autonomic modulation might be more influenced through aerobic fitness, as demonstrated in healthy and athletes [37]–[39]. It was recently demonstrated that serotonin is increased after 20 weeks of aerobic training, compared with FLEX training, in FM patients [35]. However, the mechanism underlying how exercise modifies the HRV remains unknown [10], [40], [41]. Until recently, studies have suggested that exercise influences HRV through neural stimulus by heart sympatho-vagal balance readjustments, with increased vagal modulation and reduced sympathetic modulation [42]. Few studies have focused on the acute and chronic physiological effects of strengtheningexercises on autonomic modulation in the FM [11]–[13]. It was demonstrated that after acute strengthening exercise, FM patients responded differently from the controls, as demonstrated by lower sympathetic and higher vagal modulation, likely reflecting altered autonomic responsiveness to physiological stress [12]. Only two previous studies concerning the chronic effects of STRE in FM, and the results are controversial [11], [13]. A previous study using a small sample (N = 10) demonstrated that STRE improves total power, cardiac parasympathetic tone, pain perception and muscle strength in women with FM after 16 weeks [11]. These same authors also reported that the HRV did not change after 12 weeks of strength training [13]. Other researchers have failed to demonstrate the interference of STRE in ANS in healthy people and athletes, consistent with the results of the present study, showing that the benefits of STRE are not associated with this physiological mechanism [41], [43], [44]. It is possible that, unlike aerobic training, STRE cannot modulate ANS. Beyond the type of exercise, another feasible explanation for this observation could be the high variability in the modulation of ANS in each subject [42]. Thus, a larger sample might be necessary to test this hypothesis, as high data dispersion could conceal the modulator effects of STRE training [41], [43], [44]. In conclusion, despite improvements in depression, anxiety and the quality of life in both groups, no effect of the strength training on autonomic modulation was observed, suggesting that autonomic modulation is not a target to achieve clinical benefits in fibromyalgia. BODY.SUPPORTING INFORMATION: Checklist S1CONSORT Checklist. (DOC) Click here for additional data file. Protocol S1Trial Protocol. (PDF) Click here for additional data file.

TITLE: Evaluating the Impact of Computerized Provider Order Entry on Medical Students Training at Bedside: A Randomized Controlled TrialComputerized Provider Order Entry in Medical Students Training ABSTRACT.OBJECTIVE: To evaluate the impact of computerized provider order entry (CPOE) at the bedside on medical students training. ABSTRACT.MATERIALS AND METHODS: We conducted a randomized cross-controlled educational trial on medical students during two clerkship rotations in three departments, assessing the impact of the use of CPOE on their ability to place adequate monitoring and therapeutic orders using a written test before and after each rotation. Students’ satisfaction with their practice and the order placement system was surveyed. A multivariate mixed model was used to take individual students and chief resident (CR) effects into account. Factorial analysis was applied on the satisfaction questionnaire to identify dimensions, and scores were compared on these dimensions. ABSTRACT.RESULTS: Thirty-six students show no better progress (beginning and final test means = 69.87 and 80.98 points out of 176 for the control group, 64.60 and 78.11 for the CPOE group, p = 0.556) during their rotation in either group, even after adjusting for each student and CR, but show a better satisfaction with patient care and greater involvement in the medical team in the CPOE group (p = 0.035*). Both groups have a favorable opinion regarding CPOE as an educational tool, especially because of the order reviewing by the supervisor. ABSTRACT.CONCLUSION: This is the first randomized controlled trial assessing the performance of CPOE in both the progress in prescriptions ability and satisfaction of the students. The absence of effect on the medical skills must be weighted by the small time scale and low sample size. However, students are more satisfied when using CPOE rather than usual training. BODY.INTRODUCTION: According to the Medical School Objectives Project (MSOP), established in January 1996 by the Association of American Medical Colleges to guide medical education, clinical experience is essential in creating memory connections between pathophysiological knowledge and clinical practice, enabling the student to reason flexibly around a case.[1] Bedside practice is critical during medical training as it creates experience in caregiving, diagnosis and therapeutics, and enhances the acquisition of knowledge.[1,2] This is supported by the finding that repeated written testing and testing with Standardized Patients (SPs) in simulations can enhance long-term retention of knowledge, suggesting that the same occurs with testing with real patients.[3] The widespread of Electronic Health Records (EHRs) and particularly of computerized provider order entry (CPOE) tools in University Hospitals has made them available to students during their clerkship rotations. Students have been showing a greater interest and familiarity with them than attending physicians, independently of computer literacy.[4–6] Although CPOE can facilitate medical prescription and reduce serious medication error rates by more than half [7–12], few studies have addressed the impact of CPOE on the students’ medical training and ability to place sound orders, despite the indication that it could be useful, particularly through the immediate feedback about the placed order.[13–18] These trials yielded negative results, yet they provide insights on the possible causes of failure: lack of randomization, of time and investment from the supervisors, focus on a specialty, factors of confusion such as the addition of learning material in both groups, or a method of evaluation which did not capture the beneficial effect of the intervention. We hypothesize that in vivo use of CPOE can improve medical students training and ability to formalize orders through deeper involvement in the medical practice, that an early contact with it enhances satisfaction and familiarization, and reduces errors. To assess this we conducted an educational randomized cross-controlled trial on medical students in a University Hospital. BODY.MATERIALS AND METHODS: The Georges Pompidou European Hospital is a 700 beds university hospital located in Paris, France. It is a fully computerized hospital since its opening in 2000. Students are allowed to place orders on real patients during their clerkship rotations, but these orders are systematically reviewed by a senior MD, usually the Chief Residents (CR) before delivery, being blocked by default until approval. Although this system is available in the whole hospital, it is not yet widely used for teaching purposes, as it requires additional supervising work for the CRs to supervise the students and provide feedback. We recruited CRs in three departments: Cardiology, Immunology and Internal Medicine, known to provide students good training in placing therapeutic orders. The active and willing participation was a key process in the intervention as it demanded letting the students place their own orders first, dedicating time to review the orders with the students and providing feedback and corrections. Medical students in their last undergraduate year would be included during their clerkship with these CRs in two quarter rotations from January 2012 to June 2012. The randomization was done by CR and students were once and for all assigned to a CR at the beginning of their rotation. Using a cross-over design over these two rotations, CRs were able to be their own controls. The intervention group made use of CPOE at the bedside to place real monitoring and therapeutic orders, and after the patient rounds would discuss the orders with the CR, who would provide corrections and recommendations about the placed orders. As they were actual orders, students needed to specify the drug name, posology, route of administration, hours of delivery and delivery period. The control group didn't use CPOE and bedside teaching was conducted as usual, asking verbally for what the student would do in the current situation. This requires less formalization of the order and vague answers are usually accepted (only the drug class, approximate dosages, etc.) Eight willing CRs in the three departments were recruited to participate in the study. 36 medical students in their last undergraduate year were included during their clerkship with these CRs over the two quarters of this study. Details of the distribution of CRs and students can be found in Table 1. 10.1371/journal.pone.0138094.t001Table 1BODY.CROSS-OVER SCHEMA AND DISTRIBUTION OF SUBJECTS.: QuarterInterventionDepartmentCRStudents1 Control Cardio 2 2 1 Control Int Med 2 4 1 Case Int Med 2 5 1 Case Immuno 1 7 2 Case Cardio 3 3 2 Case Int Med 2 5 2 Control Int Med 2 3 2 Control Immuno 1 7 The primary outcome measure was an increase of knowledge by the students. To evaluate it the students took a 57 items short open answers questionnaire (QROC, S1 Text). Those questions were created independently by three CRs (EP, BR, IP). Two students not included in the study took the test to check the quality of the questions. After validation, all students in the study took this test at the beginning and at the end of the three months period, without any communication of the grades or answers, so as to prevent memory bias. They had one hour to complete them. These questionnaires focused on the ability to prescribe laboratory and imaging diagnosis tests, monitoring and medications in medical situations frequently encountered in the three aforementioned specialties and were rated by two independent MD, PhD (EP and BR) blindly of the department, the student and the group, using a keyword based scale. The students also filled a 27 items satisfaction questionnaire (S2 Text, adapted from Knight 2005 [17]) at the end of the rotation, consisting of five parts: I) the general opinion of students in regard to placing orders, either on paper or using the computer, II) how they felt about their contribution to the patient’s care, III) the satisfaction with the level of commitment they were allowed to during their clerkship rotation, IV) the obstacles they encountered in placing orders and V) their preferences relating to orders. Part I and III used five points Likert scales ranging from “strongly disagree” to “strongly agree”. Part II used a five point scale from “poor” to “excellent”. Part IV used a four point scale from “no” to “a lot”. In the last part (V) students were asked about the percentage of initial orders they placed for incoming patients, the total percentage of orders they would have liked to place for all their patients, and the percentage of orders they would like to review with their resident, CR and a potential other supervisor. The orders placed during the first quarter by the students and the associated correction, if any, were extracted from the CPOE database for analysis of the cause of correction. We checked for any discrepancy in the ratings (grades above the maximum for example) and found none. We then conducted an assessment of inter-rater agreement for the two raters using intra-class correlations coefficients (ICC), both on individual questionnaires and on the students’ progress between the first and the second ones. A first bivariate analysis compared the progress between the case and control group using a two-sided t-test. A second more detailed analysis used a mixed model to compare the progress of students between the two groups. The model studied the relationship between the progress and: the intervention state as a fixed effect, the item number, the chief resident and the student as random effects. Model selection was done using ANOVA on log likelihood between the different models, choosing the most parsimonious model. p values for the fixed part were calculated using a type 3 hypotheses F test and likelihood ratio tests for the random part. Items scores in the satisfaction questionnaire were normalized between 0 and 1. We proceeded to do an exploratory factor analysis using the minres factorial method. The number of dimensions was determined using parallel analysis and items were attributed to a dimension based on their loading. Scores for each dimension were obtained by adding the normalized scores from the corresponding items. Bivariate analysis was used to compare scores on all the found dimensions between the two groups. Statistics were calculated using R version 3.1.1, the lme4 and lmerTest packages for the fitting of linear mixed models[19], and the psych package for psychometric analysis. BODY.ETHICS STATEMENT: This study was reviewed and approved by IRB #00001072 Comité de Protection des Personnes Ile de France II. All Students signed a written informed consent. The study was approved by the Medical faculty Dean of Paris Descartes University. BODY.RESULTS: 36 students in their last undergraduate year were recruited during the two rotations, with a mean age of 24.1 (+/- 0.8) and a 2:1 (female:male) sex ratio. One subject in the second quarter, Cardiology group, has been censored for two reasons. The first one was due to a large inconsistency in grades between examiners. The second one being that this subject was assigned to a CR which didn’t participate in the first quarter. This left us with 35 students total, 18 in the first quarter, 17 in the second one, with the same seven Chief Residents for each quarter. ICC(3, k) between the two examiners for each item of each questionnaire was 0.93 before censoring, and 0.94 after. ICC(3, k) between the two examiners for the difference between t1 and t2 for each item of the questionnaire was 0.87. The control group scored 69.87 points on the test at the beginning of the rotation, and 80.98 points on the test at the end of the rotation. The case group scored 64.60 points and 78.11 points on the same tests. The highest possible score for these tests was 176. The bivariate analysis shows no statistically significant difference in the progress between the two groups (+11.11 points for the control group, +13.51 points in the case group, p = 0.5562). Overall the students showed a mean progress of +12.35(±11.79) points, significantly different from 0 (4.78e-7). The density plot by group for the overall grade progress between the beginning and the end of the rotation (averaged between the two raters) is shown in Fig 1. Most of the students show a similar progress between the two measures, but one can observe a negative tail for the Control group and a positive tail for the Case group. Fig 2 shows a more detailed representation of the progress (delta in score) of students on individual questionnaire items between the beginning and the end of the rotation. Fig 3 shows the boxplots of the progress (delta in score) for each chief resident (a), intervention group (b) and individual student (c). 10.1371/journal.pone.0138094.g001Fig 1BODY.DENSITY PLOT OF THE OVERALL QUESTIONNAIRE PROGRESS BETWEEN THE BEGINNING AND THE END OF THE ROTATION, FOR EACH GROUP.: Controls are represented with a continuous line, Cases with a dotted line. 10.1371/journal.pone.0138094.g002Fig 2BODY.SCATTERPLOTS OF THE PROGRESS OF THE STUDENTS ON EACH ITEM OF THE QUESTIONNAIRE BETWEEN THE BEGINNING AND THE END OF THE ROTATION.: Plots are shown with one row for each chief resident, left column for the Control group, and right column for the Case group. Item scores have been normalized. A smoothed estimator is fitted for each subplot. 10.1371/journal.pone.0138094.g003Fig 3BODY.BOXPLOTS OF THE PROGRESS DISTRIBUTION ON ALL QUESTIONNAIRE ITEMS FOR EACH (A) CHIEF RESIDENT, (B) INTERVENTION GROUP, (C) STUDENT.: The mixed effect model finds a positive yet not statistically significant effect of the Case (vs Control) intervention state, with an estimate of 0.005 (p = 0.82). The random effect associated to the CR has an intercept of 0.0003 (p = 0.60). The random effect associated to individual items of the questionnaire has an intercept of 0.0079 (p = 3.27e-19) and the effect for individual students an intercept of 0.0027 (p = 3e-05). Other models including a random slope for the effect associated to CRs or a fixed effect for the department didn’t fit the data significantly better, thus we kept the most parsimonious model. The exploratory factor analysis of the satisfaction questionnaires identified 4 dimensions. The first one corresponds to the overall satisfaction with order practice during the rotation, including 14 items: every item from parts II and III, and the first three items of part IV. The second dimension, including 7 items, reflected the students’ opinion about the usefulness of prescribing in their training. The third dimension (8 items) showed students’ agreement with the benefits of CPOE, and the fourth one (6 items) the difficulties they encountered with it. Representative items in decreasing order of loading for each dimension are listed in Table 2. 10.1371/journal.pone.0138094.t002Table 2BODY.REPRESENTATIVE ITEMS IN DECREASING ORDER OF LOADING FOR EACH OF THE FOUR DIMENSIONS.: Overall satisfaction with order practice during the rotation III_7: I am receiving adequate preparation for being an intern Overall satisfaction with order practice during the rotation III_4: My chief resident thought it was important for me to have x Overall satisfaction with order practice during the rotation III_2: I was included in discussions about the management of my patients Overall satisfaction with order practice during the rotation IV_1 (negative): Resident or intern did not want me to write or enter orders Overall satisfaction with order practice during the rotation II_1: During this rotation, what was, in your opinion, your contribution to the medical care of patients? Opinion about the usefulness of prescribing in their training I_2: Placing orders is an important way to learn what tests and treatments are needed by patients with certain problems Opinion about the usefulness of prescribing in their training I_1: Placing orders is an important way to increase my sense that I am a caregiver for my patients Opinion about the usefulness of prescribing in their training V_3: What percentage of your patients total number of admission and follow-up orders would you like to review with your supervisor Opinion about the usefulness of prescribing in their training I_9: Medical students should be given as many opportunities as possible to place orders Opinion about the usefulness of prescribing in their training I_10 (negative): The ordering method used will have an impact on my selection of the location of my future rotations Agreement with benefit of CPOE V_1: For what percent of newly admitted patients that you picked up have you entered the complete set of admission orders? Agreement with benefit of CPOE IV_10 (negative): Computer ordering system difficult to use Agreement with benefit of CPOE I_5: Writing orders by hand encourages medical errors Agreement with benefit of CPOE I_7 (negative): Entering computerized orders encourages medical errors Difficulties encountered with CPOE IV_4–6: It took too long for the resident/chief resident/other senior to review the orders I wrote IV_9: Inadequate training on the computer ordering system Difficulties encountered with CPOE IV_7: Difficulty in finding a free computer terminal Scores in the second and third dimensions were normally distributed and compared using Student’s t test. Scores in the first and fourth dimension were not normally distributed and compared using the non-parametric Mann-Whitney U test. They were not different for the second dimension, and were significantly higher for all the other dimensions. Results are shown in Table 3. 10.1371/journal.pone.0138094.t003Table 3BODY.COMPARISON OF DIMENSION SCORES BETWEEN THE CASE AND CONTROL GROUPS.: Control groupCase groupp valueDimensionMeanSDMeanSDOverall satisfaction (14 items) 6.6 2.8 8.4 3.1 0.035* Opinion about prescribing (7 items) 4.2 1.5 5.1 1.2 0.057 Opinion about CPOE (8 items) 3.1 0.8 4.3 1 <0.001* Difficulties with CPOE (6 items) 1.3 1.3 2.4 1.6 0.019* During the first rotation of this trial, 11 students in the Case group placed 1193 orders, ranging from 28 to 201 (mean = 108.5 +/- 53) per student. 36 (3%) of these orders were urgent orders for laboratory tests or imaging examination and were not reviewed at first by the supervisor, and 97 (8%) were cancelled by the students themselves, leaving 1060 orders for review. 781 orders were for laboratory tests (71 +/- 33.5 per student), of which 108 were corrected, 188 for drugs (17.1 +/- 9), with 43 corrected and 102 for imaging examinations (9.3 +/- 8.2), with 18 corrected. Other kinds of orders included nursing (56), clinical monitoring (34), IV drugs (25), and consultations (7). A total of 202 (19%) orders were corrected during the review process, including 49 orders for oral and IV drugs. The average lag between order placement by students and validation/correction by the supervisor was 1h25 +/- 1h01. A semi-quantitative analysis of the 49 drug prescription errors corrected by CRs is presented in Table 4. 10.1371/journal.pone.0138094.t004Table 4BODY.DRUG ORDER ERRORS CORRECTED BY CHIEF RESIDENTS.: Type of modification by the supervisor of erroneous ordersNumberCancellation 9 Modification of dose 7 Modification of treatment duration 5 Modification of drug in the same therapeutic class 5 Modification of the time of delivery 4 Modification of the route of administration and dose (intravenous) 3 Modification of the time of delivery and dose 3 Modification of dose and treatment duration 2 Modification de the time of delivery and dose (intravenous) 2 Modification of the route of administration 2 Modification of drug in another therapeutic class 2 No modification 2 Cancellation because it was already prescribed 2 Modification of individual doses without changing the daily dose 1 Total 49 BODY.DISCUSSION: This study is the first educational randomized control trial with students prescribing on real patients. Overall the students showed progress in prescription skills between the beginning of the rotation, however no differences were observed between the two groups. After adjusting for questionnaire item, CR, and student, the progress was still not significant between the two groups. Although we thought the CR involvement would be crucial to the students’ progress, due to the nature of the intervention, the multivariate model shows that individual items and students baseline levels are more important. The absence of significant effect of CR on the students’ progress could be explained by the fact that the CRs were all voluntary and already particularly involved in students’ education. We can also hypothesize that the use of CPOE as an educational tool doesn’t require new pedagogical skills from the CRs and only a change of practice. If we can’t rule out a lack of power due to a relatively small sample, the small progress in prescription skills could be explained by the small time scale (3 months) and the fact that participating students were in their last year of undergrad, when little progress can be made before the residency. This could be a limitation of our study, since it induces a lack of power to observe a difference of progress between the two groups. All the questions from part II and part III of the questionnaire (feeling about the contribution to the patient’s care and satisfaction with the level of commitment they were allowed to) fell expectedly in the same dimension (overall satisfaction with order practice), as their subject is tightly related. Students in the intervention group scored better in this dimension, showing that the use of the actual tools with real patients increased their sense of belonging to the medical team and taking part in patients’ care. Scores for both groups were high in the second dimension (opinion about prescribing), indicating that students see placing orders as an important feature in medical learning, independently of having used paper or computer, albeit nearly reaching statistical significance. It is notable that students who showed a positive opinion about prescribing also wanted to have more of their prescriptions reviewed by a supervisor (correlation coefficients ranging from 0.15 to 0.54 between the corresponding items), showing that not only do they see prescribing as important but also that feedback is essential for the prescribing to be beneficial. The process of reviewing is facilitated by the CPOE as CRs can review the orders in an asynchronous fashion and from different places. The third dimension included items regarding to the perceived benefits of CPOE by the students. The intervention group also scored higher in this dimension, although they scored higher in the fourth dimension too (difficulties they encountered with CPOE). As they were the only group to use CPOE it is not surprising that they were more aware of its technical issues. However it is still interesting to see that students being exposed to CPOE showed a better opinion toward it than the other group, showing that it met their expectations. The third dimension also contained items V_1 (“For what percent of newly admitted patients that you picked up have you entered the complete set of admission orders?”) and V_2 (“What percentage of your patients’ total number of follow-up orders would you like to write or enter?”), indicating that students who were able to enter more orders perceived more benefits from the use of CPOE, although no causation link can be inferred. It could be indeed that students seeing CPOE use as beneficial were more likely to be confident enough to place orders. Nonetheless, both these items scored significantly better in the CPOE group: 0.01 (+/-0.03) vs 0.1 (+/-0.2), p = 0.032* (item V_1)), and 0.2 (+/-0.3) vs 0.6 (+/-0.4), p = 0.0051* (item V_2). Students using the CPOE system in the first part of the study mostly ordered laboratory or imaging tests and oral drugs. Although nearly a quarter of all the drug orders needed to be corrected, analysis of these corrections shows that only small adjustments were made by the CRs, such as modifications of the dose or time of delivery. A first observational study in 1993 following the introduction of a CPOE system in an academic medical center focused on the time spent on the computer placing orders, regarding this time as lost for education. It highlighted the need for an involvement of the attending physicians in the student training of CPOE use.[14] The lack of time to make use of these tools that was reported in other studies might be only a temporary adjustment to a change in routine. If at first reviewing students order can be time-consuming, the integration of this method in common practice could speed up time as well as prepare students to tools they will be using in their future. In 1995, a randomized controlled study assessed the quality of a fictitious order for an imaginary patient by comparing the orders between the beginning and the end of the rotation and found higher yet non-significant progression in scores in the group which used a computerized system.[15] We found the same not significant result in our study, where actual orders for real patients were placed. In 2001, a non-randomized controlled trial in surgery wards compared the same outcome in questionnaires about surgical procedures, providing additional educational material beside the use of computerized order entry and found no difference between the two groups.[16] Two non-randomized controlled studies in 2005 and 2012 compared the self-assessed skills and the orders quality between the two groups and found no statistically significant differences. However, students in the CPOE groups reported less occasions to place orders as their supervisors either placed the orders before them or didn’t let them by lack of time.[17,18] In our study design, as the software was already part of the routine workflow in the hospital, CRs were keener on letting students place orders using CPOE. Moreover we used a randomized cross-controlled design to avoid the bias of getting the most motivated students and CRs in the case group as well as a method of analysis taking account of the various uncontrollable factors. The most serious limitation to the cross-over design is that we couldn’t include the same students in the two quarters. However, as the intervention involved two parts we could still preserve comparable entities by randomizing on chief residents. Students showed a greater satisfaction, feeling of commitment to the patients’ care when they used CPOE compared to verbal orders, and reported more occasions to place admission and follow-up orders. We can’t however exclude the possibility of a Hawthorne effect in this case, the students knowing they were observed and in the CPOE group could have felt more motivated. In departments willing to let students make orders, they are able to do so in a supervised fashion with their orders being blocked until approval by a supervisor, ensuring the patients safety. Chief resident and residents can validate the orders and comment back on them to the students, and although this practice is time-consuming it allows for asynchronous teaching. As the teaching depends on the presence of both the professor, the student and a clinical case, it cannot be scheduled and thus asynchronous interaction could be an innovative way for students to make the most of their rotations. In our study, we show that setting up accounts with limited privileges for students in the CPOE system enabled us to try a new method of teaching and to conduct this trial with no modification on the existing infrastructure. It is a proof of concept that this method can be implemented throughout the whole hospital without any real difficulty regarding the hospital management or IT implementation standpoints. Furthermore, it readily allows for a routine monitoring of the quality of prescriptions by the students, and shows that utilization of CPOE by the students can be used to refine teaching practices. This would make possible follow-up studies on order behavior and patient care and see how students (and their CRs) differ in their practice and how this impacts on the transmission of knowledge. These different prospects are examples of how IS/IT (here the CPOE system) can be leveraged to quickly and easily implement, test and roll out new policies hospital-wide with virtually no overhead. The acceptance of CPOE and of its benefits seems to be unrelated to typing proficiency or computer literacy. The main concerns are about patient security and confidentiality, and time devoted to computer use. Ease of use, integration in the workflow and mostly training are seen as factors in their acceptance and approval.[5,20] As the opinion about CPOE/EHR follows a declining trend with the medical training[4–6], and with the push for the use of EHR in hospitals and for general practitioners, it is logical to train medical students as early as possible to use these tools. In this course of action, evaluating the (eventually positive) side effects in terms of medical learning had to be done, when concerns about losing relational skills and contact with the patient are starting to appear.[21] BODY.CONCLUSIONS: We conducted an educational randomized controlled trial with a cross-over design assessing the impact of the use of CPOE at the bedside by medical students on their training, satisfaction with patient care, and involvement in the medical team. In line with the handful of previous trials addressing the same issue, we found no statistically greater improvement in the students’ ability to place adequate monitoring and therapeutic orders in the group using CPOE. We tried to address as much caveats from the previous trials as possible, but our study has got its own shortcomings, statistical power being the main one. However, whereas we confirm that CPOE use does not allow a better acquisition of prescribing skills, it is notable that it improves students’ satisfaction. Not only does it so in regard to the tool used, but also in the larger scope of patient care and feeling like being part of the medical team. Students are also able to place admission and follow-up orders when using the CPOE, thus inherently being a training in the use and master of the computer tool. As the future is shaped towards a universal use of CPOE tools in hospitals, and with the evidence that first familiarization with these tools gets harder with professional experience, we recommend the introduction of computerized order entry as soon as possible in medical students training during their clerkship rotations. BODY.SUPPORTING INFORMATION: S1 TableBODY.DATA FOR THE SHORT OPEN ANSWERS QUESTIONNAIRE.: (CSV) Click here for additional data file. S2 TableBODY.DATA FOR THE SATISFACTION QUESTIONNAIRE.: (CSV) Click here for additional data file. S3 TableBODY.PREPARED DATA FOR THE SHORT OPEN ANSWERS QUESTIONNAIRE.: (CSV) Click here for additional data file. S1 TextBODY.SHORT OPEN ANSWERS QUESTIONNAIRE, TRANSLATED FROM FRENCH.: (DOCX) Click here for additional data file. S2 TextBODY.SATISFACTION QUESTIONNAIRE, ADAPTED FROM KNIGHT ET AL.: (DOCX) Click here for additional data file.

TITLE: The effect of text message support on diabetes self-management in developing countries – A randomised trial ABSTRACT.HIGHLIGHTS: •Report of a randomised trial on an mHealth intervention in 3 low income countries. •There was no additional effect of the text message self-management support. •Coverage, routine care and disease progression interfere with the potential impact. ABSTRACT.OBJECTIVE: mHealth interventions have the potential to facilitate self-management. This TEXT4DSM study implemented a mobile phone intervention in existing diabetes programmes in three low- and middle-income countries (Democratic Republic of Congo, Cambodia, and the Philippines). ABSTRACT.RESEARCH DESIGN AND METHODS: Sub-studies with a similar randomised controlled trial design were conducted in three different countries. Each sub-study included 480 adults with diabetes. Subjects were randomised to receive either routine care or routine care plus text message self-management support. The primary outcome was the difference in the proportion of subjects with well-controlled diabetes after 2 years. ABSTRACT.RESULTS: Baseline and 2-year HbA1c measurements were available for 781 individuals. After 2 years, the proportion of subjects with controlled HbA1c was 2.8% higher in the intervention group than in the control group (difference not statistically significant). In the logistic regression model, the odds ratio for having controlled diabetes after the intervention was 1.1, after adjusting for baseline HbA1c level, sex, receiving insulin treatment, and participating in the routine programme. The HbA1c dynamics over time differed between programmes; the number of people with controlled diabetes tended to increase in DR Congo and decrease in Cambodia. ABSTRACT.CONCLUSION: This study was the first to test the same mHealth intervention in different countries. The finding that text messages did not show an additional effect on diabetes control implied that expectations about mHealth should be cautious. The degree of coverage, the quality of the routine programme, and the progression of disease can interfere with the expected impact. Trial registration: ISRCTN registry (86247213). BODY.INTRODUCTION: Global attempts to increase access to diabetes care in low-income countries (LICs), have shown that the quality of diabetes care is important, and it should comprise dimensions of chronic care models, including self-management. Self-management is recognized as an essential component of diabetes care [1], [2], [3]. Diabetes Self-Management Education (DSME) and Diabetes Self-Management Support (DSMS) are activities to assist people in sustaining self-management behaviours. These complementary elements were shown to have beneficial effects [4], [5]. However, it remains a challenge to implement these chronic care elements into health systems in LICs. Some previous initiatives [6], [7], [8] have focused on education, but few have focused on supporting broader goals, such as realizing behaviour changes or developing coping skills. Diabetes self-management support interventions include a combination of tools designed to reach patients, including brochures, phone calls, and websites. Interventions that use mobile technology (mHealth) have the potential of facilitating self-management, education, and support [9]. mHealth applications have been limited in LICs, and they have had mixed effects on diabetes control [10], [11], [12], [13]. However, most studies have been small and of limited quality. Often, the theory underlying the intervention was not elaborated. Knowledge from behavioural sciences that can explain the pathway from knowledge and perception to intentions and actual behaviour – such as the theory of planned behaviour − has not been widely included in study designs or data evaluations. This lack of theory limits our understanding of the added value of mHealth interventions in self-management support and diabetes care programmes in LICs. This study, called the TEXT4DSM study, implemented a mobile phone DSMS intervention in three diabetes programmes that existed in the Democratic Republic of (DR) Congo, Cambodia, and the Philippines [14]. The overall aims of the study were to evaluate the effectiveness of the intervention in each country and to assess the processes and contextual factors that influenced the implementation. Previous reports analysed the health care context in the three settings and the process of implementation [15], [16]. The present study assessed the effect of the intervention after 2 years on health outcomes and on intermediate outcomes. The primary outcome was the change in the proportion of subjects with a HbA1c below 7.0% (53 mmol/mol) after 2 years. BODY.METHODS: BODY.STUDY DESIGN: This study consisted of three sub-studies in three countries. Each sub-study was designed as a two-arm, randomised controlled trial (RCT). Each sub-study included 480 adults with diabetes (type 2 or 1) that were currently participating in an existing DSME programme. Participants were randomly allocated to either self-management education, as provided by the existing programme (DSME-only; control group), or to self-management education plus a mobile phone for self-management support (DSME + DSMS; intervention group) [14]. Participants in both arms were assessed at baseline, at one year, and at two years after inclusion. The primary outcome measure was the change in the percentage of subjects with well-controlled HbA1c levels after 2 years. Outcomes were compared between the intervention and control groups. A secondary outcome measure was the change in HbA1c levels after 2 years. Again, the intervention and control groups were compared. The study design is shown in Fig. 1 (in the webannex). BODY.ETHICS APPROVAL: Medical ethics approval for this study was obtained from the Institutional Review Board of the Institute of Tropical Medicine, Antwerp (11245776); the Medical Ethics Committee of the Universitair Ziekenhuis, Antwerpen (B300201111924); the National Ethics Committee for Health Research in Cambodia (207 NECHR); the University of Kinshasa in the DR Congo (ESP/CE/050/11); and the Veterans Memorial Medical Centre in the Philippines (VMMC-2011-012). The project was reviewed by the Bridges Executive Committee. BODY.STUDY CONTEXT AND SUBJECTS: The studies took place within the ‘Kin-réseau’ programme in DR Congo, the ‘MoPoTsyo’ programme in Cambodia, and the First Line Diabetes Care (FiLDCare) project in the Philippines. Kin-réseau is a 40-year old network of faith-based primary care facilities in Kinshasa, which deliver diabetes care and education as part of their basic package. MoPoTsyo is a community-based peer educator network supported by an Non-Governmental Organisation (NGO), in which patients are recruited through community screening, receive biomedical care facilitated by the NGO, and receive self-management support in groups facilitated by a peer educator. In the FiLDCare project, trained health workers and Barangay (community) health workers provide diabetes education and support for patients with diabetes. Subjects were eligible for study participation, when they were ⩾18 years old, had been diagnosed with diabetes, were registered in a centre participating in the study, and had received at least one session in the usual care programme during the preceding year. BODY.ROUTINE PROGRAMME: BIOMEDICAL CARE AND DSME: The biomedical care consisted of periodic consultations with a doctor (ranging from every 2 months in the Kin-réseau to every half year in MoPoTsyo), which included monitoring glycaemia and risk factors and prescribing medication. Educators provided ongoing DSME. In Kin-réseau, a nurse scheduled weekly DSME sessions, with groups of about 100 patients. In MoPoTsyo, peer educators scheduled monthly DSME sessions, typically with groups of 60 patients. In FiLDCare, DSME sessions were scheduled on a regular, but not fixed basis, with groups of about 8 patients [15]. Educators used posters and booklets to convey their message, both in group sessions and in individual contacts. In preparation for the study, the DSME programme was optimised to a minimum standard, which comprised messages for about nine dimensions of diabetes self-management. The messages were related to diabetes self-care and self-management; they were based on literature, professional standards, and consultations with diabetes experts [17], [18], [19]. The nine dimensions were: 1) explanation of diabetes disease processes; 2) healthy eating; 3) physical activity; 4) monitoring; 5) medications; 6) foot care; 7) tobacco and alcohol control; 8) patient-held records; and 9) problem-solving by and empowerment of patients. BODY.THE DSME PLUS DSMS INTERVENTION: Each participant, in both control and intervention groups, was provided with a mobile phone at inclusion in the study. Contracts were negotiated with a national phone provider for buying new phones and SIM cards for all participants and for sending messages. Patients were encouraged to use the phone, for instance, to ask for advice or simply to contact other people, including fellow patients, educators, and providers. Ongoing contact with the diabetes educator provided the opportunity for patients to engage actively and to discuss the potential implementation of behaviour changes. Although participants in the control group received a mobile phone, they did not receive project-initiated phone messages. Patients in the intervention group received DSMS through automated Short Message Services (SMS) on the mobile phone. The messages were sent with the open access software, Frontline [20], in Kin-réseau and MoPoTsyo, and with the internet-based application, ‘Chikka’, in FiLDCare [21]. Messages for the DSMS were developed according to the nine dimensions of DSME. The overall protocol included a guideline about the content of messages and the underlying principles of the behaviour theory of change. The administrators of each of the three programmes developed their own protocol on the development and delivery of DSMS. These protocols took into account the local features of the organisation and context of text messaging, including the contract with the local telephone providers and the cost of sending SMS messages. These protocols specified that messages should be sent 5 times per week in Kin-réseau, 6 times per week in MoPoTsyo, and 2 times per week in FiLDCare. The message contents were developed by a team in each country, which comprised the project manager, assistant programme manager, an educator, and a general doctor with additional diabetes training. The implementation manager was responsible for sending SMS messages and for following-up any problems with coverage for the intervention. Most problems were related to technological barriers (with the phone, the subscription, or the network), contextual changes (new phone providers and people switching phone numbers), or problematic participant behaviour (people not reading their messages). Subscriptions were renewed, phone numbers adjusted and, when no other option was possible, new phones were provided. In MoPoTsyo, the manager implemented two innovations in the intervention in the 2nd year. The first was the use of voice messaging instead of SMS, due to limitations in using Khmer script; the second was the targeting of one quarter of all messages to specific groups, for instance to obese patients. The 1-year process evaluation showed that the average number of SMS messages sent to participants was 15.7 per month in Kin-réseau, 24.7 in MoPoTsyo, and 7.3 in FiLDCare, with a gradual decline over time. The decline was explained by a delay in the development of new messages [16]. BODY.THEORETICAL INTERVENTION MODEL: The theoretical framework underlying the DSMS intervention was based on the hypothesis that the messages would affect the knowledge and perceptions of patients, and this input would lead, in turn, to a change in self-management behaviour and in the utilisation of care by individual patients. The theoretical pathway to changing behaviour was largely based on the theory of planned behaviour. This theory explains how beliefs (behavioural, normative, and control beliefs) shape individual attitudes, the subjective norm, and the perceived behavioural control, which together shape the intention, and ultimately, the execution of a particular behaviour [22], [23]. The theory has been largely used to understand observed behaviour patterns, but it was also recommended as a tool in designing interventions [24]. Messages were intended to target the behavioural, normative, and control beliefs attached to each of the diabetes self-management behaviours [24], [25]. The self-management behaviours targeted were generic, but the beliefs that were addressed were specific for each context. For example, some of the messages sent were: “Keeping diabetes under control will protect your foot” (targeting behavioural beliefs), “A patient with diabetes should avoid walking barefoot” (addressing normative beliefs), and “We are trying to understand and learn how to manage diabetes on our own” (addressing control beliefs). Changing behavioural beliefs was expected to contribute to behavioural changes, when there was an opportunity and no obstacles. Changes in self-management and in the utilisation of care – which can be considered intermediate outcomes − were expected to lead to improved health outcomes over the long term (webannex, Fig. 2). The framework also indicated other factors that might impact or interfere with these pathways, such as personal or biomedical characteristics, the content of the routine programme, and the environment. BODY.RECRUITMENT: Patients and staff from the participating centres in each programme were informed about the study, and patients were invited to participate. The randomisation system used a 4 × 4 randomised block design, with the participant as the unit of randomisation. Study code numbers and randomisation envelopes were prepared prior to enrolment. After informed consent, participants were allocated to either the control or intervention trial arm. Randomisation was blinded, but the nature of the intervention informed participants about the arm they were assigned to, and educators typically learned this information through the participants. BODY.MEASUREMENTS AND MEASURES: We report on personal characteristics (sex, age, education, age of onset, time since diagnosis, travel distance to educator and doctor); on health outcomes (HbA1c, BMI, waist circumference, waist-hip ratio, blood pressure, presence of foot wounds); on the routine programme (diabetes treatment, antihypertensive treatment, an adapted version of the Patient Assessment of Chronic Illness Care score [PACICc]) [26]; on utilisation of care (contacts with educator, health care expenditure); on patient knowledge and perceptions (diabetes knowledge, feeling of control, positive and negative attitudes); and on self-management behaviour (self-monitoring glucose levels). The details on data collection were reported elsewhere [14]. Data were collected from 2012/2013 to 2014/15; they included a face-to-face interview with a predefined questionnaire, a physical examination, and a blood panel [14]. For each participant, data were collected at baseline, year 1, and year 2. Additional efforts were made to contact participants that were not present for data collection, to retrieve participants, or to obtain information about the reasons for failing to follow-up. Those reasons were recorded in a database. Patients that died, stopped the study due to diabetes-related morbidity, migrated, declined to continue in the study, or failed to follow up for unknown reasons were not included in the analysis. BODY.SAMPLE SIZE: The primary measure for calculating the sample size was the difference in the proportion of patients with well-controlled HbA1c levels (defined as HbA1C < 7.0% [53 mmol/mol]) after 2 years. The required sample size was 240 participants in each arm, in each country. This calculation was based on the following assumptions: 1) 60% of the participants had well-controlled HbA1c levels at the start; 2) a difference of 15% between the intervention and control groups was relevant, with a 2-sided significance level of 5% and a power of 80%; 3) 10% of patients would drop out over the study period [14]. BODY.ANALYSIS: Analyses of quantitative data were performed with Stata version 11. A p-value <0.05 was considered statistically significant for all tests. Continuous variables were tested for normality, and non-normal distributions were categorised. Descriptive analyses were performed for all variables. Unadjusted comparisons between study groups were performed with T-tests (for continuous variables), the Kruskal-Wallis test (for comparisons of medians), or Chi-square tests (for discrete variables). Data were analysed for confounding factors and interactions in multivariate regression analyses. We considered potential confounding effects from the following variables: HbA1c level at baseline (<7.0% vs. ⩾7.0%); sex; education level (primary or lower levels vs. secondary or higher levels); age (<45, 45–64, >64 years); time since diagnosis (<2, 2–4, 5–9, or >10 years); walking distance from the educator (⩽15 vs. >15 min); obesity at baseline (body mass index [BMI] > 30); unfavourable waist circumference at baseline (>80 cm for females and >94 cm for males in DR Congo; >90 cm for men in Cambodia/Philippines [27]); insulin treatment at baseline; and the number of SMS that an individual remembered having received in the prior month (⩾10 vs. <10). These variables were selected based on earlier evidence about determinants of HbA1c [28], our theoretical framework, and the significance of a factor based on a bivariate analyses of our own dataset (p < 0.10). We started with a simple model, which only included outcome (HbA1c status at the study end), intervention, and country (considered to be a dummy variable). We added the other variables one by one, and each variable was only retained in the model when its presence altered the OR substantially and when the difference between the two models was significant (p-value of the likelihood ratio test <0.05). After deciding which variables to retain in the model, we checked these variables for interactions. In addition to a multivariate logistic regression analysis, we analysed the change in HbA1c levels over time with longitudinal regression models. Hierarchical models were used to assess the influence of educator characteristics on the effects. We performed two secondary outcome analyses. The first analysis determined the difference between the intervention and control groups in the percentage change of individuals with well-controlled HbA1C, over the 2-year study period (see webannex, Fig. 1). The second analysis determined the individual change for each patient over 2 years. Participant HbA1C levels were evaluated at baseline and after 2 years. HbA1C levels were categorised as follows: <7.0% (53 mmol/mol); 7.0–7.9% (53–63 mmol/mol); 8.0–8.9% (64–74 mmol/mol); or >9.0% (75 mmol/mol). Participants that remained in the same category after 2 years were classified as ‘stable’. Participants that moved up one or more categories were classified as ‘deteriorating’. Participants that moved down one or more categories were classified as ‘improving’. We analysed the impact of the intervention with regression analyses. We also analysed the effect of the intervention on other, secondary outcomes and intermediate outcome variables, as indicated in the theoretical framework. Other health outcomes analysed included changes in BMI, waist circumference, waist-hip ratio, systolic and diastolic blood pressure, and the presence of foot wounds. Intermediate outcome variables were related to: 1) the utilisation of care (change in the number of contacts with the educator over the past year; direct medical and non-medical health expenditures); 2) participant knowledge and perceptions (changes in the number of correct answers on the diabetes knowledge test; feeling of control and attitude towards diabetes); and 3) self-management behaviour (self-monitoring). To capture changes in the routine programme, we also analysed changes in medication regimens and in the adapted PACICc. We performed multivariate regression analyses to check the influence of potential confounding factors on the most relevant intermediate outcome variables (knowledge, positive and negative attitudes, feelings of control, self-monitoring, and contacts with the educator); we considered the same potential confounders as those analysed for the primary outcome. We performed all analyses twice. The first analysis was performed at the aggregate level, which included all participants, with the country included as a dummy variable. The second analysis was performed at the programme level, which included the participants of each country separately. BODY.RESULTS: BODY.PARTICIPANTS AND BASELINE CHARACTERISTICS: The study included 781 participants with both baseline and 2-year follow-up HbA1c measurements. Of these, 401 were allocated to the intervention and 380 were allocated to the control group. These participants included 315 from Kin-réseau, 382 from MoPoTsyo, and 84 from FiLDCare (webannex, Fig. 3). The high loss to follow-up (LTFU) rate in FiLDCare was largely due to a discontinuation of the study in the largest of three field sites, which eliminated 317 participants. The participants in the two other field sites of FiLDCare had a LTFU rate comparable to those in Kin-réseau and MoPoTsyo. The LTFU rate due to death was larger in Kin-réseau (11%) than in the other programmes (3% and 1%). For 20 cases in DR Congo, the cause of death was recorded: 3 were directly caused by diabetes, and the other 17 were caused by various conditions, including cerebrovascular accidents, infectious diseases, and ‘old age’. Table 1 gives an overview of the differences in main characteristics between participants that completed the study and those in the LTFU group. These groups were significantly different in education levels (the LTFU group included more highly educated participants, due to differences in the FiLDCare programme). However, we found no other significant differences between groups. A previous study provided a full overview of the baseline characteristics of the study participants, the characteristics of care, physical outcomes, perceptions of care, and self-management [15]. A dataset that includes all participant data is available online (webannex 2).Table 1Differences in baseline characteristics between participants that completed the study and those lost to follow-up (*p < 0.05). Overall (n 1471)Kin-Réseau (n 506)MoPoTsyo (n 484)FildCare (n 481)n in FU (781)n LTFU (690)n in FU (315)n LTFU (191)n in FU (382)n LTFU (102)n in FU (84)n LTFU (397)Sex (male),% 29 37 33 33 33 28 21 41 Age (mean ± sd) 58 ± 10 60 ± 10 59 ± 10 63 ± 11 55 ± 9 60 ± 12 59 ± 11 63 ± 9 Education primary only or less,% 50* 26 48 51 58 58 22* 7 Time since diagnosis, med (IQR) 4 (2–8) 6 (3–12) 6 (3–10) 7 (3–7) 4 (2–7) 4 (2–7) 4 (2–10) 7 (3–13) Travel distance from educator (hour) 0.25 0.33 0.42 0.5 0.25 0.29 0.14 0.25 HbA1C at start (%, mean ± sd) 8.2 ± 2.1 8.4 ± 2.3 8.8 ± 2.3 9.1 ± 2.4 7.5 ± 1.7 7.7 ± 2.0 8.6 ± 2.7 8.2 ± 2.3 % of people with a HbA1C < 7.0% at start 35.1% 34.5% 23.2% 22.0% 44.5% 39.6% 38.1% 39.0% Treatment with insulin,% 24% 22% 49% 59% 8% 11% 9% 6% BODY.EFFECT OF THE INTERVENTION: After 2 years, an HbA1c < 7.0% (53 mmol/mol), which was considered ‘controlled diabetes’, was achieved by 33.9% of subjects in the intervention group and 31.1% in the control group (Table 1, aggregate analysis). This difference was not statistically significant (p = 0.39). In the final logistic regression model, the OR for achieving controlled diabetes after the intervention was 1.1 (95% CI 0.8–1.6). This OR was corrected for the diabetes control status at the start, sex, receiving insulin treatment, and participating in the routine programme. Testing for interactions between these variables did not change the predictive value of the model. There was no cluster effect at the educator level (webannex, Table 1). We performed a longitudinal analysis to determine whether the intervention, the country, or the time (3 time points: at baseline, 1 year, and 2 years) influenced the chance of achieving controlled diabetes after 2 years. The best predictive temporal model had a random intercept and interaction terms for intervention, time, and country. The interaction analysis showed that the intervention time had a non-significant effect in all three programmes (webannex, Table 2). BODY.DEVELOPMENT OF HBA1C OVER 2 YEARS: The secondary outcome provided information about the development of HbA1C over time at the group level. We examined the change in the proportion of patients with controlled diabetes from baseline, and we compared the intervention group to the control group (Table 2). The results were different among the programmes. In Kin-réseau, the percent increase in subjects with controlled diabetes was significantly larger in the intervention than in the control group (4.4% vs. 0.6%, p = 0.04). In MoPoTsyo, there was a decrease in subjects with controlled diabetes. In FiLDCare, diabetes control appeared to improve in the intervention group, but decrease in the control group; however, the remaining sample size was too small to demonstrate statistically significant differences.Table 2Overview of the primary outcome measure: the proportion of subjects with controlled diabetes after 2 years; and the secondary outcome measure: the difference between intervention and control groups in changes from the start to the end of the study. OverallKin-réseauMoPoTsyoFildCareDSME + DSMS (S)DSME-only (E)p, H0: ΔS = ΔEDSME + DSMS (S)DSME-only (E)p, H0: ΔS = ΔEDSME + DSMS (S)DSME-onlyp, H0: ΔS = ΔEDSME + DSMS (S)DSME-only (E)p, H0: ΔS = ΔE% of people being controlled at end (<7.0%) 33.9% 31.2% 0.39 29.4% 21.9% 13.0% 35.4% 37.5% 0.66 44.2% 36.6% 48.0% Change of% of people with controlled diabetes (p H0: Δ = 0) −1.7%, p = 0.60 −3.6%, p = 0.28 0.09  + 4.4%, p = 0.38  + 0.6%, p = 0.89 0.04* −9.1%, p = 0.06 −7.1%, p = 0.17 0.47  + 9.3%, p = 0.38 −4.9%, p = 0.65 0.43 The development of HbA1C in individual participants was evaluated by determining the different HbA1C categories (<7.0%; 7.0–7.9%; 8.0–8.9%; >9.0%) at the start and end of the study. We found that the majority of subjects did not change HbA1c categories over 2 years (webannex, Fig. 4). The proportion of subjects that remained in the same HbA1c category was larger in the control group (59.7%) than in the intervention group (50.6%). However, this difference was only significant in Kin-réseau (66.5% vs. 51.3%, p = 0.01). The proportion of subjects that improved by at least one category was larger in the intervention group than in the control group (20.9% vs. 15.5%, p = 0.05; aggregate analysis; webannex, Table 3). In the logistic regression model, the OR for improving by at least one category after the intervention was 1.4 (0.9–2.0, p = 0.10). This OR was corrected for the time since diagnosis, for remembering at least 50% of the DSMS, and for the country’s routine programme. Testing for interactions between these variables did not improve the predictive value of the model; thus, interactions were ignored. Moreover, we did not observe a cluster effect at educator level (webannex, Table 4). BODY.OTHER OUTCOMES: The number of subjects with foot wounds decreased significantly more in the intervention group than in the control group. The intervention did not significantly alter the other health outcomes, even after controlling for potential confounding factors. For some secondary health outcomes, a significant change over time was observed, but the changes were similar for both intervention and control groups. For example, both groups showed increases in waist circumference and decreases in diastolic blood pressure, in all countries. The intervention did not appear to have an effect on the intermediate outcome indicators, including patient knowledge, perceptions, and the utilisation of care (Table 2). Some indicators showed a change over time, but these changes were similar in both the intervention and control groups. For instance, all participants showed a drop in attendance to meetings with the educator in MoPoTsyo and FiLDCare. The negative attitude towards diabetes declined over time for all participants in Kin-réseau and FiLDCare. In MoPoTsyo, the number of subjects that self-monitored glucose levels significantly decreased in both the intervention and control groups. Table 2 also shows that some changes in diabetes management occurred in the routine programme, which affected both groups. In Kin-réseau, the number of participants that received insulin increased by 20% in both the intervention and control groups. In Kin-réseau and MoPoTsyo, the number of participants that received antihypertensive treatment increased in both groups, but the increase was greater in the intervention group than in the control group. In all programmes, the PACICc score declined (Table 3).Table 3Change in other health outcomes from the start to the end of the study, for intervention and control groups (pp = percentage point; headings refer to the theoretical framework shown in webannex, Fig. 2). OverallKin-réseauMoPoTsyoFildCareDSME + DSMS (S)DSME-only (E)p, H0: ΔS = ΔEDSME + DSMS (S)DSME-only (E)p, H0: ΔS = ΔEDSME + DSMS (S)DSME-only (E)p, H0: ΔS = ΔEDSME + DSMS (S)DSME-only (E)p, H0: ΔS = ΔEp(H0:start = end)p(H0:start = end)p(H0:start = end)p(H0:start = end)p(H0:start = end)p(H0:start = end)p(H0:start = end)p(H0:start = end)Other health outcomes Change in BMI, mean ± sd +0.0 ± 1.9, p = 0.92 +0.0 ± 2.4, p = 0.82 0.9 +0.3 ± 2.0, p = 0.85 +0.3 ± 2.7, p = 0.91 0.9 −0.1 ± 1.2, p = 0.70 −0.2 ± 1.6, p = 0.48 0.5 −0.1 ± 3.7, p = 0.87 +0.3 ± 4.1, p = 0.85 0.6 Change in waist circumference, mean ± sd +3 ± 8, p = 0.00* +2 ± 8, p = 0.00* 0.2 +4 ± 9, p = 0.00* +3 ± 9, p = 0.01* 0.4 +2 ± 5, p = 0.07 +1 ± 5, p = 0.14 0.4 +6 ± 12, p = 0.02 +4 ± 15, p = 0.11 0.7 Change waist hip ratio, mean ± sd 0.00 ± 0.08, p = 0.39 0.00 ± 0.10, p = 0.22 0.7 0.00 ± 0.10 0.00 ± 0.13 0.9 +0.01 ± 0.05 +0.01 ± 0.05 0.5 +0.01 ± 0.08 +0.01 ± 0.12 1 Change in systolic blood pressure mean ± sd +1 ± 24, p = 0.03* +2 ± 26, p = 0.00* 0.6 0 ± 27, p = 0.99 +2 ± 30, p = 0.57 0.5 +5 ± 17, p = 0.01* +5 ± 18, p = 0.02* 0.9 −9 ± 32, p = 0.14 −7 ± 33, p = 0.24 0.7 Change in diastolic blood pressure −4 ± 14, p = 0.00* −3 ± 13, p = 0.00* 0.5 −4 ± 16, p = 0.03* −3 ± 15, p = 0.04* 0.8 −2 ± 10, p = 0.02* −2 ± 10, p = 0.01* 0.9 −10 ± 19, p = 0.00* −5 ± 17, p = 0.11 0.2 Change in people with foot wound, pp −3.5%, p = 0.02* −1.3%, p = 0.38 0.1 −9.0%, p = 0.01 −3.9%, p = 0.21 0.1 +0.5%, p = 0.65 +0.5%, p = 0.65 0.96 −2.3%, p = 0.64 0.0%, p = 1 0.33 

 Utilisation of care Change in number of contacts with educator (last year), median (IQR) −2 (−9; +2), p = 0.00* −2 (−9; +2), p = 0.00* 0.7 0 (−8; +6), p = 0.96 0 (−12; +4), p = 0.98 0.8 −4 (−9; 0), p = 0.00* −3 (−9; 0), p = 0.00* 0.5 −2 (−8; 0), p = 0.00* −1 (−3; 0), p = 0.00* 0.3 Change in direct health expenditure (USD), median (IQR) +0.50 (−3.14; +4.31), p = 0.39 +0.25 (−3.81; +3.52), p = 0.04* 0.2 −0.76 (−6.83; 7.02), p = 0.45 −2.01 (−8.17; +3.81), p = 0.01* 0.2 +0.75 (−0.88; +3.02), p = 0.00* +0.75 (−0.63; +3.02), p = 0.06 0.8 −11.59 (−23.18; +44.83), p = 0.00* −11.59 (−42.88; +40.56), p = 0.00* 0.5 Change in direct non-medical health expenditure, median (IQR) 0 (−3.27; +1.76), p = 0.39 0 (−3.27; +1.51), p = 0.01* 0.9 −7.08 (−47.92; 3.22), p = 0.25 −6.29 (−38.06; 3.81), p = 0.87 0.7 0 (−1.26; +1.26), p = 0.43 0 (−1.26; +1.26), p = 0.01 0.6 0.00 (−8.11; +0.00) (n = 10) −9.73 (−16.22; −8.81) (n = 7) 0.01* 

 Patient knowledge & perceptions Change in correct diab knowledge, mean +0.3 ± 4.0, p = 0.16 +0.7 ± 4.3,p = 0.00* 0.2 +0.3 ± 5.2 p = 0.53 +0.4 ± 4.3, p = 0.33 0.8 +1.0 ± 2.92, p = 0.00* +1.4 ± 2.98, p = 0.00* 0.3 −2.7 ± 5.2, p = 0.00* −1.4 ± 6.1, p = 0.11* 0.3 Change in feeling of control, mean 0.3 ± 3.2, p = 0.35 +0.2 ± 3.4, p = 0.46 0.8 −0.2 ± 3.4, p = 0.36 −0.7 ± 3.0, p = 0.06 0.3 0.3 ± 2.6, p = 0.31 +0.1 ± 2.9, p = 0.70 0.6 +2.0 ± 4.7, p = 0.00* +3.6 ± 4.5, p = 0.00* 0.1 Change in positive attitude, mean +0.3 ± 3.8, p = 0.11 0.0 ± 4.1, p = 0.94 0.2 +0.3 ± 4.2, p = 0.48 −0.5 ± 4.3, p = 0.14 0.1 −0.1 ± 3.1, p = 0.63 0 ± 3.5, p = 0.95 0.7 +2.5 ± 4.5, p = 0.00* 1.8 ± 5.1, p = 0.02* 0.5 Change in negative attitude, mean −1.8 ± 6.4, p = 0.00* −1.3 ± 6.5, p = 0.00* 0.3 −3.1 ± 6.3, p = 0.00* −2.1 ± 6.2, p = 0.00* 0.2 +0.2 ± 5.6, p = 0.65 +0.7 ± 5.7, p = 0.13 0.4 −6.6 ± 6.5, p = 0.00* −7.1 ± 6.7, p = 0.00* 0.8 

 Self-management behaviour Change in people self-monitoring, pp −15.0%, p = 0.00* −10.4%, p = 0.00* 0.1 +3.4%, p = 0.50 +5.5%, p = 0.27 0.4 −31.3%, p = 0.00* −28.3%, p = 0.00* 0.51 −8.7%, p = 0.38 +9.2%, p = 0.38 0.94 

 Indicators about the routine programme Change in people on insulin treatment, pp +7.4%, p = 0.01* +11.2%, p = 0.00* 0.1 +20.3%, p = 0.00* +18.6%, p = 0.00* 0.7 +0.2%, p = 0.93 +6.7, p = 0.06 0.00* 1.4%, p = 0.84 +2.5%, p = 0.69 0.7 Change in people on antihypertensive treatment, pp +15.8%, p = 0.00* +10.4%, p = 0.00* 0.03* +22.2%, p = 0.00* +13.9%, p = 0.02* 0.1 +16.3%, p = 0.00* +8.0%, p = 0.13 0.02* −10.5%, p = 0.29 +8.8%, p = 0.43 0.2 Change in PACIC, mean −6.3 ± 12.8, p = 0.00* −6.3 ± 18.8, p = 0.00* 1 −2.6 ± 12.8, p = 0.01* −1.8 ± 12.2, p = 0.07 0.6 −7.2 ± 10.5, p = 0.00* −7.8 ± 11.8, p = 0.00* 0.6 −15.1 ± 18.0, p = 0.00* 16.2 ± 21.5, p = 0.00* 0.8 BODY.DISCUSSION: BODY.SUMMARY OF FINDINGS: The DSMS intervention in our study did not increase the number of subjects with controlled diabetes after 2 years. The multivariate regression analyses showed that the most important determinant for whether a subject had controlled diabetes after 2 years was starting with controlled diabetes at baseline. Consistent with the lack of a DSMS effect, the HbA1c analysis for individual participants showed that more than half of the participants remained in the same HbA1c category. However, the general tendencies in HbA1c dynamics over time were different between programmes. For all participants, the number with controlled diabetes increased in Kin-réseau, but decreased in MoPoTsyo. Moreover, the favourable development in Kin-réseau was significantly larger for the intervention group than for the control group. There were no significant effects of the intervention on the intermediate outcomes or on other outcomes. For intermediate outcomes, the intervention and control groups showed similar decreases in negative attitude towards diabetes and similar increases in the number of subjects that received insulin and anti-hypertension treatment, in Kin-réseau and FiLDCare. The baseline characteristics of the participants in the MoPoTsyo programme, such as HbA1C and BMI, were more favourable than those of participants in the other programmes. Nevertheless, participants in the MoPoTsyo programme displayed many indicators that worsened over the study period. Coincident with the reduction in the frequency of contact with the educator, we observed reduced percentages of subjects that performed glucose self-monitoring. These changes might be explained by the scaling up that occurred in the routine MoPoTsyo programme, within the national strategy; this process led to uncertainty among staff and patients and delays in payments [29]. BODY.LIMITATIONS: The limitations of our study included the design and rate of the LTFU variable, the heterogeneity of the implementation of the intervention and the tools used for data collection, and the lack of intention-to-treat (ITT) analysis. The LTFU rate was different in each programme. Some of the subjects that were LTFU had died or became seriously ill, which implied that these subjects had worse health outcomes than those that completed the study. However, the LTFU rates were comparable between the intervention and control groups; therefore, the LFTU rates were unlikely to have influenced the estimated intervention effects. Nevertheless, the particularly high LTFU rate in FiLDCare, which reflected the discontinuation of the study at the largest site, made it difficult to interpret findings from that programme. Nevertheless, the LTFU rates for the two remaining FiLDCare study sites were comparable to the LTFU rates in the other two programmes, both in number and in causes. These differences in LTFU rates justified the separate analyses for each programme. This limitation was foreseen, and it was accounted for in the original sample size calculation. An ITT analysis would have further reduced the differences between intervention and control groups. Although we anticipated a risk of contamination between the intervention and control groups, through contacts between patients of different groups, the fact that contamination occurred was another study limitation [14]. Programme managers and educators reported that patients of different groups exchanged messages. In addition, some contamination probably stemmed from educators that were aware of the contents of DSMS text messages, and incorporated similar content into their routine DSME protocol; thus, the DSMS content reached both groups. In Kinshasa, patients visited the educator to ask additional questions; this activity contributed to greater interactions. However, this contamination can also be regarded as a (welcome) strengthening of the routine programme. The implementation of the intervention was more problematic than foreseen. Technological barriers limited the abilities to target messages to specific individuals, to tailor text messages to the (different) patient’s needs, and to reach all the participants. Moreover, the commercialisation of the SMS market resulted in subjects becoming overwhelmed with messages, which led to a degree of lethargy in reading them. The organisational capacity required to develop and send messages regularly varied across programmes. This variation led to differences in message frequency and coverage efficacy across the three programmes [16]. The coverage was best in Kin-réseau, where more than half of the patients remembered receiving most messages. Better coverage appeared to be linked to better results. Although we used a theoretical framework in designing the intervention, it was difficult to target behavioural beliefs in the message texts. For data collection, we implemented a scale from the Diabetes Care Profile, which measured overarching concepts, such as the feeling of control and the attitude towards diabetes, instead of specific behavioural beliefs. Most of these scales produced homogeneous scores between groups. Thus, these instruments had limited value for detecting differences between contexts or over time. BODY.THIS STUDY FROM A LARGER PERSPECTIVE: Two meta-analyses have reported that mobile phone interventions and automated text messages have had mixed effects on glycaemic control in patients with type 2 diabetes [11], [13]. A number of studies in developing countries demonstrated that SMS messaging had a positive impact on HbA1C [30], [31]. However, most previous studies only lasted 6 months to a maximum of 1 year; in contrast, our study lasted 2 years. Moreover, our study included a wide variety of patients (e.g., different diabetes durations, different antidiabetic therapies), in contrast to many other studies, which selected more homogeneous cohorts, for instance, only patients on oral treatment [30]. Some previous studies combined SMS with other tools [32], and only few studies reported on intermediate outcomes. Thus, it has been difficult to unravel the mechanisms of change [13]. The SMS messaging in our study had been conceptualised as behavioural support, based on other studies that had provided some evidence of success [9]. However, if the messages were not perceived as support, their potential effect would have been influenced [33]. Our theory of change stated that DSMS works through the mechanism of increasing patient knowledge and perceptions. Therefore, an absence of measurable changes in these indicators could explain the lack of effect on the final outcomes. It is also possible that other, unrevealed covariates interfered with the relationship between the intervention and HbA1c. Personal and disease-related characteristics of the patient, and characteristics of the routine care could have had stronger influences than expected for health outcomes. From a process analysis, we learned that the number of participants that used their phone on their own initiative to call or send SMS messages about issues related to diabetes increased in all three countries. Patients most frequently contacted their educator, their doctor, and other patients. In-depth analyses of the interdependency between the intervention goals, the intermediate outcome, and the final outcome might explain some elements of this study [34]. Our study was performed in three different programmes, and in each programme, participants had different baseline characteristics and health indicators. It is known that HbA1C gradually increases over time, by an average of roughly 1% (10.9 mmol/mol) over 2 years. However, it is less clear how the duration of disease, quality of care, and different phenotypes of diabetes impact this trend [35], [36]. Another explanation for the absence of an effect might be that we did not measure alternative indicators. That is, potential effects of the DSMS intervention might have been realised, not through the mechanism of increased knowledge or better coping skills, but through patients receiving improved care. For example, more people may have started taking insulin or received improved education on routine care. The fact that similar studies have shown a positive effect on HbA1c without a substantial change in behaviour also suggested that other pathways might have influenced the effects [37]. These effects would most likely spill over into the control group, which would reduce our ability to detect differences. BODY.CONCLUSIONS: This study reported results from a RCT that tested an mHealth intervention for diabetes control in three different LMICs, with a long-term follow-up of 2 years. Our study did not show a benefit of adding the mHealth intervention to existing care and self-management programmes. The absence of an effect might be explained by the variety of patients and disease-related characteristics, the inhomogeneous implementation of the intervention, and/or the influences that the routine programme might have had on the outcomes. BODY.AUTHOR CONTRIBUTIONS: JVO was the coordinating investigator of the study and drafted the first version of the manuscript. KC and JDM contributed to the data analyses. GMK was in charge of the study in the Philippines; MVP was in charge in Cambodia; and JCK and CD were in charge in the DR Congo. HH performed the field-work coordination in Cambodia, including the development of SMS messages. KVA contributed substantially to the interpretation of the results, and thus, to the Discussion section. DK and BM contributed to the adaptation of the intervention and to the interpretation of the results, which were important in writing this paper. FS supervised the drafting of this manuscript and commented on all phases, from setting-up the paper to the final version. GK was the principal investigator of the study from the start. He also contributed to the Methods section and the interpretation of results. BODY.DUALITY OF INTEREST AND ACKNOWLEDGEMENT: KVA was the chair of the International Working Group on the Diabetic Foot of the International Diabetes Federation. MVP was executive director of MoPoTsyo. HH was an employee of MoPoTsyo. CD was a volunteer at Memisa Brussel. JCK was employed by Memisa DR Congo. GMK was the initiator of the FiLDCare project. FS had no conflict of interest. GK was the principal investigator of the TEXT4DSM study, but he had no financial conflict of interest. JVO was the coordinating investigator of the TEXT4DSM study. JVO takes full responsibility for the contents of the article. BODY.FUNDING: The TEXT4DSM study was supported by a BRIDGES grant from the International Diabetes Federation. BRIDGES, an International Diabetes Federation project, is supported by an educational grant from Lilly Diabetes. The grant was used to pay part of the salaries of JVO, HH, JCK, and GMK.

TITLE: Lactobacillus gasseri SBT2055 suppresses fatty acid release through enlargement of fat emulsion size in vitro and promotes fecal fat excretion in healthy Japanese subjects ABSTRACT.BACKGROUND: Lactobacillus gasseri SBT2055 (LG2055) has been shown to prevent abdominal adiposity, and suppression of lipid absorption is considered a possible mechanism, detail of which, however, are poorly understood. In the present study, we evaluated the effects of LG2055 on fat hydrolysis by determining pancreatic lipase activity and fat emulsion properties in vitro. We also examined whether LG2055 influences fecal fat excretion in humans. ABSTRACT.METHODS: Pancreatic lipase activity was investigated in vitro using an artificially prepared fat emulsion and 4-methylumbelliferyl oleate (4-MUO) as substrates. The concentrations of free fatty acids and 4-methylumbelliferone were quantified. Fat emulsion droplet size was measured using a particle size analyzer. The clinical study was performed as a double-blind, randomized, placebo-controlled trial. Subjects consumed 100 g of fermented milk (FM)/d, either with or without LG2055 supplementation, for seven days. Fecal samples were collected during three-day pre-observational and FM intake periods and fecal fat levels were determined. ABSTRACT.RESULTS: LG2055 dose-dependently suppressed lipase activity in the fat emulsion assay but not in the 4-MUO assay. LG2055 dose-dependently increased fat emulsion droplet size. The effects of LG2055 on lipase activity and fat emulsion properties were increased compared with four other tested strains (Lactobacillus gasseri SBT0317, Lactobacillus gasseri JCM1131T, Lactobacillus. delbrueckii subsp. bulgaricus JCM1002T and Streptococcus thermophilus ATCC19258T). In our clinical study, fecal fat level after FM intake was significantly increased compared with that observed before FM intake in the LG2055-containing active FM group but not the control FM group lacking LG2055. ABSTRACT.CONCLUSIONS: LG2055 increased fat emulsion droplet size, resulting in the suppression of lipase-mediated fat hydrolysis. The influence of LG2055 on the physicochemical properties of fat emulsion provides a mechanism for the probiotic-mediated suppression of lipid absorption and promotion of fecal fat excretion in humans. ABSTRACT.TRIAL REGISTRATION: UMIN000015772 BODY.BACKGROUND: Lactobacillus gasseri SBT2055 (LG2055), a probiotic lactic acid bacterium originating in the human intestine [1,2], has an ability to improve the intestinal environment [3] and exerts anti-obesity effects in rats [4-6] and mice [7]. Our previous clinical studies in Japanese adults with overweight exhibited a significantly decreased visceral fat area, body weight, body mass index (BMI), and waist and hip circumferences following consumption of fermented milk containing LG2055 at 200 g/d for 12 weeks [8,9]. Suppression of lipid absorption in the small intestine has been proposed as a potential mechanism for the anti-obesity effects of LG2055. Hamad et al. evaluated lymphatic lipid content in rats with permanent cannulation of the thoracic duct [4]. They demonstrated that rats fed a diet containing fermented skim milk supplemented with LG2055 showed a lower maximal rate of lymphatic lipid absorption compared with rats fed a diet containing non-fermented skim milk; these findings were supported by the observation of increased fecal fatty acid excretion [4]. Furthermore, our recent study showed that Japanese hypertriacylglycerolemic subjects who consumed fermented milk containing LG2055 at 200 g/d for 4 weeks demonstrated significantly decreased postprandial serum lipid concentrations after the intake of oral fat-loading test meals [10]. Serum lipid concentrations are influenced by not only lipid absorption via diet but also lipid metabolism. Therefore, estimation of fecal lipid excretion is a more direct index to examine dietary lipid absorption. Dietary lipid digestion undergoes several complex processes prior to mucosal absorption in the small intestine. Pancreatic lipase is a key enzyme for lipid absorption because the majority of lipolysis is carried out in the duodenum by pancreatic lipase that is secreted from the pancreas and hydrolyzes dietary lipid-derived triacylglycerol into glycerol and fatty acids [11]. Dietary triacylglycerol, the major source of dietary fat, is not directly absorbed in the intestine unless it has been hydrolyzed by pancreatic lipase. Therefore, suppression of lipase-mediated fat hydrolysis is an effective approach for suppression of dietary triacylglycerol absorption [12]. Suppression of lipase-mediated fat hydrolysis is mainly associated with two mechanisms: the first is direct enzymatic inhibition by an inhibitor like orlistat, which is a potent pancreatic lipase inhibitor used as a pharmaceutical agent for the management of obesity [13]; the second is associated with modification of fat emulsion properties. Fat emulsion interface properties, including droplet size and specific surface area, influence the effects of lipase-mediated fat hydrolysis on dietary fat absorption [14]. As fat emulsion particle diameter increases, specific surface area decreases. Thus, lipase-mediated fat hydrolysis is sensitive to fat emulsion size [15], and the change in fat emulsion droplet size is critical for modification of fat digestion and absorption. However, the mechanism of LG2055-mediated suppression of lipid absorption is unclear. In this study, we investigated the mechanism associated with suppression of lipid absorption by the probiotic bacterium LG2055. We determined LG2055 effects on pancreatic lipase-mediated hydrolysis of an artificial fat emulsion or synthetic substrate (lipase activity) and measured fat emulsion droplet size in vitro using a simple oil-in-water emulsion as a physiological model. We also examined whether intake of LG2055 altered fecal fat excretion in healthy Japanese subjects. BODY.METHODS: In vitro study BODY.MATERIALS: Triolein, taurocholic acid, pancreatic lipase (type VI-S, from porcine pancreas), orlistat, and 4-methylumbelliferyl oleate (4-MUO) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) was purchased from Dojindo Laboratories (Kumamoto, Japan). Lecithin from egg, NEFA C Test Wako, and catechin mixture from green tea (product number 032–18231) were purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan). BODY.PREPARATION OF LG2055 AND OTHER BACTERIA: Lactobacillus gasseri SBT2055 (LG2055), a bacterial strain derived from a fecal specimen of a healthy adult originally isolated by Fujiwara et al. [3] was deposited in the International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba, Ibaraki 305–8566, Japan). Lactobacillus gasseri SBT0317 (LG0317) was isolated from a dairy product and stocked at Megmilk Snow Brand Co. Ltd. [16]. Lactobacillus gasseri JCM1131T (LG1131T) and Lactobacillus delbrueckii subspecies (subsp.) bulgaricus JCM1002T (LB1002T) were obtained from the Japan Collection of Microorganisms. Streptococcus thermophilus ATCC19258T (ST19258T) was obtained from the American Type Culture Collection (Manassas, VA, USA). Each strain was grown at 37°C for 16 h in de Man, Rogosa and Sharpe (MRS) broth (Becton-Dickinson and Company, MD, USA). Harvested cells of each strain were washed twice with saline and once with sterilized water, then lyophilized using a freeze dryer (FDU-2200, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). BODY.MEASUREMENT OF PANCREATIC LIPASE ACTIVITY: Pancreatic lipase activity using fat emulsion as a substrate was determined using the method described by Han et al. [17]. A fat emulsion was prepared by sonication of lipid suspensions composed of triolein (80 mg), lecithin (10 mg), and taurocholic acid (5 mg) in 9 ml of TES buffer (0.1 M TES, 0.1 M NaCl, pH 7.0) for 25 min. Bacterial cells of each strain were suspended in TES buffer. For the enzyme reaction, 100 μl of a bacterial cell suspension or orlistat solution and 50 μl of pancreatic lipase (10 units) were added to 100 μl of sonicated substrate suspension (fat emulsion) in a total volume of 250 μl, and the reaction mixture was incubated at 37°C for 30 min. After completion of the reaction, the solution was heated in a boiling water bath for 2 min for enzyme inactivation. The blank of each sample was heated in a boiling water bath for 2 min immediately following addition of the enzyme solution for inactivation. The concentration of released fatty acids was measured using a NEFA C Test Wako. To examine dose-dependency, LG2055 suspensions were prepared at final concentrations ranging from 1–100 μg/ml. The effects of five bacterial strains (LG2055, LG1131T, LG0317, LB1002T and ST19258T) were compared using preparations at final concentrations of 100 μg/ml. Pancreatic lipase activity of each sample was calculated using fatty acid production in the absence of sample as 100%. Lipase activity using 4-MUO as a substrate [18] was determined using the method described by Nakai et al. [19]. LG2055 cells were suspended in distilled water. Twenty-five microliters of a LG2055 suspension or an orlistat solution and 50 μl of a 0.1 mM 4-MUO solution dissolved in Tris buffer consisting of 13 mM Tris–HCl, 150 mM NaCl, and 1.3 mM CaCl2 (pH 8.0) were mixed in the well of a microtiter plate, and 25 μl of the lipase solution (50 U/ml) in Tris buffer was then added to initiate the enzyme reaction. After incubation at 25°C for 30 min, 100 μl of 0.1 M sodium citrate (pH 4.2) was added to terminate the reaction. The amount of 4-methylumbelliferone released following the lipase reaction was measured using a fluorometrical microplate reader (Varioskan™ Flash, Thermo Fisher Scientific, Inc., MA, USA) at an excitation wavelength of 327 nm and an emission wavelength of 449 nm. The LG2055 suspensions and orlistat solutions were prepared at final concentrations ranging from 1–100 μg/ml, and 0.1–100 μg/ml, respectively. Pancreatic lipase activity of each sample was calculated using 4-methylumbelliferone production in the absence of sample as 100%. BODY.MEASUREMENT OF PARTICLE SIZE OF FAT EMULSION: LG2055 was suspended in 1.2 ml of TES buffer and added to 8 ml of the fat emulsion preparation. After addition of LG2055, the pH of the suspension was adjusted to 7.5 using NaHCO3, to reflect the in vivo condition, in which pancreatic juice containing alkaline sodium (sodium bicarbonate (NaHCO3)) is secreted into the small intestine for maintenance of a neutral pH. TES buffer was used as a negative control, whereas a catechin mixture derived from green tea (Wako) was used as a positive control [20]. The prepared final suspensions were incubated at 37°C with constant shaking (100 strokes/min) for 3 h [20]; next, the size distribution and mean fat emulsion droplet sizes were measured using a particle size analyzer (Microtrac ® MT3000II, Nikkiso Co., Ltd, Tokyo, Japan). Suspensions of the five bacterial strains (LG2055, LG1131T, LG0317, LB1002T and ST19258T) were prepared at a final concentration of 100 μg/ml. BODY.HUMAN STUDY: BODY.SUBJECTS: Thirty healthy adults (12 men and 18 women) between 27–69 years of age were enrolled in the study. Subjects with a frequency of defecation less than five days per week and severe internal organ disorders, including coronary heart disease, respiratory impairment, endocrinopathy, or alimentary allergy were excluded. None of the subjects consumed special health-promoting foods, took medications known to alter lipid metabolism, or regularly ingested fermented milk. BODY.STUDY DESIGN: The study was performed as a double-blind, randomized, placebo-controlled clinical trial, according to the guidelines established in the Declaration of Helsinki. All procedures involving human subjects were approved by the institutional review board of the Miyawaki Orthopedic Clinic (Eniwa City, Hokkaido, Japan) prior to initiation of the study. All subjects provided written informed consent prior to study participation. The study was conducted from January 2014 to February 2014 by a contract research organization, New Drug Research Center, Inc. (Minato-ku, Tokyo, Japan). The clinical trial was registered at the University Hospital Medical Information Network Clinical Trials (No. UMIN000015772). BODY.PREPARATION OF THE TEST FERMENTED MILK: Two types of fermented milk (FM) were prepared: the active FM containing LG2055 and the control FM lacking LG2055. The active FM was prepared using lactic acid bacteria starter cultures (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) commonly used for conventional yogurt production and viable LG2055 cells. An FM mixture consisting of approximately 11% skim milk powder with a small amount of flavor, agar, and sucralose as a non-caloric artificial sweetener was inoculated with the yogurt starter cultures and LG2055 cells, then cultured at 40°C for 3.5–4 h. On the initial day, the viable cell count of LG2055 was approximately 5 × 109 CFU/100 g of FM. The control FM was prepared in the same manner, except that LG2055 cells were excluded. These FM preparations were equivalent in energy (146.4 kJ), protein (3.7 g), fat (0.1 g), carbohydrate (4.9 g), and sodium content (40 mg) per 100 g and were indistinguishable in taste. The test FM preparations were kept in cold storage and delivered weekly. BODY.STUDY SCHEDULE AND PROTOCOL: The study length was fourteen days, comprising a seven-day pre-observational period followed by a seven-day FM intake period. Subjects were randomized into the control and active FM groups. To equalize energy and fat intakes, subjects were provided the same diets throughout the entire study period (14 days), consisting of different menus at each meal, each day of the study. Energy intake was set at approximately 8368 and 9623.2 kJ per day in women and men, respectively; the amount of fat intake was set at approximately 70 and 85 g per day (31.5 and 33.3 percent energy) in women and men, respectively. After the seven-day pre-observational period, both groups started consuming the test FM for seven days. The subjects consumed FM at 100 g/d with meals (at either breakfast, lunch or dinner) and they were asked to maintain their normal lifestyle habits, including exercise routines. All subjects maintained a detailed dietary record during the entire study period. Fecal samples were collected during the final three days of the pre-observational (days 5–7) and FM intake periods (days 12–14). Fecal samples were weighed and immediately frozen for storage. Body weight, body fat percentage, blood pressure, pulse rate, and fasting blood parameters (triacylglycerol, total cholesterol, high density lipoprotein cholesterol, NEFA, glucose, total protein, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transpeptidase) were determined at the beginning of the test period (day 1) and the day after the end of the experimental period (day 15). Physician interviews were also performed at each time point (day 1 and 15). Information regarding subjective symptoms such as headache, nausea, and abdominal pain were obtained through a physician interview at each time point (day 1 and 15). BODY.MEASUREMENT OF FECAL FAT CONCENTRATION: Fecal fat level was determined according to the methods described by van de Kamer et al. [21]. BODY.PHYSICAL CHARACTERISTICS AND BLOOD SAMPLE ANALYSES: Blood analyses were performed by SRL, Inc. (Shinjuku-ku, Tokyo, Japan). The blood samples were centrifuged at 188 × g for 10 min at 4°C, and the supernatant was stored at a temperature below −30°C until analysis. The concentration of serum NEFA was measured using a biochemical autoanalyzer JCA-BM6010 (JEOL Ltd., Tokyo, Japan). The concentrations of other serum parameters (triacylglycerol, total cholesterol, high density lipoprotein cholesterol, glucose, total protein, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transpeptidase) were measured using a biochemical autoanalyzer AY5400 (Beckman Coulter Inc., CA, USA). BODY.STATISTICAL ANALYSIS: All in vitro experiments were performed three times and data were expressed as means with standard deviation (SD). The Tukey-Kramer post-hoc test was used for multiple comparisons between strains. In the human study, the differences between the pre- and post-FM intake periods, and the differences between the control and active FM groups, were evaluated using Student’s paired t-test and Student’s unpaired t-test, respectively. A P value < 0.05 was considered statistically significant. BODY.RESULTS: BODY.EFFECTS OF LG2055 ON PANCREATIC LIPASE ACTIVITY :in vitro We first examined the effect of LG2055 on pancreatic lipase activity using a fat emulsion as a substrate in vitro (Figure 1). The lipase inhibitor orlistat [13] strongly inhibited lipase activity in a dose-dependent manner (0.001–1 μg/ml); LG2055 also suppressed lipase activity in a dose-dependent manner (1–100 μg/ml) (Figure 1A). All the examined lactic acid bacterial species and strains significantly suppressed lipase activity at a final concentration of 100 μg/ml compared with the control lacking bacteria. Furthermore, LG2055 strongly suppressed lipase activity compared with the other four strains (LG1131T, LG0317, LB1002T, and ST19258T) (Figure 1B).Figure 1 Suppressive effect of lactic acid bacteria on pancreatic lipase-mediated hydrolysis of triolein in an emulsion. Substrate suspensions (fat emulsion) were incubated with pancreatic lipase (200 U/ml) and lactic acid bacterial cells or orlistat for 30 min at 37°C. After boiling for 2 min, released fatty acids were quantified. The suppressive activity was calculated for fatty acid production, in which the activity in the absence of sample was represented as 100%. (A) Lactobacillus gasseri SBT2055 (LG2055) suppressed the release of fatty acids from fat emulsion in a dose-dependent manner. (B) Comparison of the capacity of various lactic acid bacteria in suppressing fatty acid release. Each of the five bacterial strains (LG2055, Lactobacillus gasseri SBT0317 (LG0317), Lactobacillus gasseri JCM1131T (LG1131T), Lactobacillus delbrueckii subsp. bulgaricus JCM1002T (LB1002T), and Streptococcus thermophilus ATCC19258T (ST19258T)) was added to the lipase reaction solution at 100 μg/ml. Values are means with standard deviations for triplicate experiments. Statistical differences between the strains were analyzed using the Tukey-Kramer post-hoc test and significant differences (P < 0.05) are indicated using different letters. When lipase activity was measured using 4-MUO as a substrate (Figure 2), orlistat was strongly inhibitory, whereas LG2055 had no effect at concentrations ranging from 1–100 μg/ml. Thus, LG2055 did not suppress lipase activity using 4-MUO as a substrate. The other four strains (LG1131T, LG0317, LB1002T, and ST19258T) did not also suppress the lipase activity using 4-MUO (data not shown).Figure 2 Effect of Lactobacillus gasseri SBT2055 (LG2055) on pancreatic lipase activity using 4-methylumbelliferyl oleate (4-MUO) as substrate. Various concentrations of LG2055 and orlistat, and 0.1 mM 4-MUO solution were mixed in the well of a microtiter plate, and the lipase solution (50 U/ml) was added. After incubation at 25°C for 30 min, 0.1 M sodium citrate (pH 4.2) was added to terminate the reaction. The suppressive activity was calculated based on the amount of 4-methylumbelliferone, in which the activity in the absence of sample was represented as 100%. Values are means with standard deviations for triplicate experiments. BODY.EFFECTS OF LG2055 ON FAT EMULSION DROPLET SIZE :in vitro The average fat emulsion droplet size remained mostly constant following incubation for 3 h in the absence of LG2055 (shortly after preparation: 2.00 ± 0.41 μm, after incubation: 2.21 ± 0.87 μm), indicating that the fat emulsion preparation was stable for a minimum of 3 h. The catechin mixture (1000 μg/ml) increased fat emulsion droplet size, as previously reported [20]. LG2055 significantly modified droplet size distribution at the same concentration as the catechin mixture (Figure 3A). Enlargement of the fat emulsion by LG2055 was also observed in phase-contrast micrographs (Figure 3B).Figure 3 Effect of Lactobacillus gasseri SBT2055 (LG2055) on fat emulsion droplet size in vitro. LG2055 or a catechin mixture was added to the fat emulsion preparation. The suspension was incubated at 37°C with constant shaking for 3 h and the size distribution of fat emulsion was measured using a particle size analyzer. No sample (only TES buffer) was added to the negative control and the catechin mixture was used as a positive control. (A) Changes in emulsion droplet size distribution after incubation with 1000 μg/ml of LG2055 or catechin mixture. The distributions are an average of three determinations. (B) Phase-contrast micrographs (scale bar: 50 μm). Black and white arrows indicate the representative fat emulsions and LG2055 cells, respectively. LG2055 dose-dependently increased the average fat emulsion droplet size at concentrations ranging from 1–100 μg/ml (Figure 4A). All five strains of lactic acid bacteria (100 μg/ml) significantly increased the average fat emulsion droplet size compared with the control lacking bacteria. In addition, LG2055 significantly increased droplet size compared with the other strains (Figure 4B).Figure 4 Increased effects of Lactobacillus gasseri SBT2055 (LG2055) on fat emulsion droplet size. (A) The dose-dependency of LG2055 on the average fat emulsion droplet size. (B) Comparison among lactic acid bacteria at 100 μg/ml. Fat emulsion droplet size was measured in the same manner as described in Figure 3. Values are means with standard deviations for triplicate experiments. Statistical differences between the strains were analyzed using the Tukey-Kramer post-hoc test and significant differences (P < 0.05) are indicated using different letters. BODY.CLINICAL STUDY OF FECAL FAT EXCRETION: The physical and biochemical blood characteristics of the subjects were not significantly different between groups (Table 1). No subject dropped out during the study.Table 1 Baseline characteristics of the study subjects Parameters Control Active Number of subjects 15 15 Gender   Male 6 6   Female 9 9 Age (years) 40.1 ± 9.5 42.6 ± 10.8 Height (cm) 162.2 ± 6.4 163.6 ± 9.4 Body weight (kg) 57.3 ± 6.4 58.8 ± 9.6 Body fat percentage (%) 24.9 ± 6.0 25.4 ± 6.4 BMI (kg/m2) 21.8 ± 2.5 21.9 ± 2.8 Systolic blood pressure (mm Hg) 116.6 ± 13.4 120.9 ± 15.5 Diastolic blood pressure (mm Hg) 69.0 ± 9.4 77.0 ± 13.0 Pulse rate (beats/min) 67.2 ± 10.3 69.1 ± 7.3 Triacylglycerol (mg/dl) 65.1 ± 22.0 68.0 ± 30.1 Total cholesterol (mg/dl) 197.1 ± 24.7 203.7 ± 31.0 Non-esterified fatty acid (μEq/l) 398.5 ± 169.5 393.3 ± 173.7 Glucose (mg/dl) 82.4 ± 6.6 86.3 ± 5.0 Values are means ± SD. The effects of the control and active FM preparations on physical and biochemical blood parameters are listed in Table 2. Body weight, body mass index (BMI), and total cholesterol levels significantly decreased similarly in both groups; alanine aminotransferase (ALT) and gamma-glutamyl transpeptidase (γ-GTP) significantly decreased in the control FM group, and pulse rate significantly decreased in the active FM group but remained within the normal range. Based on daily records and physician interviews, no irregularities in daily life or adverse events related to FM consumption were observed during the study period, (data not shown).Table 2 Initial (day 1) and final (day 15) values of physical characteristics and biochemical blood parameters (n = 15) Parameters Control Active day 1 day 15 day 1 day 15 Body weight (kg) 57.4 ± 6.5 55.8 ± 6.0** 58.4 ± 9.4 57.2 ± 9.2** Body fat percentage (%) 24.2 ± 6.4 24.2 ± 5.6 25.1 ± 6.6 25.1 ± 6.9 BMI (kg/m2) 21.9 ± 2.6 21.3 ± 2.4** 21.8 ± 2.8 21.4 ± 2.9** Systolic blood pressure (mm Hg) 117.3 ± 15.5 117.9 ± 13.6 115.9 ± 14.4 118.6 ± 15.2 Diastolic blood pressure (mm Hg) 68.7 ± 10.1 69.1 ± 10.4 71.9 ± 11.2 73.0 ± 11.6 Pulse rate (beats/min) 67.0 ± 5.9 65.9 ± 10.6 71.9 ± 9.0 67.0 ± 8.3* Triacylglycerol (mg/dl) 67.5 ± 33.5 59.5 ± 21.3 67.2 ± 37.2 66.5 ± 32.3 Total cholesterol (mg/dl) 188.5 ± 17.9 176.1 ± 27.4* 192.7 ± 35.4 179.7 ± 28.0* HDL cholesterol (mg/dl) 68.1 ± 10.3 68.7 ± 11.1 66.5 ± 11.8 65.6 ± 11.5 Non-esterified fatty acid (μEq/l) 331.8 ± 105.5 372.1 ± 135.1 413.1 ± 223.8 462.9 ± 169.2 Glucose (mg/dl) 85.7 ± 10.0 82.5 ± 5.8 85.3 ± 6.0 84.0 ± 6.2 Total protein (g/dl) 7.4 ± 0.3 7.4 ± 0.5 7.5 ± 0.4 7.5 ± 0.4 AST (U/l) 19.8 ± 4.6 19.1 ± 4.1 19.4 ± 4.5 19.1 ± 4.6 ALT (U/l) 20.3 ± 11.6 18.2 ± 9.4* 15.7 ± 5.6 14.3 ± 5.0 ALP (U/l) 185.4 ± 35.1 176.2 ± 35.7 185.1 ± 38.6 188.3 ± 30.4 γ-GTP (U/l) 23.2 ± 14.1 18.6 ± 9.1* 21.3 ± 10.5 18.5 ± 8.8 Abbreviations: HDL high density lipoprotein, AST aspartate aminotransferase, ALT alanine aminotransferase, ALP alkaline phosphatase, γ-GTP gamma-glutamyl transpeptidase. Values are means ± SD. Significantly different from the initial values within the group; *P < 0.05, **P < 0.01. There were no statistically significant differences in wet weight of feces between groups (270.1 ± 159.0 g in control FM group vs. 336.8 ± 116.9 g in active FM group before FM intake; 264.9 ± 151.0 g in control FM group vs. 304.4 ± 128.7 g in active FM group after FM intake). After FM intake, fecal fat levels were significantly increased compared with those observed before FM intake in the active FM group, whereas no increase was observed in the control FM group (Figure 5A). The amount of change between pre- and post-FM intake periods increased in the active FM group compared with the control FM group, although the difference was not statistically significant (P = 0.086).Figure 5 Effect of intake of FM containing Lactobacillus gasseri SBT2055 (LG2055) on fecal fat excretion in humans. (A) Fecal samples were collected during the final three days of the pre-observational period (before) and FM intake period (after), and fecal fat levels were determined. (B) The amount of change in the fecal fat levels in the control and active FM groups. Values were calculated by subtracting fecal fat levels at pre-observational period from those at FM intake period. Values are means with standard deviations. Statistical analysis was performed using paired Student’s t-test (A) and unpaired Student’s t-test (B). An asterisk indicates a significant difference (P < 0.05). BODY.DISCUSSION: The suppression of pancreatic lipase-mediated fat hydrolysis is an effective strategy for prevention of obesity and hyperlipidemia. Pancreatic lipase inhibitors such as orlistat are currently used clinically as pharmaceutical agents [22]. In addition, it has been reported that dietary compounds, including chitosan [17], saponin [23], and oolong tea polyphenols [19], inhibit pancreatic lipase, consequently suppressing dietary lipid absorption. In this study, we observed that LG2055, a probiotic bacterium displaying anti-obesity properties, suppressed fatty acid release from fat emulsion in vitro (Figure 1A). Moreover, it was reported that LG2055 suppresses lipid absorption in the small intestine in vivo [4]. Taken together, these findings suggest that suppression of pancreatic lipase-mediated fat hydrolysis could represent a mechanism by which LG2055 mediates suppression of lipid absorption. Consistent with our findings, Matsumura [24] and Zhou et al. [25] reported that certain strains of Lactobacillus inhibit pancreatic lipase. The present study further demonstrated that all five examined lactic acid bacterial strains, including LG2055, had an ability to suppress lipase activity (fatty acid release from fat emulsion) to a certain extent (Figure 1B); however, LG2055 significantly suppressed fatty acid release from fat emulsion compared with the other strains, indicating a more potent effect. These results suggest that many strains of lactic acid bacteria likely have the ability to suppress lipase activity. However, LG2055 could represent a specific strain with an increased potential for lipase suppression among lactic acid bacteria. The suppression of fat hydrolysis is mainly classified into two types: the first is suppression mediated by direct interaction between the inhibitor and enzyme, whereas the second is associated with modification of fat emulsion properties [26,27]. If LG2055 directly inhibits lipase-mediated hydrolysis, its suppressive effect should be observed when using a synthetic substrate such as 4-MUO. We observed that orlistat inhibited lipase activity using 4-MUO as a substrate, consistent with results using fat emulsion. Conversely, LG2055 was not inhibitory (Figure 2), indicating that LG2055 does not directly inhibit pancreatic lipase. We also confirmed that all four tested strains (LG1131T, LG0317, LB1002T, and ST19258T) did not inhibit lipase activity using 4-MUO as a substrate (data not shown). These results imply that LG2055 acts on the fat emulsion of a substrate rather than directly on the enzyme, leading to suppression of lipase-mediated hydrolysis. Fat emulsion interface properties, namely droplet size and specific surface area, control lipase-mediated fat emulsion hydrolysis [14,28,29]. Therefore, we examined whether LG2055 modifies fat emulsion properties by measuring fat emulsion droplet size upon mixing with LG2055. LG2055 promoted fat emulsion enlargement (Figure 3B) and dose-dependently increased fat emulsion droplet size (Figure 4A). An increase in droplet size is known to be associated with a decrease in specific surface area, resulting in delayed lipase-mediated hydrolysis. It has been reported that green tea extract [15], catechin mixture [20], and green coffee bean extract [20] increase fat emulsion droplet size, resulting in suppression of lipid absorption. However, there are no reports evaluating the effect of lactic acid bacteria on fat emulsion droplet size. Matsumura [24] and Zhou et al. [25] demonstrated that certain Lactobacillus strains inhibited pancreatic lipase activity but did not examine the mechanisms involved. To our knowledge, this is the first report describing an enlargement of fat emulsion by probiotics. With the exception of LG2055, the four tested strains also promoted an increase in fat emulsion droplet size (Figure 4B). However, LG2055 significantly increased droplet size compared with the other strains. These results are consistent with the strong suppressive effects of LG2055 on lipase-mediated fat hydrolysis. It remains unclear how LG2055 increases fat emulsion droplet size. One possibility is it that an interaction between LG2055 and bile acids (e.g. taurocholic acid) could contribute to the modification of fat emulsion properties. Interestingly, it was reported that certain Lactobacillus strains have an ability to bind and deconjugate taurocholic acid [30]. LG2055 also has an ability to deconjugate taurocholic acid [16]. Bile acids have amphipathic properties and are critical for the emulsification of dietary lipids in the intestine [31]. As our preliminary study demonstrated, LG2055 did not increase fat emulsion droplet size without inclusion of taurocholic acid prepared by the method reported by Shishikura et al. [15] (data not shown). LG2055 potentially interacts with bile acids and destabilizes the fat emulsion, resulting in its coalescence. Usman et al. previously reported that LG0317, a strain included in the present study, had a decreased capacity to deconjugate taurocholic acid compared with LG2055 [16]. In our study, LG0317 had a decreased effect on fat emulsion enlargement. These findings indicate the importance of the interaction between taurocholic acid and lactic acid bacteria for promotion of increased fat emulsion droplet size. We recently reported that consumption of FM containing LG2055 decreased postprandial serum triacylglycerol and non-esterified fatty acid (NEFA) concentrations in peripheral blood after intake of oral fat-loading test meals compared with consumption of FM without LG2055 [10]. Although triacylglycerol and NEFA concentrations in peripheral blood are important biomarkers of fat absorption, they are affected by metabolism in organs such as the liver [32]. Thus, we evaluated fecal lipid excretion to assess more directly the effect of LG2055 on lipid absorption in humans. Our data show that consumption of FM containing LG2055 increased fecal fat excretion (Figure 5A/B). Given that LG2055 suppressed pancreatic lipase-mediated fat hydrolysis in vitro, its effects on fecal fat excretion are likely associated with suppression of lipase-mediated fat hydrolysis, resulting in decreased lipid absorption. In this study, we observed that BMI and total serum cholesterol concentration were significantly decreased, but remained within the normal range, after FM intake in both the control and active FM groups (Table 2). We consider that this is because energy intake by the subjects was moderately controlled, or beneficial effects of fermented milk on lipid metabolism [33] were taken. Consumption of LG2055 for seven days did not significantly influence body fat percentage. However, constant ingestion of LG2055 over a prolonged period could reveal more pronounced anti-obesity effects, as we previously demonstrated when LG2055 was consumed daily for 12 weeks [9]. BODY.CONCLUSIONS: In conclusion, our findings clearly demonstrate that LG2055 modifies the physicochemical properties of fat emulsion by increasing droplet size and suppressing fatty acid release from fat emulsion. These data provide a mechanism by which LG2055 mediates the suppression of lipid absorption and an increase in fecal fat excretion in humans.

TITLE: The effects of listening to preferred music on pain intensity after open heart surgery ABSTRACT.BACKGROUND:: Pain is a common phenomenon after surgery. Cardiac surgeries are no exception and patients generally experience acute pain after these surgeries. Inadequate pain management after cardiac surgery predisposes patients to many complications. Therefore, the aim of this study was to determine the effects of listening to preferred music on pain intensity after open heart surgery. ABSTRACT.MATERIALS AND METHODS:: This study was a randomized clinical trial (RCT) conducted in open heart intensive care unit (ICU) of a university hospital in Sari, Iran. A total of 60 patients who were scheduled to undergo open heart surgery were randomly allocated in two groups. Patients in the intervention group (n = 30) listened to their preferred music by headphones for 30 minutes, whereas those in the control group (n = 30) did not listen to music. Using a Numerical Rating Scale (NRS), pain intensity was measured among the patients before the intervention, and immediately, 30 minutes and one hour after the intervention. Data was analyzed by Chi-square test, student's t-test and repeated measures analysis of variance (ANOVA). ABSTRACT.FINDINGS:: Mean pain intensity in the intervention group before, immediately after, 30 minutes and one hour after the intervention were 5.8, 3.1, 2.5 and 2.4, respectively. Corresponding numbers in the control group were 4.7, 4.7, 4.8 and 4.9, respectively. Repeated measures ANOVA showed music to significantly reduce pain intensity (p = 0.0001) ABSTRACT.CONCLUSIONS:: Music can be effective as a non-pharmacological, inexpensive, non-invasive and side effect free method for pain management after open heart surgery. BODY.I:NTRODUCTION Today, cardiac diseases are the most common causes of mortality worldwide. They would be the main cause of death worldwide in 2020, according to a prediction made by the World Health Organization (WHO).[1] In the Middle East countries including Iran, cardiac diseases are turning into major health and social problems.[2] Despite technological developments in the treatment of cardiovascular diseases, such as thrombolytic therapy, angioplasty and atherectomy, the only alternative in many patients is still cardiac surgery.[3] While approximately 686000 open heart surgeries are performed in the USA annually,[4] the number decreases to more than 40000 in Iran.[5] Despite the frequency of cardiac surgeries and increased knowledge regarding pain and pain management, patients experience considerable pain during the postoperative period[6] and pain management remains inadequate after the cardiac surgery.[7] Gelinas showed that more than 75% of cardiac surgery ICU patients mentioned an experience of moderate to severe pain in the ICU.[8] Therefore, pain is a common complaint of patients expressed to nurses,[9] and as a result, it is considered as a nursing priority.[10] Inadequate postoperative pain management leads to inability in coughing and effective movement which in turn predisposes patients to complications such as pneumonia, atelectasis, and deep veins thrombosis (DVT).[11] Schwann et al. showed that a considerable number (13%) of cardiac surgery patients develop DVT despite maximal thromboprophylaxis.[12] In addition, postoperative pain can keep patients from participating in expected activities such as deep breathing and getting out of bed which improve and reinforce the patient's better feeling and wellness.[13] Moreover, postoperative pain has a negative psychological impact on patients and delays the postoperative recovery.[11] There are several options for postoperative pain control including systemic medications (narcotics and non-narcotics), regional anesthesia (epidural) and non-pharmacological interventions. Systemic medications can lead to complications such as insufficient treatment, respiratory depression and excessive sedation. Epidural anesthesia may also result in hypotension, itching, nausea, vomiting and urinary retention.[14] Furthermore, medications cannot be used in all patients and impose expenses to the health care system.[15] Therefore, the recent tendency towards non-pharmacological strategies for relieving pain is growing fast.[16] In addition, a balanced combination of pharmacological and non-pharmacological methods provide up to 23% better pain management.[17] One of these methods, named as music therapy, involves listening to pleasant stimulating sounds.[16] Music is a source of pleasure for many people which has been used since ancient years to influence human health and alleviate sickness and suffering.[6] The application of music therapy to reduce discomfort has a long history. Egyptians used music therapy to cure infertility in women and Iranians used the sound of “ood” (a traditional Iranian musical instrument) to treat many diseases.[18] In nursing practice, music is considered as an effective and noninvasive intervention in relieving pain, stress and anxiety. It is also believed to increase comfort and immunity and to improve vital signs.[19] There are limited numbers of published studies on the effect of music on pain after open heart surgery.[7] In addition, music has a cultural implication[17] and insufficient studies have been conducted in many countries such as Iran. Meanwhile, the success of music therapy may be greatly enhanced by determining patient's preference, familiarity and cultural context.[20] While some studies have demonstrated pain to decrease after music,[421–26] others found music to be ineffective on pain.[27–33] Many previous investigations have been limited in a way. For instance, some employed a small sample size,[2434] some evaluated different types of surgeries and anesthesia in one study,[35] and some used a type of music not selected by patients.[19222430323637] Moreover, while a systematic review recommended a minimum duration of 30 minutes for music therapy to be effective in clinical practice,[38] a number of studies played music for less than 30 minutes.[73437] On the other hand, the constant presence of the researcher during the intervention[36] might have affected patient's response. However, no similar study has been conducted in Iran. Therefore, considering the above mentioned facts and according to the cultural, social and economic differences in Iran, we tried to perform a study without the aforesaid limitations to investigate the effects of preferred music on pain intensity among patients after open heart surgery in Mazandaran Heart Center, Sari, Iran. BODY.M:ATERIALS AND METHODS This study was a randomized clinical trial (RCT) performed on patients undergoing open heart surgery (coronary artery bypass graft (CABG) and valvular surgery) admitted in open heart ICU of Mazandaran Heart Center (a university affiliated teaching hospital in Sari, Iran) during May-August 2010. A sample size of 30 subjects per group was selected. Sample size calculations were based on mean and standard deviation (SD) of a similar study[26] and considering a 95% level of significance and a power of 80%. The inclusion criteria included willingness to participate in the study, non-emergency open heart surgery, first time open heart surgery, hemodynamic stability (systolic blood pressure higher than 90 mmHg, lack of life threatening dysrhythmia and a heart rate of 60-100 bpm).[3940] Patients were excluded if they were connected to a ventilator at the time of intervention, had a history of mental disorders, chronic pain, and hearing disorders, used narcotic drugs 4 hours before intervention at the ICU, had a history of playing music, needed intra-aortic balloon pump (IABP), or used other alternative methods for pain reduction such as massage therapy during the intervention. In addition, with any complications during the operation or anesthesia or willingness to quit the study, the patient was omitted from the study. After obtaining approval from the Ethics Committee of Mazandaran University of Medical Sciences, the researcher went to the heart surgery ward of the hospital. Then, patients who were supposed to undergo surgery on the same day and met the inclusion criteria were randomly selected. Afterwards, the study procedure was explained for the subjects and written informed consents were taken. The participants were assigned into two groups of intervention and control. Each group consisted of 30 patients. The subjects were instructed about as a result, it is considered as a nursing priority.[10] Inadequate postoperative pain management leads to inability in coughing and effective movement which in turn predisposes patients to complications such as pneumonia, atelectasis, and deep veins thrombosis (DVT).[11] Schwann et al. showed that a considerable number (13%) of cardiac surgery patients develop DVT despite maximal thromboprophylaxis.[12] In addition, postoperative pain can keep patients from participating in expected activities such as determining their pain intensity using a 0–10 point Numerical Rating Scale (NRS). In the intervention group, the patients were provided with a list of music pieces. After listening to approximately one minute of the pieces patients were interested in, using a portable computer, they were asked to choose their favorite piece. Demographic characteristics of the patients including age, gender, education, marital status, diabetes, pervious surgery, place of residence and profession were obtained by referring to medical records of the patients and also by asking the patients. As mentioned before, pain intensity was scored using a self-reporting 0-10 NRS in which 0 indicated lack of any pain and 10 represented most severe pain. This scale has extensively been used in the studies related to pain[792436] and its validity and reliability have been approved.[4142] In the intervention group, while the patients were staying at the ICU for the first 24 hours after the surgery, their pain intensity was assessed and recorded. Then, the music pieces the patients selected previously were played by an MP3 player with special headphones for 30 minutes. All relaxation music pieces were selected by a music expert considering the cultural conditions of the society and the type of recommended music in the literature, i.e. with a tempo of 60-80 beats (or even less) per minute.[2028] The musical tempo was evaluated by a Metro-Tuner (Musedo MT-30, China). Pain intensity was again evaluated immediately, 30 minutes and 1 hour after the music was completely played. However, in the control group, headphones were used without playing any music. This study was conducted between 3 to 6 P.M. of the day after surgery because the traffic of hospital staff was lower and patients’ routine care was completed and other people were less likely to be present and to interfere with the process. Evaluation of pain intensity was done by nurses with sufficient knowledge in this context who were blinded to the study groupings. The anesthesia was administered according to a single protocol recommended by the institution for all subjects. If, for some reason, the protocol could not be used for a subject, the patient was excluded from the study. In addition, other factors affecting pain intensity, such as incision method, type and extent of incision, having chest tube, and type of analgesic drugs used, were the same for all patients. Data was analyzed using the Statistical Package for Social Sciences (SPSS) and descriptive-inferential statistics. Descriptive statics, i.e. mean, SD, and proportions, were used to determine the demographic data of all study subjects. Chi-square test and student's t-test were used to respectively compare qualitative and quantitative data between the two groups. Repeated measurements were conducted to determine the impact of preferred music on pain intensity at different times of intervention. An α = 0.05 was the level of significance used for all analyses. BODY.F:INDINGS All randomized patients were included in the analysis and there were no drop outs. A total of 60 persons participated in the study, 56.6% of whom were female. While mean age of all patients was 57.83 (10.62) years, it was 57 (11.6) in the intervention group and 58.6 (9.6) in the control group. The majority of patients (98.3%) were married and the two groups did not significantly differ in this regard (p = 0.9). While 40% (n = 24) of the subjects were housewives, 1.7% (n = 1) were unemployed and there was no significant difference between two the groups (p = 0.3). Most subjects (91.7%) underwent a CABG (83.4% (n = 25) in the intervention group and 86.7% (n = 26) in the control group (p = 0.7)). A combination of an internal mammary artery (IMA) graft and a saphenous vein graft (SVG) was used for all patients undergoing CABG. Others demographic and clinical characteristics of the patients are shown in Table 1. As it is seen, there were no significant differences in baseline demographic and clinical characteristics of the participants among the two study groups. The mean and standard deviation of pain intensity in the intervention and control groups, before intervention, immediately, 30 minutes and 1 hour after completion of the intervention are shown in the Table 2. There was no significant difference regarding baseline pain intensity among the two groups (t = 1.746; p = 0.08; and df = 58). Repeated measures analysis of variance (ANOVA) indicated music to significantly reduce pain intensity (p < 0.0001; F = 16.31). Table 1Frequency distribution of the subjects in the intervention and control groups base on some demographic and clinical characteristics Table 2Mean and standard deviation of pain intensity among patients in the intervention and control groups before, and immediately, 30 minutes and one hour after the intervention BODY.D:ISCUSSION The present study examined the effects of listening to preferred music on pain intensity after open heart surgery As it is observed, the two groups (intervention and control) were similar at the baseline of the study in terms of variables studied, confirming the random nature of the groups. Based on the results of this study, music significantly decreased postoperative pain in patients after open heart surgery. Two studies that focused on patients’ pain experience in the ICU after cardiac surgery reported more than 70% of patients to recall pain after surgery and therefore emphasized on pain as a major problem in patients after cardiac surgery.[843] Like our study, both Sendelbach et al.[7] and Hatem et al.[22] showed that music reduced patients’ pain after cardiac surgery. Voss et al. studied patients during chair rest after open heart surgery and reported lower pain levels in the music intervention groups compared with the control groups.[4] Similarly, Tse et al. showed lower pain ratings and decreased pain over time in patients who listened to music immediately after nasal surgery.[44] Nilsson et al. stated patients exposed to soft relaxing music intraoperatively to have significantly lower pain scores compared with the control group patients on the first day after the surgery.[4546] In contrast, Allred et al. evaluated patients undergoing knee arthroplasty and did not find any significant pain reduction (p = 0.337) in the case group (music) as compared with the control group (resting on bed).[28] Likewise, the effect of music on pain reduction after caesarian section was not statistically significant in a study conducted in Iran.[32] Music ineffectiveness on pain reduction in these studies could be attributed to the type of the music listened by the patients, i.e. one type of Spanish guitar music was played for all participants without considering the local and national culture of the patients. However, it has been suggested that preferred music, as opposed to prescribed music, is a critical factor in the effectiveness of music therapy.[47] The results of Stratton and Zalanowski showed a significant correlation between the degree of relaxation and liking the music.[48] Since the emotional responses differ from culture to culture, a type of music related to the cultural features of the subjects should be selected.[49] Therefore, the success of music interventions may be greatly enhanced by determining preferences, familiarity and cultural contexts of the patients. Additionally, preferred music can reestablish a sense of control in an environment that may be unfamiliar to patients.[20] However, Cepeda et al. reported music not to reduce alfentanil requirement and pain intensity in patients undergoing extracorporeal shock wave lithotripsy (ESWL).[50] Similarly, another study conducted by Nilsson et al. on patients after cardiac surgery did not reveal any significant differences in pain between the group that listened to music and the control group.[36] The reason might again be using only one type of music for all the patients. In other words, greater effects could have been observed if patients had selected their favorite type of music. BODY.C:ONCLUSION The results of this study support the beneficial effects of preferred music on pain control after open heart surgery while the patients are in the ICU. Since listening to music is a simple, inexpensive, and side effect free intervention which could be simply provided by an MP3 player and headphones, we recommend extensive use of music therapy in patients undergoing open heart surgery for postoperative pain reduction.

TITLE: Sequential compression pump effect on hypotension due to spinal anesthesia for cesarean section: A double blind clinical trial ABSTRACT.BACKGROUND: Spinal anesthesia (SA) is a standard technique for cesarean section. Hypotension presents an incident of 80–85% after SA in pregnant women. ABSTRACT.OBJECTIVE: To determine the effect of intermittent pneumatic compression of lower limbs on declining spinal anesthesia induced hypotension during cesarean section. ABSTRACT.METHODS: This double-blind clinical prospective study was conducted on 76 non-laboring parturient patients, aged 18–45 years, with the American Society of Anesthesiologist physical status I or II who were scheduled for elective cesarean section at Razi Hospital, Ahvaz, Iran from December 21, 2015 to January 20, 2016. Patients were divided into treatment mechanical pump (Group M) or control group (Group C) with simple random sampling. Fetal presentation, birth weight, Apgar at 1 and 5 min, time taken for pre-hydration (min), pre-hydration to the administration of spinal anesthesia (min), initiation of spinal to the delivery (min) and total volume of intravenous fluids, total dose of ephedrine and metoclopramide were recorded. Data were analyzed by SPSS version 19, using repeated measures of ANOVA and Chi square test. ABSTRACT.RESULTS: Heart rate, MPA, DAP and SAP changes were significantly higher in off-pump group in the baseline and 1st-minute (p<0.05), and in the other times, this change was significantly different with control groups. ABSTRACT.CONCLUSION: This research showed the suitability of the use of Sequential Compression Device (SCD) in reducing hypotension after spinal anesthesia for cesarean section, also this method can cause reducing vasopressor dosage for increased blood pressure, but the approval of its effectiveness requires repetition of the study with a larger sample size. ABSTRACT.TRIAL REGISTRATION: The trial was registered at the Iranian Registry of Clinical Trials (http://www.irct.ir) with the IRCT ID: IRCT2015011217742N3. ABSTRACT.FUNDING: The authors received no financial support for the research, authorship, and/or publication of this article. BODY.1. INTRODUCTION: Spinal anesthesia (SA) is a standard technique for cesarean section (1, 2). Hypotension is defined by a systolic pressure drop by more than 20% of the baseline, or a pressure drop by more than 90 –100 mm/Hg. This complication presents an incident of 80–85% after SA in pregnant women (3). The hypotension is related to venous pooling secondary to progesterone-induced decrease in vascular tone, and is dangerous to mother, fetus, and newborn (4). The rigorousness of hypotension is dependent on the altitude of the block, the arrangement of the patient, the volume, and type of surgery. The procedures that decrease hypotension consist of volume loading, inotropic agents, prevention of aortocaval compression, and regular blood pressure monitoring. Early detection and management of hypotension can significantly reduce the risk to mother and fetus. Hypotension due to SA is caused by an increase in venous pooling as a consequence of parasympathetic over-activation, causing vasodilation in the lower part of the body. This situation is more severe in pregnant women, due to aortocaval compression. Under normal conditions, hypotension in pregnant women is compensated by increased cardiac output; however, after SA-induced sympathectomy, this compensation is attenuated or sometimes eliminated, and thus leads to a heart rate drop, followed by a cardiac output decline (6). Since uterine blood flow is pressure-dependent (due to the lack of a placental auto-regulation), prolonged maternal hypotension is harmful to the fetus (due to lower Apgar scores and fetal acidosis). It is also frequently associated with nausea and vomiting for the mother (7). As stated earlier, there are several methods for decreasing SA-induced hypotension, including the use of Sequential Compression Device (SCD). In this method, the pressure is intermittently put above the knee. According to previous studies, almost 125 cc of blood is moved in compression phase. Although there is no comparative study into the blood volume among pregnant and non-pregnant women, it is well known that there is a high blood volume in the lower extremities at term, which is further increased by SA through vasodilatation (8). Therefore, theoretically, the SCD method mobilizes more blood to the central [active] blood volume, and thus decreases hypotension in a more stable form; whereas, other methods are relatively short-term and temporary. Due to the limited amount of relevant research, this study intended to determine the effect of intermittent pneumatic compression of lower limbs on declining SA-induced hypotension during C-section. BODY.2. MATERIAL AND METHODS: BODY.2.1. TRIAL DESIGN AND PARTICIPANTS: This study was a randomized clinical trial that was conducted from December 21, 2015 to January 20, 2016 in a governmental educational hospital (Razi Hospital, Ahwaz, Iran). BODY.2.2. SELECTION CRITERIA: The inclusion criteria for participation in this study were: 1) non-laboring parturient, 2) anesthesiologist physical status I or II, 3) elective cesarean section, and 4) age between 18–45 years. The exclusion criteria included: 1) patients in whom spinal anesthesia was contraindicated, 2) emergency cesarean section, 3) multiple gestation, 4) pregnancy pathology, 5) heart diseases, 6) history of hypertension or pregnancy-induced hypertension, 7) body mass index (BMI) >40, and 8) gestational diabetes. BODY.2.3. INTERVENTIONS: This double-blind clinical prospective study was conducted on 76 non-laboring parturient patients. After approval of the ethics committee of Ahvaz Jundishapur University of Medical Sciences and obtaining written informed consent, patients were divided in to treatment mechanical pump (Group M) or control group (Group C) with simple random sampling. The following variables were noted: body mass index, gestational age and medical history. In the operating room, all patients were situated at 15 degrees left lateral position and routine monitoring was applied for continuous monitoring. Baseline systolic, diastolic and mean arterial pressures (SAP, DBP and MAP) and heart rate (HR) were recorded and then pre-hydration with 10ml/kg lactated ringer solution was administrated. Two groups had appropriate sized sleeves applied to the lower limbs. Discontinuous compression started with a pressure of 50 mmHg in M group patients. Spinal anesthesia was performed in the sitting position using a 25 G Sprotte needle in the L3–4 or L4–5 interspace. All patients received 10 mg of bupivacaine 0.5%. Subsequently, the patients were positioned supine with a 15-degree left lateral angle. Five minutes after SA, the upper sensory level of anesthesia was measured. The arterial blood pressure was measured and recorded each minute until delivery and thereafter, every 3 minutes until 10 minutes, and then every 5 minutes until arrival to recovery room, and finally, every 10 minutes until discharge from recovery room. Hypotension was defined either as a decrease in any MAP measurement to less than 20% of the base line, or SAP less than 90 mmHg. If Hypotension occurred, it was treated by an anesthesia nurse, who was blinded to the protocol, with 5mg increments of intravenous ephedrine boluses until corrected and intravenous fluid administration simultaneously. BODY.2.4. OUTCOMES: The primary outcome of the study was defined as the incidence of hypotension. Secondary outcomes that we compared, included serial changes in blood pressure, nausea or vomiting, HR, and Apgar scores at 1 and 5 min. Nausea and vomiting were treated with metoclopramide. Total dose of ephedrine and metoclopramide was recorded BODY.2.5. SAMPLE SIZE: The sample size was calculated based on the results of previous studies (1, 9) by assuming the test power of 80% and a confidence level of 95% and using the following formula: n = (Z1−α/2+Z1−β)2×((P1×(1−P1)+P2×(1−P2)) / (P1−P2)2; Where: n = Sample size, Z1−α/2 = 1.96 when α = 5% for two-tailed hypothesis, Z1−β = 0.842 when β = 20% (test power = 80%), P = Probability of the main outcome. BODY.2.6. RANDOMIZATION AND BLINDING: Patients were divided into treatment mechanical pump (Group M) or control group (Group C) with simple random sampling. Then, the participants in each of the groups were assigned randomly to receive either mechanical pump or control on a 1:1 ratio. Randomization was done by one of the researchers who did not have a role in the treatment of the participants. BODY.2.7. STATISTICAL METHODS: Statistical analyses were performed using SPSS version 19 (SPSS Inc., Chicago, Illinois, USA). The results are expressed as mean ± (SD), median (range) or number (%). p<0.05 was considered as significant. The comparison of normally distributed continuous variables between the groups was performed using students’ T test. Nominal categorical data between the groups were compared using the Chi square. Serial data were analyzed by repeated measures of ANOVA. The incidence of hypotension was compared using the Chi square test. BODY.2.8. RESEARCH ETHICS: This research was approved by the Pain Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (Proposal No. PAIN-9312). In this study, for ethical considerations, the participants were informed about the objective and nature of the study, and each participant provided her written consent in her native language (Persian) prior to the study. Also, we were committed to keeping all of the participants’ information confidential. BODY.3. RESULTS: Seventy-six patients were randomly assigned between two groups M and C. Three patients in group M were excluded (one patient because of inadequate spinal block and two patients because severe shivering prevented accurate measurement of blood pressure). Patient characteristics and surgical times were similar between two groups (Table 1). There is no difference in hypotension between the two groups (p=0.17). The MAP changes were significantly higher in the C group only at the baseline (p<0.05). In addition, the lowest MAP belonged to the C group in the 35th-minute, and the highest MAP belonged at the baseline in this group. These changes were not statistically significant (Figure 1). The SAP changes were significantly higher in the C group only at the baseline, but at other times, with no significant difference between two groups. In addition, the lowest SAP belonged to the C group in the 3rd- and 15th-minute, and the highest SAP belonged to the C group at the baseline. These changes were not significant. DAP changes were significantly higher in the C group in the baseline and 1st-minute (p<0.05). These changes were significantly higher in the M group in the 5th-minute. In addition, the lowest DAP belonged to the C group in the 5th- and 35th-minute, and the highest DAP belonged to the C group at the baseline. These changes were not statistically significant (Figure 1, Table 2). Heart rate changes were significantly higher in the M group in the first and third minutes (p<0.05). In addition, the lowest heart rate belonged to the C group in the third-minute, and the highest heart rate belonged to the M group in the 15th-minute. These changes were not statistically significant (Figure 2). The occurrence of nausea (24 patients in the M group versus 48 in the C group, p=0.005) and vomiting (4 patients in the M group versus 68 in the C group, p=0.001) was significantly lower in the M group. The total administered dosage of ephedrine was significantly lower in the M group (median 4.1mg vs. 17.1mg, p=0.001). There was no difference in neonatal Apgar scores at 1 and 5 minutes between groups. BODY.4. DISCUSSION: According to the findings section, hypotension in the pneumatic compression group or M group is not significantly different from the control group, but the administrated dosage of ephedrine was significantly lower in the pneumatic compression group. In addition, the incidence of nausea and vomiting was considerably lower in the pneumatic group and literature review produced almost similar results. For example, Sujata randomly divided 100 pregnant women, undergoing elective C-section under spinal anesthesia, in pneumatic compression and control groups. The protocols of standard pre-surgery fluid therapy and spinal anesthesia were similar in both groups. Hypotension was defined by a systolic pressure drop by more than 20% of the baseline and was cured with 6 mg intravenous bolus ephedrine. The incidence of hypotension was considered as the primary outcome and the median dosage of the administered ephedrine was determined. The hypotension rates were 25.5% and 60% in the treatment and control groups, respectively (p=0.001). This was inconsistent with the findings of our study; however, the median ephedrine dosages in their study were 12 [0–24] mg in the treatment group and 0 [0–12] mg in the control group (p<0.001). On the other hand, there was no difference between groups in the time to the onset of hypotension (9), which was almost consistent with our findings. In addition, Adsumelli randomly divided 50 pregnant women, undergoing C-section into two groups of SCD and control, each with 25 subjects. Similarly, a standard fluid therapy method and spinal anesthesia was applied to all patients. Hypotension was defined by a systolic pressure drop by more than 20% of the baseline. The baseline indices of systolic pressure, average blood pressure, diastolic blood pressure, pulse pressure, and heart rate were recorded after spinal block, every minute until the delivery of newborn. Median hypotension drop was observed in 52% of the SCD and 92% of the control groups (p=0.004). There was no significant difference between the two groups in terms of systolic pressure, diastolic pressure, heart rate, and pulse pressure (1). This finding agrees with ours, as the difference was significant only in a few minutes. Bishnu et al. divided 100 pregnant women, undergoing C-section under spinal anesthesia, into SCD and control groups. They then compared the two groups in terms of the rate and duration of hypotension drop, mean dosage of required ephedrine, average amount of blood loss, and sensory block level. In 24% of SCD and 54% of control groups, hypotension drop was significant (p=0.002). The mean dosages of administered ephedrine were 10 mg and 3.15 mg in SCD and control groups, respectively (p=0.008). There was no difference between groups in the time to the onset of hypotension drop. The average blood loss and sensory block level were comparable between the two groups (5). BODY.5. CONCLUSIONS: This research showed the suitability of the use of Sequential Compression Device (SCD) in reducing hypotension after spinal anesthesia for cesarean section, also this method can cause reducing vasopressor dosage for increased blood pressure, but the approval of its effectiveness requires repetition of the study with a larger sample size.

TITLE: Communication and Low Mood (CALM): a randomized controlled trial of behavioural therapy for stroke patients with aphasia ABSTRACT.OBJECTIVE:: The aim was to evaluate behavioural therapy as a treatment for low mood in people with aphasia. ABSTRACT.DESIGN:: A randomized controlled trial comparing behavioural therapy plus usual care with a usual care control. Potential participants with aphasia after stroke were screened for the presence of low mood. Those who met the criteria and gave consent were randomly allocated. ABSTRACT.SETTING:: Participants were recruited from hospital wards, community rehabilitation, speech and language therapy services and stroke groups. ABSTRACT.SUBJECTS:: Of 511 people with aphasia identified, 105 had low mood and were recruited. ABSTRACT.INTERVENTIONS:: Behavioural therapy was offered for up to three months. Outcomes were assessed three and six months after random allocation. ABSTRACT.MAIN MEASURES:: Stroke Aphasic Depression Questionnaire, Visual Analog Mood Scales ‘sad’ item, and Visual Analogue Self-Esteem Scale. ABSTRACT.RESULTS:: Participants were aged 29 to 94 years (mean 67.0, SD 13.5) and 66 (63%) were men. Regression analysis showed that at three months, when baseline values and communication impairment were controlled for, group allocation was a significant predictor of the Stroke Aphasic Depression Questionnaire (P < 0.05), visual analogue ‘sad’ (P = 0.03), and Visual Analogue Self-Esteem Scale (P < 0.01). At six months, group alone was a significant predictor of the Stroke Aphasic Depression Questionnaire (P < 0.05), and remained significant when baseline values were controlled for (P = 0.02). Mean Stroke Aphasic Depression Questionnaire 10-item hospital version scores decreased from baseline to six months by six points in the intervention group as compared with an increase of 1.9 points in the control group. ABSTRACT.CONCLUSIONS:: Behavioural therapy seemed to improve the mood of people with aphasia. BODY.INTRODUCTION: Mood disorders are common after stroke (see ref. 1, pp.283–284). They include depression and anxiety, but also general psychological distress, which is not so severe that it leads to a clinical diagnosis but nevertheless has a negative effect on recovery and quality of life. Depression is well recognized following stroke, with an average prevalence of 33%,2 and is associated with worse rehabilitation outcomes, increased carer strain, and higher mortality (see ref. 1, p.284). Anxiety disorders also occur in about a third of those with stroke, and post-traumatic stress disorder in between 5% and 30% (see ref. 1, pp.290–291). Patients with aphasia are highly susceptible to depression,3 and the severity of communication difficulties may be associated with emotional distress.4 Antidepressants may reduce depression, but also increase adverse events,5 and are not appropriate for all patients. Some studies have evaluated psychological interventions after stroke, but a Cochrane review reported no evidence of benefit.5 One trial found that a brief psychosocial–behavioural intervention plus an antidepressant reduced depression in patients recruited within four months following stroke.6,7 Motivational interviewing early after stroke was found to have a beneficial effect on mood at 12 months.8,9 However, both studies excluded patients with severe communication difficulties.6,8,9 In a trial of cognitive behavioural therapy,10 secondary analysis found that the severity of communication difficulties was associated with less improvement in mood,11 although only patients with mild communication problems were included. Most studies of psychological interventions are ‘talk based’ and are not accessible for patients with aphasia. Behavioural therapy is an approach which does not require intact communication and can be adapted for people with aphasia.12 Behavioural approaches are based on the behavioural model of depression, in which depression is considered to arise from a lack of positive response contingent social reinforcement from the environment. The aim of therapy is to increase the level of activity, particularly the frequency of pleasant events, in order to improve mood. Behavioural approaches are effective at treating depression in older adults13 and in people with dementia,14 and require evaluation in people with aphasia. The aim of this study was to evaluate behavioural therapy for low mood in people with aphasia following stroke. BODY.METHODS: The Communication and Low Mood (CALM) study was a multicentre randomized controlled trial comparing behavioural therapy with usual care (ISRCTN56078830). Ethical approval was obtained from Nottingham Research Ethics Committee 1. Stroke patients with aphasia were identified on hospital wards, and asked if they were willing to be contacted after discharge from hospital. In addition, referrals were sought from community stroke and rehabilitation services and speech and language therapists. People attending stroke and communication groups in the community were also invited to take part. Potential participants were identified in six centres (Nottingham, Mansfield, Chesterfield, Sheffield, Lincoln and Leicester), between 28 April 2008 and 12 January 2011. Aphasia was confirmed by a speech and language therapist for potential participants recruited through hospital and community services. For potential participants recruited through the voluntary sector, the presence of aphasia was confirmed using the Sheffield Screening Test for Acquired Language Disorders.15 People were excluded if they were blind or deaf, had dementia documented in their medical notes, were unable to speak English prior to stroke, or were receiving any treatment for depression at the time of their stroke. Potential participants were given an information sheet and invited to consent to have their mood screened. Mood was assessed using the ‘sad’ item of the Visual Analog Mood Scales16 and the Stroke Aphasic Depression Questionnaire 10-item hospital version,17 completed by a nurse, relative or carer. Those who were identified as having low mood on either the visual analogue ‘sad’ item (cut-off >50) or the Stroke Aphasic Depression Questionnaire (cut-off >6)18 were then invited to consent to take part in the randomized trial. Informed consent from patients or assent from a relative or carer was obtained. Participants completed further baseline measures, which included measures of language impairment (Sheffield Screening Test), the Frenchay Aphasia Screening Test reading and writing subtests,19 Barthel Index,20 and a picture-based measure of self-esteem (Visual Analogue Self-Esteem Scale).21 Demographic and stroke details were also recorded. Participants were randomly allocated to one of two groups: behavioural therapy or usual care, using a computer-generated pseudo-random list (1:1 ratio) with permuted blocks of varying sizes, generated by a clinical trials unit with no other involvement in the trial. Randomization was stratified according to recruitment centre and whether the patient was recruited in hospital or in the community. The assistant psychologist providing treatment accessed the allocation by logging into a secure computer server, thus ensuring concealment of allocation. Patients allocated to either group received all other services that were available to them as local practice. After randomization, participants allocated to receive behavioural therapy received up to 20 sessions of treatment over three months, with each session lasting approximately 1 hour. Sessions took place at the participant’s place of residence. Therapy was delivered by an assistant psychologist supervised weekly by a clinical psychologist. There was an assistant psychologist based in each of four centres. The additional two centres (Mansfield and Lincoln) were covered by assistants from Chesterfield and Nottingham. In addition, all assistant psychologists attended a joint monthly supervision meeting with a consultant clinical neuropsychologist (JM). The assistant psychologists received training in supported communication from speech and language therapists and were provided with a therapy manual. The manual had been developed from studies of cognitive behavioural therapy for depression after stroke10 and with older adults,22 and guidelines on conducting cognitive behavioural therapy with people with aphasia.12 The intensity of therapy was left to the discretion of the assistant psychologist. Treatment strategies focused on maximizing mood-elevating activities and included education, activity monitoring, activity scheduling, and graded task assignments. The intervention was tailored to the individual’s needs, and communication resources, such as pictures, photographs and letter charts, were used. The delivery of therapy was monitored by observation of therapy sessions by the chief investigator (ST). The content of therapy was documented using record forms completed by the assistant psychologist after each session. The primary outcome measure was the Stroke Aphasic Depression Questionnaire 21-item hospital version17 at six months after randomization. This scale is an observational measure of mood, which was completed by a relative or carer. This was also completed at three months after randomization. Secondary outcomes were completed by an independent assessor, blind to the participant’s group allocation, at three and six months after randomization. These included the visual analogue ‘sad’ item, Visual Analogue Self-Esteem Scale, and Nottingham Leisure Questionnaire23 at three and six months after randomization. In addition, the Carer Strain Index24 and patient and carer versions of a 100 mm visual analogue Satisfaction with Care Rating scale (patient and carer versions, adapted from Lincoln et al.25) were also completed at six months. The secondary outcome measures were included to assess whether the intervention also improved self-reported mood, leisure activities and carer strain, and these measures are suitable for stroke patients with aphasia. The independent assessor recorded whether the patient was taking antidepressant medication at each outcome assessment. The sample size estimate was based on the primary outcome measure, the Stroke Aphasic Depression Questionnaire 21-item hospital version, at six-month follow-up. A sample size of 76 in each group was estimated to give 90% power (5% two-sided significance) to detect a difference in mean score of five points on the primary outcome measure, assuming a common standard deviation of 9.42 based on a pilot study (unpublished data) with 22 patients. Allowing for 15% attrition (based on previous stroke rehabilitation studies), the aim was to recruit 176 participants. Outcomes were analysed by intention to treat. The analysis plan was peer reviewed by two independent stroke experts and an independent statistician before the analysis was conducted. Primary and secondary outcome measures were continuous and analysed using hierarchical multiple linear regression. This approach analyses the effect of continuous and categorical variables on a continuous outcome and has been used in other controlled trials of complex interventions.26 For each regression model, the group allocation (behavioural therapy or usual care) was entered in the first block, and baseline values of the outcome variable and Sheffield Screening Test score were entered in the second block. For the Stroke Aphasic Depression Questionnaire model, the baseline score on the 10-item version of the scale was entered in the second block. The primary analysis was conducted on participants who had complete data for that outcome measure. Sensitivity analyses were conducted to examine the robustness of the primary analysis. Regression models were first repeated including all participants by replacing missing outcome data using the last observation carried forward method,27 as the assumption was made that there would be no change in mood or activities over time in the absence of treatment. Second, the regression models were repeated using per protocol data, with participants allocated to receive behavioural therapy included if they received a minimum of three sessions. Outcomes between the intervention and control group were also compared using area under the curve to analyse the differences between repeated measurements,28 using Mann–Whitney U-tests. A two-sided significance level of 0.05 was used for all analyses. Analyses were performed using SPSS (version 19) for Windows. BODY.RESULTS: Of the 511 patients identified, 281 (55%) were excluded and 230 (45%) consented to have their mood assessed. The reasons for exclusion are shown in Figure 1. Of the 230 assessed, 128 (56%) were identified as having low mood and invited to take part in the trial. Of these, 23 refused and 105 were randomized: 54 to usual care and 51 to behavioural therapy. Patient flow through the trial is shown in Figure 1. Figure 1.Trial profile. Groups were similar on baseline characteristics. Results are shown in Table 1. Patients were predominantly male, married, and living with their spouse; 23 (21%) had had a previous stroke and 29 (28%) were receiving antidepressant medication. Table 1.Baseline demographics and stroke characteristics CharacteristicUsual care (n= 54)Behavioural therapy (n = 51)n%n%Gender – male 37 69 29 57 Setting where recruited  Hospital 7 13 5 10  Community 47 87 46 90 Marital status  Single 7 13 4 9  Married/partnered 33 61 32 63  Widowed/divorced/separated 14 26 15 29 Place of residence  Independent housing 49 91 49 96  Residential or nursing home 5 9 2 4 Living arrangements  Alone 10 19 13 25  With spouse/partner 33 61 32 63  With relatives 7 13 3 6  Other 4 7 3 6 Side of lesion  Left 38 70 34 67  Right 5 9 3 6  Bilateral 3 6 0 0  Unknown 8 15 14 28 Previous stroke 12 22 11 22 Currently receiving antidepressant medication 14 26 15 29 MeanSDMeanSD Age (years) 65.5 13.9 68.5 13.1 Months post stroke (median, interquartile range) 9.0 (4.9–39.0) – 8.7 (4.1–26.1) – Barthel Index (mean, SD) 14.4 (6.0) 6.0 14.8 (4.0) 4.0 Sheffield Screening Test  Receptive 5.5 2.4 5.2 2.5  Expressive 6.2 3.9 6.1 4.1 Frenchay Aphasia Screening Test  Reading 3.6 1.5 3.5 1.7  Writing 2.1 1.9 1.6 1.8 Stroke Aphasic Depression Questionnaire Hospital version 10 9.5 4.4 11.2 5.8 Prorated Stroke Aphasic Depression Questionnaire Hospital version 21 20.1 9.0 23.4 12.2 Visual Analog Mood Scale – ‘sad’ 40.6 28.7 48.1 29.0 Visual Analogue Self-Esteem Scale 35.7 8.2 32.8 7.8 Of the 51 patients randomized to behavioural therapy, 48 (94%) agreed to receive therapy and 44 (86%) completed the course of therapy. Patients who completed therapy received a mean of 9.07 sessions (range 3–18, SD 2.63) and the mean duration of each session was 58 minutes (range 30–89, SD 10.71). At three months and six months after randomization 89 patients completed the outcome assessments. Reasons for non-completion are shown in Figure 1. Complete outcomes were obtained on 85% of participants. Scores on outcome measures are shown in Table 2. The baseline Stroke Aphasic Depression Questionnaire 10-item version scores were prorated to obtain baseline Stroke Aphasic Depression Questionnaire 21-item version scores for the purpose of evaluating progress over time. Table 2.Scores on outcome measures MeasureTime in monthsUsual careBehavioural therapynMeanSDnMeanSDStroke Aphasic Depression Questionnaire Hospital version 21 3 44 19.2  9.6 39 16.9 10.2 6 42 21.9  9.5 39 17.4 10.0 Visual Analog Mood Scale – ‘sad’ 3 48 36.3 28.4 41 26.5 22.3 6 46 32.1 29.3 43 25.5 21.5 Visual Analogue Self-Esteem Scale 3 46 33.2  7.4 41 35.4  6.7 6 44 33.3  7.9 43 34.3  7.3 Nottingham Leisure Questionnaire 3 48 15.7  6.9 41 17.1  6.7 6 46 15.9  6.8 43 17.0  7.6 Carer Strain Index 6 36  6.3  3.6 37  6.6  3.1 Satisfaction with Care – Patient  Emotional support 6 45 58.0 30.0 41 61.5 31.9  Communication support 6 45 58.2 34.2 41 63.5 31.7  Hospital and community services 6 45 65.3 30.5 41 68.9 26.6 Satisfaction with Care – Carer  Emotional support 6 38 55.1 30.5 37 65.7 27.1  Communication support 6 38 58.6 31.3 37 68.5 28.3  Hospital and community services 6 38 62.8 27.9 37 73.9 21.9 The results of the regression analyses on those who completed outcome measures are shown in Table 3. At three months, group allocation alone was not a significant predictor for any of the outcome measures. When baseline values and communication impairment were controlled for, the effect of allocation became significant for the Stroke Aphasic Depression Questionnaire (P = 0.05), visual analogue ‘sad’ item (P = 0.03), and Visual Analogue Self-Esteem Scale (P = 0.002), but not for the Nottingham Leisure Questionnaire. At six months, group alone was a significant predictor of the primary outcome measure, the Stroke Aphasic Depression Questionnaire (P = 0.045), and remained significant when baseline values were controlled for (P = 0.022). There was no significant effect of group allocation alone on the visual analogue ‘sad’ item, Visual Analogue Self-Esteem Scale, Nottingham Leisure Questionnaire or Carer Strain Index when baseline values and communication impairment were controlled for. Table 3.Regression analysis of outcome measures for patients with outcome data 3 months6 monthsBBeta95% CIPBBeta95% CIPStroke Aphasic Depression Questionnaire Hospital version 21 Unadjusted difference −2.26 −0.12 −6.69 to 2.17 0.313 −4.50 −2.23 −8.89 to −0.11 0.045 Difference adjusted for baseline SADQH−10 and SST −3.91 −0.20 −7.81 to 0.01 0.050 −6.13 −0.31 −9.95 to −2.30 0.002 Visual Analog Mood Scale – ‘sad’ Unadjusted difference −9.79 −0.19 −20.65 to 1.07 0.077 −6.58 −0.13 −17.47 to 4.31 0.233 Difference adjusted for baseline VAMS ‘sad’ and SST −11.65 −0.22 −22.30 to −0.99 0.033 −7.32 −0.14 −18.34 to 3.71 0.190 Visual Analogue Self-Esteem Scale Unadjusted difference 2.24 0.16 −0.78 to 5.27 0.144 0.96 0.06 −2.31 to 4.24 0.560 Difference adjusted for baseline VASES and SST 3.90 0.28 1.14 to 6.39 0.002 2.51 0.17 −0.39 to 5.40 0.089 Nottingham Leisure Questionnaire Unadjusted difference 1.14 0.11 −1.44 to 4.30 0.326 1.04 0.07 −1.98 to 4.07 0.495 Difference adjusted for baseline SST 1.57 0.12 −1.23 to 4.40 0.275 1.30 0.09 −1.63 to 4.16 0.387 Carer Strain Index Unadjusted difference 0.29 0.04 −1.27 to 1.85 0.713 Difference adjusted for baseline SST 0.25 0.04 −1.31 to 1.82 0.749 SADQH-10, Stroke Aphasic Depression Questionnaire 10-item hospital version; SST, Sheffield Screening Test for Acquired Language Disorders; VAMS, Visual Analog Mood Scales; VASES, Visual Analogue Self-Esteem Scale. Comparison of the area under the curve revealed significant differences between the groups for visual analogue ‘sad’ item (P = 0.015), Visual Analogue Self-Esteem Scale (P = 0.005) and the Stroke Aphasic Depression Questionnaire (P = 0.003) but no significant differences for the Nottingham Leisure Questionnaire (P = 0.551). Sensitivity analysis at three months, replacing missing data using the last observation carried forward on the assumption of no change, showed that, after adjustment for baseline values, group allocation no longer significantly predicted Stroke Aphasic Depression Questionnaire 10-item hospital version (P = 0.052), but all other results from three months remained consistent. At six months, the analysis replacing missing data using last observation carried forward showed that group allocation alone remained a significant predictor of Stroke Aphasic Depression Questionnaire scores. After controlling for baseline values, group allocation was now a significant predictor of Visual Analogue Self-Esteem Scale scores (P = 0.02) and the visual analogue ‘sad’ item, but the Nottingham Leisure Questionnaire and Carer Strain Index remained non-significant. Per protocol analysis at three and six months showed the same results as the primary intention to treat analysis. At three months, 15 (28%) of the control group and 11 (22%) of the intervention group were receiving medication for mood problems and this difference was not statistically significant (chi-square = 0.15, P = 0.70). At six months, 15 (28%) of the control group and 14 (27%) of the intervention group were receiving medication for mood problems, and this was not significant (chi-square = 0.00, P = 1.0). Satisfaction with Care ratings for both patients and carers were higher for the behavioural therapy group compared with usual care, but these differences were not significant (P = 0.06–0.61). BODY.DISCUSSION: In patients with aphasia and low mood after stroke, allocation to behavioural therapy compared with usual care significantly predicted better self-reported mood, self-esteem, and observer-rated mood three months after randomization. Six months after randomization there was a significant benefit for observer-rated mood. Overall summary scores using the area under the curve showed significant differences in self-reported mood, observer-rated mood, and self-esteem. There was no significant effect of behavioural therapy on leisure activities or carer strain. Both patients and carers in the behavioural therapy group reported higher satisfaction with emotional support, communication support, and hospital and community services, although this did not reach statistical significance. Behavioural approaches were suitable for patients with aphasia, a group who have previously been largely excluded from studies evaluating psychological treatments for low mood. Differences between the behavioural therapy and usual care groups cannot be attributable to concurrently receiving antidepressant medication, as this was comparable between the groups. Behavioural therapy was shown to have beneficial effects with an average of nine sessions per participant. The optimum intensity and duration of therapy are unknown and it was left to the therapist’s discretion to decide how much treatment to provide. No patients required the maximum 20 sessions allowed, indicating that the intervention did not need to be delivered as intensively as expected. However, the three-month intervention period did not allow flexibility to provide follow-up therapy visits to support the maintenance of benefits, as might be provided in a clinical service. Mitchell et al.6 found benefit at 12 months’ follow-up of a psychosocial–behavioural intervention given for only eight weeks early after stroke, although patients with receptive or global aphasia were excluded. It is possible that a longer duration than the three months provided in this study may be required for some patients to sustain the early benefits of therapy, but exploration of patient characteristics affecting outcome would be needed to inform changes to therapy intensity and duration. As we did not include an attention control group, we cannot draw conclusions about the ‘active ingredients’ of therapy or the role of non-specific attention. As noted by Watkins et al.,9 it is difficult to identify an appropriate attention control. This next stage is required in order to ascertain the essential components of the therapy provided. The absence of evidence of benefit on leisure activities may reflect that the Nottingham Leisure Questionnaire was not sensitive enough to detect changes in leisure activities following therapy. As the Nottingham Leisure Questionnaire was not completed at baseline it was not possible to assess improvement from before the intervention. Also, it may not only be the level of leisure activities themselves that is relevant, but the value that the patient places on activities that they are able or unable to take part in. The generalizability of the findings may be suggested by the fact that that the trial was multicentre, open to patients with any severity of aphasia, and followed a therapy manual, and the intervention was delivered by multiple therapists, who reflected the experience level of NHS assistant psychologists who could deliver the therapy in clinical practice under the supervision of a clinical psychologist. There was no restriction on how long ago the patients had had their stroke, while other studies of psychological interventions have recruited patients within 28 days,8,9 two months29 and four months6 after stroke. Unlike single centre studies, the results cannot be attributable to a specific therapist or local setting, although we cannot be sure of the comparability of usual care across study sites. The randomization was stratified by study site, so any differences in usual care would not have masked any benefits of therapy. Acceptability of the intervention to participants was not evaluated. The content of the therapy varied between participants, and therefore it is difficult to define the intervention precisely. However, this reflects clinical practice, and indicates that the overall approach is useful rather than any specific techniques. Participants were recruited from a variety of sources at a wide range of times since stroke and are likely to be representative of those who would be referred in clinical practice. However, in clinical practice, patients would be referred for behavioural therapy based on the severity of mood problems and suitability for the intervention, and therefore some people may have been included whom clinical therapists would not have expected to benefit. The recruitment rate was lower than planned, and so the intended sample size was not achieved in the study recruitment period due to the time taken to identify and visit potential participants from a variety of sources. Future studies should increase the number of recruiting centres. The rate of low mood was 60%, which is high, but probably reflects the fact that participants were being recruited for a treatment study and so the referring clinicians may have been biased to identifying potential participants with low mood. Few assessments are available to screen for mood problems in patients with aphasia. The Visual Analog Mood Scales ‘sad’ item and Stroke Aphasic Depression Questionnaire were used, with patients meeting the criteria on one or both measures to be eligible. The use of these criteria may have led to under-representation of patients with severe aphasia, and some patients with low mood may have been missed. The mean scores on the ‘sad’ item were below the cut-off of 50, indicating that the sample included some patients with no self-reported severe mood problems, although mood did still improve at three months. There are limitations to both observer-rated and visual analogue scales to assess mood in those with communication problems, and therefore the strategy was to consider low mood as reflected in either type of measure. In addition, the Stroke Aphasic Depression Questionnaire 10-item hospital version was used at baseline, whereas the 21-item version of the scale was used at follow-up. This occurred because the 10-item version was in routine clinical use on the wards where patients were recruited, whereas the 21-item version has been shown to be more sensitive and therefore was considered a more appropriate outcome measure. An additional problem of the Stroke Aphasic Depression Questionnaire at outcome was that the relative or carer completing the scale knew whether the participant had received treatment. However, as the results are consistent between observer-rated and independently assessed measures, this suggests the findings are not simply due to observer bias. The study was limited by the small sample size, which may not have had adequate power to detect significant effects across all analyses. The differences in leisure activities and satisfaction were in the predicted direction, but the study may not have had sufficient power to detect significant differences on these measures. However, the findings are promising. Future studies of behavioural therapy should recruit a larger sample size, identify patients with more severe mood problems, and investigate whether treatment duration should be extended to facilitate the maintenance of treatment gains. Overall the results of this preliminary study suggest that behavioural therapy improved the mood of stroke patients with aphasia and low mood. Further evaluation of this treatment strategy is therefore warranted. BODY.CLINICAL MESSAGES: Mood problems are common in people with aphasia. Behavioural treatments are appropriate for those with low mood and communication problems. In a randomized trial, behavioural therapy for an average of 10 sessions improved participants’ mood.

TITLE: Randomised Trial of Chloroquine/Sulphadoxine-Pyrimethamine in Gambian Children with Malaria: Impact against Multidrug-Resistant P. falciparum Multi-drug resistant malaria ABSTRACT.OBJECTIVES:: In the Gambia, the combination of chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) has replaced CQ monotherapy for treatment of malaria caused by Plasmodium falciparum. We measured the efficacy of the combination CQ/SP, and the prevalence of parasites carrying alleles associated with resistance to CQ or SP. ABSTRACT.DESIGN:: We conducted a single-blind, randomised, controlled trial to compare the efficacy of CQ/SP to that of SP or CQ alone. ABSTRACT.SETTING:: The study took place in the town of Farafenni and surrounding villages in the Gambia. ABSTRACT.PARTICIPANTS:: Participants were children aged 12 mo to 10 y presenting as outpatients with uncomplicated P. falciparum malaria. ABSTRACT.INTERVENTIONS:: 500 children were randomised to receive CQ, SP, or CQ/SP as supervised treatment and actively followed over 28 d. ABSTRACT.OUTCOME MEASURES:: Primary outcome was parasitaemia at any time during follow-up. Secondary outcomes were PCR-confirmed recrudescent infections among treatment failures, and clinical failure requiring rescue medication by day 28. Pretreatment parasite isolates from 161 patients were tested for the presence of resistance-associated genetic markers. ABSTRACT.RESULTS:: The prevalence of parasitological failure by day 28 for the CQ group was 60.3%, compared to 17.6% for SP (odds ratio [OR], 0.106; 95% confidence interval [CI], 0.057–0.194; p < 0.001) and 13.9% for CQ/SP (OR versus CQ, 0.140; 95% CI, 0.078–0.250; p < 0.001). There was no difference between the SP and CQ/SP groups (OR, 1.324; 95% CI, 0.705–2.50). The projected prevalence of PCR-corrected treatment failure was 30.2, 6.06, and 3.94% in the CQ, SP, and CQ/SP groups, respectively. The pfdhfr-triple mutant and pfdhps-437G mutation were common, with prevalences of 67.4 and 51.2%, respectively. Pretreatment carriage of pfdhps-437G and of multidrug-resistant parasite genotypes was associated with treatment failure in the SP group, but not in the CQ or CQ/SP groups. ABSTRACT.CONCLUSIONS:: The combination of CQ/SP was an efficacious treatment for uncomplicated malaria in Gambian children in this study, but the frequent occurrence of multidrug-resistant parasites suggests that this observed efficacy is not sustainable. ABSTRACT.EDITORIAL COMMENTARY: Background: Throughout Africa, the readily available and cheap antimalarial drug, chloroquine, has been failing as a good first-line treatment for uncomplicated malaria. This is mainly due to the malaria parasite becoming resistant to treatment. Many African countries are now adopting newer artemisinin-based combination therapies as first-line treatment. However other countries have put interim solutions in place before introducing artemisinin-based drugs. The combination of chloroquine and sulfadoxine-pyrimethamine was officially adopted as first-line therapy in the Gambia in 2004. However, little data exists on the efficacy of this combination in that setting. It is also not known whether parasites resistant to sulfadoxine-pyrimethamine, as well as chloroquine, are common in the Gambia. In this trial, conducted in a rural town in the Gambia in 2001, the researchers randomized 500 young children presenting with uncomplicated P. falciparum malaria to receive either chloroquine, sulfadoxine-pyrimethamine, or the combination of both treatments. The researchers then looked for the presence of parasites in the blood of treated children over 28 days of follow-up. Infections following treatment were defined as either the same infection that was originally treated (a recrudescence) or a new infection. The need for rescue medication, indicating clinical failure, was also examined. What the trial shows: In this trial, both sulfadoxine-pyrimethamine and the combination of chloroquine and sulfadoxine-pyrimethamine were more effective at clearing parasites from the blood than chloroquine alone. However the combination of chloroquine and sulfadoxine-pyrimethamine appeared to be equivalent to sulfadoxine-pyrimethamine in clearing parasite infection. When the researchers distinguished parasite infections into recrudescences or new infections, they found that either treatment option containing sulfadoxine-pyrimethamine prevented a recurrence of the original infection better than treatment with chloroquine. Clinical failure was also higher amongst children treated with chloroquine than those treated with sulfadoxine-pyrimethamine alone or the combination. In children treated with chloroquine and sulfadoxine-pyrimethamine, parasite infection was found in 14% of participants at 28 days of follow-up. A number of children carried parasites predicted to be resistant to both chloroquine and sulfadoxine-pyrimethamine. However, these children were not significantly more likely to fail treatment. Strengths and limitations: The trial took place in an area where there is, to date, little data on the efficacy of the different treatment regimens studied. The trial is also part of an ongoing series of other studies, which will ultimately allow researchers to detect changes in the efficacy of different treatments over time, providing information about parasite selection and the useful therapeutic life of particular treatments. However, the loss to follow-up in this trial was high (between 25 and 32%, depending on the treatment arm) due to logistical constraints around the time of the Gambian national election. It is not possible to tell if data from the participants lost to follow-up would have changed the overall conclusions. Contribution to the evidence: This study provides additional data about the efficacy of chloroquine / sulfadoxine-pyrimethamine treatment in the Gambia, as compared to chloroquine or sulfadoxine-pyrimethamine alone. Prior evidence was not strong enough to confirm the advantage of chloroquine / sulfadoxine-pyrimethamine over chloroquine alone. The identification of parasites resistant to both chloroquine and sulfadoxine-pyrimethamine indicates that new combination drugs are needed in this part of Africa. BODY.INTRODUCTION: Malaria control in Africa has until recently relied heavily on chemotherapy with chloroquine (CQ), a cheap and safe antimalarial drug [1,2]. CQ remains widely distributed and readily available even in the most remote villages in sub-Saharan Africa. In the Gambia, each village with a population of 400 or more has a trained village health worker who is provided with chloroquine and other basic drugs [3]. Chloroquine-resistant strains of Plasmodium falciparum, first observed in East Africa in 1987, have now been reported in all countries of tropical Africa [4]. Studies between 1994 and 2000 found that 65–73% of Gambian children treated with chloroquine were parasitaemic at some point over 28 days of follow-up [5,6], demonstrating that as in much of Africa, CQ had ceased to be a satisfactory first-line treatment for uncomplicated P. falciparum malaria and an alternative was urgently required [7]. There have been a number of trials in sub-Saharan Africa to measure the efficacy, effectiveness, and impact on transmission of a variety of combination antimalarial regimens, including those incorporating a member of the artemisinin family [6,8–14]. Although the use of these newer combinations as first-line treatment for malaria is being adopted as policy in many African countries, interim solutions have been urgently sought by some prior to the full-scale introduction of artemisinin-based combination therapy. In the Gambia, the combination of CQ and sulfadoxine-pyrimethamine (SP) has been shown to be a more effective symptomatic treatment than SP alone for malaria [8]. CQ/SP has therefore been used increasingly in the face of spreading resistance to CQ, and the combination was officially adopted as an affordable alternative frontline therapy in 2004. However, few reliable data are available on the efficacy of the combination CQ/SP in the Gambia, and although recent work has described the contribution of mutations in the CQ-resistance-associated loci pfcrt and pfmdr1 to CQ treatment failure, to enhanced transmission to mosquitoes, and to an excess of severe malaria among rural Gambian children (15–17), there are no recent Gambian studies examining the prevalence of mutations associated with SP resistance, in the pfdhfr and pfdhps genes, nor of their effect on treatment outcome. This paper reports the result of a randomised controlled trial conducted in 2001 to evaluate the efficacy of CQ/SP compared to SP or CQ alone. We also measured the pretreatment prevalence of parasites carrying resistance-associated alleles of four genes previously implicated in treatment failure of CQ and SP, and evaluate their contribution to therapeutic outcome. BODY.METHODS: BODY.PARTICIPANTS: The study took place from September to December 2001 in Farafenni, a rural town on the north bank of the Gambia River. It is located on the Senegal border about 170 km from the Atlantic coast. The town has been the site for clinical trials to determine the efficacy of antimalarial drug combinations with gametocyte carriage and transmission as major endpoints since 1998 [6,10,11,13,14]. The climate is characteristic of the sub-Sahel with a short rainy season from mid-June to mid-October. Malaria is thus seasonal with most clinical episodes occurring during a limited period of 8 to 10 wk at the end of the rains. Recruitment took place at the Maternal and Child Health clinic according to an established protocol [6]. Briefly, children 0.5–10 y of age living within 15–20 km radius of Farafenni who presented to the Maternal and Child Health clinic or the General Hospital with history of fever and/or current fever (axillary temperature, ≥37.5 °C) and other symptoms suggestive of acute malaria infection, a carriage of P. falciparum parasites at a density ranging between 500 and 250,000 parasites/μl of blood, and a packed cell volume (PCV) ≥20% were enrolled in the study after obtaining the free and informed consent of their parents or guardians. Excluded from the study were children with anaemia (PCV, <20%), any signs or symptoms of severe malaria, inability to take drugs orally or any evidence of chronic disease, malnutrition or any other acute infection, including non-falciparum malaria. If there was evidence of treatment with any antimalarial drug within the past 2 wk, either from notes on the clinic card carried by children under 5 y of age, or after questioning of the caregiver, the child was excluded. The study protocol was reviewed and approved by the Medical Research Council/Gambian Government Joint Ethics Committee, and the Ethics Committee of the London School of Hygiene and Tropical Medicine. BODY.INTERVENTIONS: We performed a single-blind, randomised, controlled trial of oral treatment of uncomplicated falciparum malaria with CQ, SP, and the combination CQ/SP. A thorough clinical history (including demographic data) was taken, and a medical examination was performed by a study clinician on the day of screening, day 0. At screening and at each visit, body temperature was measured with a digital thermometer (Toshiba, Tokyo, Japan). The treatments used were CQ alone (25 mg of CQ base per kilogram of body weight over a 3-d period: 10 mg/kg on days 0 and 1; 5 mg/kg on day 2), SP alone given in a single dose (25 mg/kg sulphadoxine/1.25 mg/kg pyrimethamine), or the combination of CQ/SP. The group sizes were 130:180:200, respectively. This ratio was derived a priori as likely to ensure sufficient power for both efficacy and transmission endpoints of the trial, taking into account that children randomised to receive SP were further divided into three groups according to the day of the week on which they were recruited, as part of the accompanying study of post-treatment transmission [18]. These three SP-treated groups differed in their follow-up schedule, and only SP-treated patients recruited on Thursday or Friday received identical follow-up to children receiving CQ or CQ/SP. Therefore, only these patients are included in the analysis of clinical efficacy (Figure 1). Treatment allocation and prescription of the required medication were performed by a study clinician, and medicines were administered by a study nurse. Treatment was performed in a separate room but was not deliberately obscured from the view of the study team. Older children chewed or swallowed the tablets whole with water. For younger children, the tablets were crushed together in a cup and mixed with water before administration. Subsequent doses, for those in chloroquine groups, were supervised by trained field assistants in the patients' homes. All treated children were observed for 30 min, and any child who vomited was administered a replacement dose. Children who vomited both the initial and the repeat medication were excluded from the study and either given parenteral CQ, which at the time of the study was standard care in the Gambia for children with uncomplicated malaria who could not retain oral medication, or admitted to the paediatric ward of Farafenni Hospital. Figure 1BODY.CONSORT FLOWCHART: A total of 1,366 children was screened, and 500 were randomised. Children randomised to receive SP on Thursday or Friday (N = 79), were scheduled for standard follow-up and thus fulfilled the criteria for efficacy evaluation. Follow-up data are shown for these 79, but not for the other SP-treated children. Shaded boxes represent gametocyte screening days [18]. In addition to the study drugs, oral paracetamol (10 mg/kg, three times daily) was given together with the study medication in the clinic, and additional doses (to last for 3 d) were given to parents/guardians for administration to their children at home. BODY.OBJECTIVES: The principal objective was to assess the efficacy of SP and CQ/SP for the treatment of uncomplicated falciparum malaria in children, compared to the efficacy of CQ monotherapy. A secondary objective was to estimate the prevalence of alleles of parasite genes associated with resistance to CQ and SP. BODY.OUTCOMES: The primary outcome was parasitaemia at any time during follow-up. Secondary outcomes were PCR-corrected group estimates of treatment failure, and clinical failure requiring rescue medication by day 28. Pretreatment parasite isolates from 161 patients were tested for the presence of resistance-associated genetic markers. BODY.SAMPLE SIZE: The sample size required in each treatment group was estimated with transmission endpoints in mind. We expected to enroll approximately 500 patients, based on data from 1998–2000. In our previous studies, the observed prevalence of gametocyte carriage 7 d after treatment with SP was >70% (excluding those who had gametocytes on day 0), and 40% after treatment with CQ [13]. We therefore expected that the prevalence of gametocytes after SP/ CQ would be at least 28% (i.e., 40% × 70%). Based on these figures, and to ensure that at least 30 mosquito feeds were performed in each treatment group, we set out to enroll (a) 200 subjects in the CQ + SP group, (b) 50, 60, and 70 subjects in the three groups receiving SP and being screened for gametocytes on days 7, 10, and 14, respectively, and (c) 130 subjects in the CQ group. All CQ- and CQ/SP-treated children were to be screened for gametocytes on day 7 only. This sample size was expected to provide sufficient power at the 90% confidence level to detect a 10-fold difference in infectiousness at day 7 between gametocyte-positive children treated with SP alone, and those treated with SP + CQ [18]. This was not expected to provide sufficient power to detect any difference in parasitological efficacy between SP and CQ/SP, which was estimated a priori to be approximately 90% for both regimens. BODY.RANDOMISATION—SEQUENCE GENERATION: Eligible patients were randomly assigned to treatment using a predetermined computer generated randomisation list. The list was generated by a statistician not otherwise engaged in the study. BODY.RANDOMISATION—ALLOCATION CONCEALMENT: Allocation was not concealed, but taken from a list with 550 allocations on it, in the order of recruitment. Access to this list was restricted. BODY.RANDOMISATION—IMPLEMENTATION: Patients underwent initial clinical and parasitological screening to confirm a diagnosis of malaria before being asked for informed consent by a field worker able to speak Wolof, Mandinka, and Fulani. This person did not have access to the allocation list. If consent was given, a clinical examination, treatment allocation, and prescription of the required medication were performed in a separate room by a study clinician who had access to the randomisation list. BODY.RANDOMISATION—BLINDING: Only the study clinician responsible for initial recruitment and treatment, and the field assistants visiting the children during follow-up, had knowledge of drug allocations. Field assistants did not have access to the full allocation list, but knew the treatment group of children in their care. All laboratory staff, microscopists, entomologists, and the principal investigator were blinded to treatment allocations. BODY.POST-TREATMENT FOLLOW-UP: Field assistants visited children at home on days 1 and 2 to supervise treatment (to those in CQ groups) and on days 3, 14, and 28 to enquire from the caretaker about the child's condition of health and to collect finger prick samples that provided thick blood films for microscopy and filter paper blood spots for parasite typing by PCR. If on any of these visits the caretaker was concerned about the child's health, he or she was asked to attend the clinic as soon as possible. Parents/guardians were also requested to bring their children to the Maternal and Child Health clinic at any time in the event of clinical aggravation. If a parent or guardian reported that a child was unwell, the child was examined by a study clinician. If the child had parasitaemia and fever (axillary temperature, ≥37.5 °C) or recent history of fever and did not have other conditions that could explain the symptoms, and fewer than 29 d had elapsed since recruitment, the child was considered a clinical treatment failure and given rescue treatment as follows: those in the CQ treatment group received SP, and those in the other two groups received a standard course of oral quinine. Children presenting with persistent symptoms of malaria within 3 d of enrollment with >90% reduction in parasitaemia were not considered to be clinical treatment failures, and did not receive rescue treatment. They were monitored until complete recovery. On day 7 after treatment, the children were collected from their homes and taken to the Medical Research Council Field Station in Farafenni where they were clinically examined and finger prick blood samples were obtained for thick blood film preparation and PCV estimation. The blood films were stained with Field's stain and read immediately for malaria parasites. Some children identified as gametocyte carriers at this time contributed to transmission experiments described elsewhere [18]. BODY.BLOOD SAMPLING AND LABORATORY MEASUREMENTS: At screening and at day 3, 7, 14, and 28 follow-up visits, blood samples were obtained by finger prick for thick blood smears for malaria microscopy. At day 7, or any time that the child had symptoms consistent with clinical malaria, two thick smears were made; the first was stained with Field's stain and read immediately. The second was dried overnight, stained with Giemsa, and read later by two experienced laboratory assistants to provide definitive parasite counts. One hundred high-power fields were read before a slide was declared negative. At screening and at day 7, blood samples were collected in heparinised capillary tubes and spun using a micro-haematocrit centrifuge (Hawksley, West Sussex, United Kingdom) for PCV determination. Staff performing all laboratory investigations were blinded to treatment regimens. BODY.MOLECULAR GENOTYPING: P. falciparum genotypes circulating in children with detectable asexual parasitaemia during posttreatment follow-up were compared with those present in the same child prior to treatment. DNA was extracted from dried blood spots using a Chelex-based method [19]. Alleles of the polymorphic locus pfmsp2 were compared between pretreatment and posttreatment parasite isolates by PCR [11,20]. The procedure of Cattamanchi et al. [21] was followed, in that indeterminate samples in which a majority of novel bands appeared in the posttreatment infection were scored as new infections. Resistance-associated loci encoding amino acid positions 72–76 in pfcrt (sensitive allele, Cys-Val-Met-Asn-Lys [CVMNK]; resistant allele, Cys-Val-Ile-Glu-Thr [CVIET]), position 86 in pfmdr1 (resistant allele, Asn [N]; sensitive allele, Tyr [Y]), positions 51, 59, and 108 in pfdhfr (resistant alleles, Ile, Arg, and Asn [I, R, N], respectively; sensitive alleles, Asn, Cys, and Ser [N, C, S], respectively), and positions 437 and 540 in pfdhps (sensitive alleles, Ala and Lys [A, K], respectively; resistant alleles, Gly and Glu [G, E], respectively) were identified as previously described [15,22,23]. Briefly, fluorescent-labeled oligonucleotide probes specific for each allele of interest were hybridised to PCR products spotted in 12 × 8 arrays on nylon membranes, and hybridisation signals were detected by chemiluminescence. BODY.STATISTICAL METHODS: All data were double entered and verified using Epi-Info, version 6 (Centers for Disease Control and Prevention, Atlanta, Georgia, United States) and transferred to Stata 7.0 (Stata Corporation, College Station, Texas, United States) for statistical analysis. All children with follow-up data were included in the primary analysis. Any child presenting with danger signs on days 0–3 in the presence of malaria parasites or fever/history of fever plus parasitaemia any time from days 4 to 28 was given rescue medication and treated as a clinical therapeutic failure. Proportions were compared using the χ2 statistic for parasitological data (denominators >50 in each case) or using Fisher exact test for clinical failure data and molecular genotyping data, because in both these analyses expected proportions in some cells of 2 ×11]. Confidence intervals around projected relative risk estimates were calculated from the variance of the log relative risk as described [24], after modification to allow for each risk being the product of two proportions. BODY.RESULTS: BODY.RECRUITMENT AND PARTICIPANT FLOW: A total of 1,368 children was screened. Two records were without parasitaemia data and were therefore excluded from further analysis. Of the 1,366 children with complete information on malaria infection, 844 (61.8%) were positive for malaria parasites according to microscopy at the recruiting clinic, and were therefore eligible for enrolment into the study. Of these, 500 were enrolled into the trial; 126, 181, and 193 in the CQ alone, SP alone, and CQ/SP groups, respectively. The most common reasons for ineligibility for enrollment were low density parasitaemia (<500/μl of blood) (n = 114), refusal to give parental consent (n = 40), anemia (PCV, <20%) (n = 34), residence outside the clinic catchment area (n = 24), high density parasitaemia (≥250,000 parasites/μl of blood), and/or other severe signs of malaria (n = 18) and presence of concomitant diseases and other reasons (n = 51). A trial profile is shown in Figure 1. Loss to follow-up by day 28 was 25.4, 31.5, and 32.6% in the CQ, SP, and CQ/SP treatment groups, respectively. BODY.BASELINE DATA: There were no differences in baseline demographic characteristics or malaria infection between the treatment groups (Table 1). Table 1Baseline Demographic and Clinical Characteristics of Patients Randomised to CQ Alone, SP Alone, or CQ/SP Treatment BODY.OUTCOMES AND ESTIMATION: BODY.CLINICAL OUTCOMES AND ADVERSE ADVENTS.: The frequency of posttreatment, parasitologically confirmed clinical malaria (clinical failure) among evaluable study patients in each treatment group is shown in Table 2. By day 28, as expected from previous studies, more children in the CQ treatment group had experienced an episode of clinical malaria posttreatment than in either of the other groups. There was weak evidence that clinical failure was more common among SP-treated children than among those receiving CQ/SP (OR, 2.68; 95% CI, 0.764–9.34). This was apparently due to persistence of symptoms during the first 3 d (Table 2), consistent with the findings of Bojang et al. [8]. However, clinical endpoints were not the main focus of the trial, and we did not collect sufficient data to make a valid comparison with earlier results [8]. There were no serious adverse events reported during follow-up, and no deaths among the 500 recruited participants during the study period. Table 2Clinical Failure Rates in Children Treated with CQ, SP, or CQ/SP for Uncomplicated P. falciparum Malaria BODY.PARASITOLOGICAL OUTCOMES.: The point prevalence of parasitological treatment failure for each treatment group at each day of follow-up is shown in Figure 2. This is a per protocol analysis. At each time point, parasitological failures were most common among CQ-treated children, with an uncorrected cumulative treatment failure rate over 28 d of 60.3% for CQ, compared to 17.6% for SP (OR, 0.106; 95% CI, 0.057–0.194; p < 0.001) and 13.9% for CQ/SP (OR compared to CQ, 0.140; 95% CI, 0.078–0.250; p < 0.001). To compare the parasitological outcomes of each group in more detail, the ratio between arithmetic mean parasite densities at each day of active follow-up was calculated, and the statistical significance of this difference tested by negative binomial regression, including those with zero parasites (Table 3). We have previously used this method to compare gametocyte densities among treatment groups [11,14]. We found that the parasitological efficacy of SP was significantly higher than that of CQ at days 7, 14, and 28, and that of CQ/SP was significantly higher than that of CQ at days 3, 7, and 14 (Table 3). No significant difference in mean parasite density was found between the SP and CQ/SP groups at any point (data not shown). Figure 2BODY.POINT PREVALENCE OF :Plasmodium falciparum Asexual Parasitaemia 3, 7, 14, and 28 Days after Treatment of Children with CQ, SP, or CQ/SP Error bars represent the upper 95% confidence limit of the proportion. Denominators for these data are (for CQ, SP, and CQ/SP groups, respectively): day 3, 111, 160, 168; day 7, 107, 52, 152; day 14, 98, 134, 143; and day 28, 94, 124, 130. Table 3Parasitological Benefit of SP and CQ/SP Treatment Compared to CQ Treatment at Each Point of Follow-Up Figure 3BODY.PREVALENCE OF RESISTANCE-ASSOCIATED LOCI AMONG 90 PRETREATMENT PARASITE ISOLATES: Genotypes at seven loci in four P. falciparum genes were determined, as was the prevalence of the pfdhfr triple-mutant IRN, and of the putative multidrug-resistant genotype TYRG. BODY.CORRECTION OF PARASITOLOGICAL FAILURE RATES BY MSP2 GENOTYPING: Alleles of msp2 in pretreatment DNA samples were compared to those in posttreatment DNA samples to distinguish recrudescent parasites from parasites newly emergent from the liver during follow-up, as previously described [11]. PCR was performed for paired isolates from 20 patients with posttreatment parasitaemia from each treatment group, and interpretable data obtained for 16, 15, and 17 pairs from the CQ, SP, and CQ/SP groups, respectively. Results are tabulated in Table 4, and used to estimate the true failure rate in each group by extrapolation. We found a corrected failure rate for CQ of 30.2%, for SP of 6.06%, and for CQ/SP of 3.94%. Using these estimates, we also projected a “virtual” relative risk of posttreatment recrudescence in each treatment group (Table 4). Both treatment groups containing SP were substantially better than CQ monotherapy in this analysis, whereas there was no significant benefit identified for CQ/SP over SP alone. Table 4Cumulative 28-d Parasitological Failure Rates Corrected by msp2 Genotyping BODY.BASELINE PREVALENCE OF RESISTANCE-ASSOCIATED ALLELES: The prevalence of drug resistance-associated mutations at seven loci in four genes was measured among 90 day 0 samples randomly selected from among all trial participants (Figure 3). The prevalence of the pfdhfr triple mutant 51-I, 59-R, 108-N (IRN) was also measured, as was prevalence of the combination genotype pfcrt-76T, pfmdr1-86Y, pfdhfr-59R, and pfdhps-437G (TYRG), a possible multidrug-resistant genotype. Combination genotypes IRN and TYRG were assigned to infections in which each allele was present. However, in some such individuals, wild-type alleles were also present at some of the loci in the combination, and so it is possible that IRN or TYRG did not occur as a single haplotype. Of the 60 patients designated as IRN, 49 (81.7%) unequivocally harboured true triple-mutant haplotypes, whereas the other 11 patients were of mixed genotype at two or three of the dhfr resistance-associated loci. Of the 17 patients who harboured parasites carrying the mutations pfcrt-76T, pfmdr1-86Y, pfdhfr-59R, and pfdhps-437G, the resistant allele alone was detected at three or four of these loci in 11 cases (64.7%). Thus, we are certain that parasites of the haplotype TYRG must occur in these infections. Mixed sensitive and resistant alleles were detected at two loci in four further isolates, and at three and all four loci in the remaining two isolates, respectively. Thus, in these latter six isolates, the haplotype TYRG may occur, but this is not certain. All were retained in subsequent analysis of associations with treatment failure. BODY.RESISTANCE-ASSOCIATED ALLELES AND TREATMENT FAILURES: The risk of treatment failure associated with resistant genotypes at presentation was investigated by examining resistance-associated loci in pretreatment isolates from 100 patients who subsequently failed treatment. These comprised 44 children who received CQ, 31 who received SP, and 25 who received CQ/SP, of which four, three, and three children, respectively, required rescue treatment at the time of failure due to recurrent or persistent clinical signs of malaria. The prevalence of each marker in these pretreatment samples was compared to the prevalence among 61 isolates from children who were successfully treated. These children, 7, 22, and 32, respectively, from the CQ, SP, and CQ/SP groups, were among the 90 randomly selected isolates described in Figure 3. Because 29 of these children subsequently failed treatment, only 61 were used for the purposes of estimating the odds of failure associated with each pretreatment genotype. A major purpose of these exploratory analyses was to identify a haplotype adequately representing “multidrug resistance” that could be defined at a single locus for each gene and so did not require using data at all seven loci. Estimates of the association between treatment failure and both single locus and selected multilocus genotypes is presented in Table 5. The only statistically significant association with treatment failure for any single locus was in the SP treatment group, where children presenting with the pfdhps-437G allele were more likely to fail treatment than those carrying the wild-type 437A allele (Table 5). Other weak associations could be discerned in the data, but given the multiple testing performed, these findings were not considered reliable. The combined dual locus genotype TY defined at pfcrt-76 and pfmdr1-86, previously associated with CQ treatment failure in this population [15], appeared to associate with treatment failure in the SP group only. The combined dual-locus genotype RG defined at pfdhfr-59 and pfdhps-437 was strongly associated with treatment failure, also in the SP group only, suggesting that the combination of these two alleles comprise the dominant SP-resistant haplotype in this study area. The four locus genotype combining both of these two allele genotypes, TYRG, was also strongly associated with treatment failure only in the SP treatment group (Table 5). Table 5Odds of Subsequent Treatment Failure for Each Single-Locus Genotype, the pfdhfr Triple Mutant, and the Four-Locus Genotypes TFRG and TYRG at Day 0, in Each Treatment Group BODY.DISCUSSION: BODY.INTERPRETATION: In this study, we have examined the efficacy of the combination CQ/SP for treating uncomplicated falciparum malaria in Gambian children, compared to either CQ or SP alone. Pairwise comparisons of parasitological outcomes between regimens at each point of follow-up demonstrated a sustained benefit of treatment with SP or CQ/SP compared to CQ alone, but no difference in efficacy was found between the two SP-containing treatment groups at any time point. Cumulative failure rates, after correction by msp2 genotyping to estimate rates of recrudescence, did not differ between the SP- and CQ/SP-treated groups of patients. The clinical failure rate of CQ monotherapy over 28 d was 22.6% in this study, and thus CQ is no longer useful as first-line treatment for malaria in children. The combination CQ/SP is an efficacious treatment for uncomplicated malaria in Gambian children, but the high prevalence of CQ-resistant parasites coupled with a small but measurable SP failure rate strongly suggest that this level of efficacy is unsustainable in our study area. These data were collected in late 2001, and continued use of CQ/SP as first-line treatment may have further eroded the efficacy of this combination since that time. However, subsequent studies show that crude parasitological efficacy for CQ/SP in Farafenni has remained good at 88% in 2002 [11] and 86% in 2003 (P. Milligan and S. Dunyo, unpublished data). CQ/SP has been the first-line antimalarial regimen in the Gambia since 2004. In 1995, Bojang et al. [8] measured the parasitological efficacy of SP and CQ/SP in children aged 1–10 y presenting to health facilities at Sibanor and Basse, the Gambia. These authors found the cumulative (uncorrected) parasitological failure rate at day 28 was 10% for SP (15 of 150) and 5% (7 of 141) for CQ/SP. These compare with uncorrected rates of 17.6% (29 of 165) and 13.9% (24 of 173) in the present study. Assuming the methodologies used are compatible, this suggests that children treated with SP in 2001 had a relative risk of parasitological failure of 1.31 (p = 0.053) compared to children treated with SP in 1995. Children treated with CQ/SP in 2001 had a relative risk of failure of 1.47 (p = 0.009). This exploratory comparison does suggest that the efficacy of both SP and CQ/SP has diminished in the 5-y period between these two studies. Bojang et al. did find that CQ/SP provided more rapid resolution of malaria symptoms than treatment with SP alone, but this was not tested in our study. We used msp2 genotyping in a subset of parasitological treatment failures to estimate the recrudescence rate in each treatment group. This method is prone to underestimate the true number of recrudescent infections in areas of stable transmission, because the PCR method will not pick up minority genotypes that may be present in the pretreatment sample at low abundance. A multiplicity of infection is seen in most infected patients in sub-Saharan Africa, and, among children enrolled in our study, there were on average four genotypes per infection prior to treatment (R. Ord and C. J. Sutherland, unpublished data). Minority genotypes that are resistant may flourish under drug selection and become dominant during follow-up. These will appear to be a “new” infection, but are actually recrudescent [20]. Thus PCR correction must be interpreted carefully, and for this reason we prefer to project a comparative estimate of the true failure rate among the treatment groups, rather than assign each posttreatment parasitaemia its own status as a recrudescent or a new infection. In our study area, addition of SP to CQ provided a therapeutic efficacy of over 90% after PCR correction [6; this study], and is currently the recommended regimen in the Gambia. SP alone also provides parasitological efficacy of approximately 90% over 28 d [9; this study]. We were therefore surprised to find high prevalences of both the pfdhfr “IRN” triple mutant and the pfdhps-437G allele in our pretreatment population. The absence of the pfdhfr-164L and pfdhps-540E mutations in this study area (R. Hallett, R. Ord, and A. Randall, unpublished data) may explain why a reasonable level of SP efficacy is retained, although a lack of correlation between mutations and outcomes in individual patients is frequently observed in clinical trials of antimalarial treatment [25,26]. Nevertheless, the single marker pfdhps-437G was strongly associated with SP treatment failure in univariate analyses (Table 5). BODY.GENERALIZABILITY: Our study suffered from a high dropout rate due to logistic constraints exacerbated by national elections in the Gambia in October 2001, which meant that many staff and participants traveled away from the study area. Nevertheless, estimates of efficacy were consistent with other studies in the area in 1998 and 1999 (SP) [13], in 1998 and 2000 (CQ) [6,13], and in 2002 and 2003 (CQ/SP) [11]. Therefore, finding that SP and CQ/SP are efficacious treatments despite the common occurrence of resistance-associated genotypes in four genes of interest is likely to be generally true. Multidrug-resistant parasite genotypes have recently been described in a single case report from South Africa [27], but have not been widely investigated in African parasite populations, nor has the impact of such genotypes on treatment outcomes been measured in clinical trials. We found that P. falciparum parasites with the multidrug-resistant genotype TYRG, defined at the four loci pfcrt-76, pfmdr1-86, pfdhfr-59, and pfdhps-437 respectively, are relatively common among Gambian children presenting with uncomplicated malaria. The TYRG genotype was associated with therapeutic failure after treatment with SP, but not with the combination CQ/SP. Unexpectedly, there was also a weak association between SP treatment failure and the carriage of CQ resistance-associated alleles of pfcrt and pfmdr1. This may reflect the relatively low power of our study, and the exploratory nature of our analyses, which did not correct for multiple testing. Furthermore, the high prevalence of pfcrt-76T in the parasite population is likely to have masked associations between CQ-resistance loci and treatment outcomes. Nevertheless, these preliminary results do demonstrate the need for carefully designed studies to measure the contribution of multidrug-resistant parasites to inadequate treatment of uncomplicated malaria in African children as combination treatments become more widely deployed. Inadequate treatment is likely to increase risk of progression to severe disease, particularly severe malarial anaemia [17], and thus short-term gains in terms of improved treatment efficacy achieved with combinations such as CQ/SP and amodiaquine/SP may be quickly eroded if multidrug-resistant genotypes enjoy a selective advantage in the treated host. In the accompanying paper, we investigate the nature of that selective advantage in Anopheles gambiae mosquitoes experimentally fed on gametocytes from children who had received the CQ/SP combination [18]. The results suggest that children harbouring multidrug-resistant parasites are significantly more infectious to mosquitoes than other CQ/SP-treated children. BODY.OVERALL EVIDENCE: A recent systematic review found a poor evidence base for the therapeutic efficacy of CQ/SP [28], yet this drug combination has been first-line therapy for uncomplicated malaria in the Gambia since 2004. We have identified multidrug-resistant parasite genotypes of P. falciparum carrying alleles implicated in resistance to both CQ and SP, and found these to be common in our study area in 2001. Nevertheless, these parasites did not substantially challenge the therapeutic efficacy of SP or CQ/SP in this study, which were found to have 82.4 and 86.1% efficacy against recurrent parasitaemia, respectively. The prevalence of these genotypes suggests they are advantageous to the parasite. Therefore, continued use of CQ/SP may favour an increase in the prevalence of SP resistance-associated alleles, and should the absent pfdhps-540E mutation be introduced to the population the efficacy of the combination may then drastically decline [25]. The risk of this occurring is heightened by the prevalence of CQ-R parasites in the Gambia [15–17; this study], such that SP has been added to a drug that is already failing. It has been demonstrated that the addition of artesunate to CQ produces a poor therapeutic combination [6]. A possible interim solution in the Gambia for the period leading up to implementation of ACTs may be amodiaquine plus SP, a combination found to work very well in other settings where CQ resistance is high but amodiaquine remains efficacious [29]. BODY.SUPPORTING INFORMATION: CONSORT ChecklistClick here for additional data file. (50 KB DOC) Trial ProtocolClick here for additional data file. (88 KB DOC)

TITLE: A Chinese Mind-Body Exercise Improves Self-Control of Children with Autism: A Randomized Controlled TrialMind-Body Exercise Improves Self-Control of Autism Self-control problems commonly manifest as temper outbursts and repetitive/rigid/impulsive behaviors, in children with autism spectrum disorders (ASD), which often contributes to learning difficulties and caregiver burden. The present study aims to compare the effect of a traditional Chinese Chan-based mind-body exercise, Nei Yang Gong, with that of the conventional Progressive Muscle Relaxation (PMR) technique in enhancing the self-control of children with ASD. Forty-six age- and IQ-matched ASD children were randomly assigned to receive group training in Nei Yang Gong (experimental group) or PMR (control group) twice per week for four weeks. The participants’ self-control was measured by three neuropsychological tests and parental rating on standardized questionnaires, and the underlying neural mechanism was assessed by the participants’ brain EEG activity during an inhibitory-control task before and after intervention. The results show that the experimental group demonstrated significantly greater improvement in self-control than the control group, which concurs with the parental reports of reduced autistic symptoms and increased control of temper and behaviors. In addition, the experimental group showed enhanced EEG activity in the anterior cingulate cortex, a region that mediates self-control, whereas the PMR group did not. The present findings support the potential application of Chinese Chan-based mind-body exercises as a form of neuropsychological rehabilitation for patients with self-control problems. Chinese Clinical Trial Registry; Registration No.: ChiCTR-TRC-12002561; URL: www.chictr.org. BODY.INTRODUCTION: Executive dysfunction, which refers to difficulties in working memory, attention, planning, response inhibition, mental flexibility, and/or self-monitoring, is a typical cognitive deficit associated with autism spectrum disorders (ASD). Of these executive dysfunctions, response disinhibition and mental inflexibility are relatively more common in individuals with ASD [1]–[3]. It has also been suggested that executive dysfunctions accounts for typical autistic features, such as uncontrollable behavioral and emotional reactions, repetitive behaviors, strong need for sameness, restricted interests, and inappropriate social communication and interaction. As there is not effective pharmacological intervention for enhancing the executive control of emotions and behaviors in individuals with ASD, the majority of the interventions are primarily behavioral [4]–[5]. Despite the reported effectiveness of some of the behavioral interventions in reducing behavioral or emotional disturbances, the intervention programs tend to be very intensive and time consuming [6]–[7]. For instance, although applied behavioral analysis was found to be effective in reducing various autistic symptoms, it requires intensive training for at least 20 hours per week for at least two years [7]. Furthermore, such positive outcomes are less prominent for children with limited mental ability or more severe autistic symptoms, who are not able to follow the rules and requirements of the training [8]. In view of the limitations of the conventional interventions, some researchers have been exploring various alternative methods, such as massage [9]–[11], acupuncture [12]–[13], music therapy [14]–[16], and diet/nutrition [17]–[19]. Although some preliminary data supports the positive effects of these novel interventions, the treatment efficacies remain largely inconclusive [20]. Our research team has been studying the effects of a traditional Chinese Shaolin medical concept (called Chanyi) in reducing some of the executive dysfunctions in individuals with ASD for the past years [21]–[24]. The present study further examines one component of this intervention approach, i.e., the mind-body exercise, as a possible intervention to improve the self-control of children with ASD. According to the National Center for Complementary and Alternative Medicine (NCCAM) established by the US government, a mind-body intervention is defined as any intervention which “focuses on the interactions among the brain, mind, body and behavior, with the intent to use the mind to affect physical functioning and promote health”. The fundamental assumption of these interventions is that because the mind and the body are inter-connected, individuals have the internal ability to change their own thoughts and behaviors to enhance their mental and physical health. Mind-body interventions have long been used in China to improve both mental and physical health, and are now becoming increasingly popular in Western countries. Moreover, a range of scientific studies have examined the therapeutic effects of mind-body interventions on depression [25]–[26], anxiety [27], insomnia [28], chronic pain [29], and cardiovascular problems [30]. Although the existing studies have found encouraging empirical evidence for the treatment efficacy of mind-body interventions, the majority of the studies have examined adult or elderly populations. As a result, the effects and applicability of mind-body interventions on the pediatric population are relatively less understood. A few studies have explored the effects of relaxation training and mind-body interventions on the psychological and cognitive functions of children, and the results have so far been positive [31]–[38]. It should be noted that many of the mind-body techniques, such as meditation and Tai Chi, requires either a high degree of mental control or involves slow movements that might not be easily mastered by children with particular neurodevelopmental disorders. Nevertheless, the emerging positive outcomes of certain forms of mind-body training for normally developed children have provided insights into their potential effects on children with ASD. The present study investigated the efficacy of the mind-body exercise, Nei Yang Gong, which is one component of a newly developed mind-body intervention. The intervention, which is based upon a Chinese Chan tradition named Chanwuyi (i.e., Zen, martial arts and healing) from the Sanhuangzhai monastery [39], is also known as the Dejian Mind-Body Intervention (DMBI), as named after the Grand Master of Chanwuyi – Shi Dejian (a Shaolin monk). Recent empirical studies on this intervention have shown that it has positive effects in improving physical health, mood and cognitive function in community-dwelling adults [40], children with autistic/Asperger’s disorder [22]–[24], and individuals with brain damage [41] and depression [25]–[26]. While the previous studies examined the effects of the holistic approach of Chanyi, which includes psychoeducation, mind-body exercises and diet modification, the present study examined the effect of a single component of the model, the mind-body exercises, as a possible intervention for children with ASD. Somewhat like Tai Chi, Nei Yang Gong involves sets of slow movements that emphasize smooth, gentle, and calm movements. The basic principles and practices of Nei Yang Gong have been elaborated in two published books [39], [42] and on the website of a charity foundation (www.chanwuyi.org). The practice of Nei Yang Gong has two primary functions. First, it aims to foster self-awareness and mental self-control to help restore a calm and relaxed state. Second, it helps to reduce stress, increase flexibility of the limbs, and improve the circulation of Qi and blood. For example, the “shoulder relaxation” movement helps to relax the neck and the shoulders while improving self-control and self-awareness. It should be noted that Nei Yang Gong has been developed on the basis of the Chan medical model, which emphasizes the maintenance of a natural and relaxed attitude to achieve smooth circulation of Qi and blood. In this way, Nei Yang Gong differs from some of the other mind-body techniques, such as mindfulness and meditation, which require a high degree of conscious mental awareness and self-control. Given its required “natural” attitude, it is anticipated that Nei Yang Gong will be easily mastered by and feasibly applied to children with ASD. Our hundreds of clinical cases of children with neurodevelopmental disorders reveal that even children with moderate mental retardation are able to learn and benefit from Nei Yang Gong. Our previous randomized controlled study revealed the possible neural mechanism underlying the therapeutic effect of Nei Yang Gong [43]. It was found that regular practice of Nei Yang Gong was able to foster a simultaneously relaxed and attentive brain state, as reflected in increased electroencephalographic (EEG) alpha asymmetry and intra- and inter-hemispheric EEG theta coherence indices. In contrast, this altered brain activity was not found among the participants of the comparison group, who practiced Progressive Muscle Relaxation (PMR). As relaxation and attentiveness are both significant factors for improving performance, Nei Yang Gong can be regarded as both a cognitive enhancement method and a relaxation exercise. Therefore, it is anticipated that the children who practice Nei Yang Gong (i.e., the experimental group) will show greater improvement in self-control (as measured by standardized neuropsychological tests) and greater reduction in related behavioral and social problems (as measured by parental reports on daily behaviors and social communication) than children who practice PMR (i.e., the control group). Derived from the work of Jacobson [44], PMR is a conventional and well-established behavioral treatment technique. It was selected for the comparison group because of the repeated empirical support for the effectiveness of PMR in alleviating anxiety, physiological arousal, and behavioral disturbances [45]–[47]. Furthermore, given the concreteness and simplicity of PMR, it can be applied to children with ASD at a younger age and with a lower functioning level. Scientific evidence supports the applicability and effectiveness of PMR in reducing aggression, increasing self-control and inducing relaxation in children as young as age 6 [48], [49] and in children with learning disabilities [50], [51]. Moreover, a few studies have reported that PMR alone or as a component of Cognitive Behavioral Therapy has positive effects in reducing anxiety and disruptive behavior of individuals with ASD [52]–[54]. The present study also compared the neural activity patterns of the two groups as measured by EEG assessment during an inhibitory control task (i.e., a Go/No-Go task) to explore the possible underlying neurological mechanism relating to improved self-control. The EEG test was adopted because it is non-invasive, practical, and easy to administer to children with ASD who have difficulty sitting still. The source of the scalp-EEG activity was localized using the standardized low-resolution brain electromagnetic tomography (sLORETA) analysis method [55] with the anterior cingulate cortex (ACC) as the region of interest. The ACC plays a significant role in inhibitory control [56]–[58] and reduced activity levels in the ACC have been found in children with ASD when performing an inhibitory task [59]–[61]. Accordingly, it is anticipated that if the children practicing Nei Yang Gong show improvement in self-control, then the source activity levels in the ACC during an inhibitory control task will be elevated, whereas this neurophysiological change will not be found in those practicing PMR. BODY.MATERIALS AND METHODS: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. BODY.STUDY DESIGN: The study was designed as a randomized controlled parallel trial. The recruited children with ASD were randomly and equally assigned into the experimental group (who received training on Nei Yang Gong), or the control group (who received training on Progressive Muscle Relaxation). Their ability to exercise self-control was measured before and after the one month of intervention. The progress of the participants throughout the trial is depicted in Figure 1. 10.1371/journal.pone.0068184.g001Figure 1BODY.CONSORT FLOW DIAGRAM.: The diagram shows the progress of the participants through the enrollment, allocation, follow-up, and data analysis phases of the trial. BODY.ETHICS STATEMENT: This study was conducted in accordance with the Helsinki Declaration of the World Medical Association Assembly. The research protocol was approved by the Human Subjects Ethics Sub-committee (HSESC) of the Hong Kong Polytechnic University (Ref. No.: HSEARS20120517001) and the Chinese Clinical Trial Registry (Registration No.: ChiCTR-TRC-12002561). Written consent was obtained from the parent of each participant prior to the commencement of the study. BODY.PARTICIPANTS: Forty-eight children with ASD aged between 6 and 17 voluntarily participated in the study with their parents’ written consent. Prior to the recruitment, the a priori sample size was estimated based on the effect size (0.7) of our previous research on this Chinese mind-body intervention for autism; with α = 0.05, power = 0.8, and the usual attrition rate in our previous studies of about 25%, the minimum required samples in each group was 24. The children were recruited from three primary schools and one secondary school in Hong Kong using an online advertisement and application platform, and the existing database at the Neuropsychology Laboratory of the Chinese University of Hong Kong. Except for two individuals, all participants received formal diagnosis of Autistic Disorder or Pervasive Developmental Disorders Not Otherwise Specified (PDD-NOS) by a clinical psychologist through a standard clinical interview with their parents based on the DSM-IV-TR criteria [62]. The clinical psychologist also assessed the severity of each child’s autistic symptoms in relation to social interaction, communication and repetitive/stereotypical behaviors using the Autism Diagnostic Interview-Revised (ADI-R) [63]. The interview included detailed questions about a child’s early development and current functioning, with higher scores indicating more severe autistic symptoms. Children with other neurodevelopmental, psychiatric or neurological comorbidities, or were on prescribed psychiatric medication, were excluded from the study. The remaining 46 children were matched into pairs according to their age and level of intelligence. The individuals in each matched pair were then randomly assigned into the experimental or the control group with equal probability using the drawing lots method. The random assignment was performed by a research assistant who was blind to the experimental design. Before the end of the intervention, six children in total dropped out of the study, leaving 20 children each in the control and experimental groups. Table 1 presents the demographic and clinical characteristics of the two groups. The children in both groups were matched on age, t(38) = 1.01, p = 0.32, gender, χ2(1) = 1.11, p = 0.29, and severity of autistic symptoms as measured by the four ADI-R subscales, with t ranging from −0.80 to 0.97, and p ranging from 0.34 to 0.87. The two groups also demonstrated comparable levels of general intelligence, t(38) = 0.36, p = 0.72, which was assessed by the research assistant using the short form of the Chinese version of the Wechsler Intelligence Scale for Children-Fourth Edition (Hong Kong) (WISC-IV(HK)) [64] or the Stanford–Binet Intelligence Scale–Fourth Edition (SB-FE) [65] for non-verbal children and those who showed a floor effect in the WISC-IV(HK). Six children (i.e., 30% of the participants) in each group had limited intelligence with an IQ score below 70. 10.1371/journal.pone.0068184.t001Table 1BODY.BASELINE DEMOGRAPHIC AND CLINICAL CHARACTERISTICS OF PARTICIPANTS IN THE CONTROL AND EXPERIMENTAL GROUPS.: Characteristics Control Group Experimental Group t/ χ2 P value (n = 20) (n = 20) Age, years 12.42 (3.25) 11.28 (3.90) 1.01 0.32 Gender-Male (%) 85.00 95.00 1.11 0.29 IQ 80.50 (18.51) 78.35 (18.85) 0.36 0.72 Diagnosis 0.17 0.68 Autistic Disorder (%) 100 85 3.24 0.07 PDD-NOS (%) 0 15 Severity of Disorder ADI-R Social Interaction 24.60 (4.64) 23.20 (4.48) 0.97 0.34 ADI-R Communication 18.65 (4.06) 19.20 (4.25) −0.42 0.68 ADI-R Stereotyped Behavior 6.60 (2.60) 6.45 (2.94) 0.17 0.87 ADI-R Abnormal <36 months 3.15 (1.73) 3.60 (1.82) −0.80 0.43 Notes. ADI-R = Autism Diagnostic Interview-Revised; IQ = Intelligence quotient as assessed by the Chinese version of the Wechsler Intelligence Scale for Children-Fourth Edition (Hong Kong) or the Stanford-Binet Intelligence Scale-Fourth Edition; PDD-NOS = Pervasive Developmental Disorders Not Otherwise Specified. Standard deviations are in parenthesis. BODY.PROCEDURES: Prior to the baseline assessment, the children and their parents were briefed on the assessment procedure and informed consents were obtained from the parents. The assessment was conducted at the Hong Kong Polytechnic University. The children were individually assessed in a quiet room by trained research assistants on their intellectual functioning, self-control, and scalp EEG activities. During the assessment, the children’s parents were interviewed by a clinical psychologist on their developmental and medical histories using a structured clinical interview. The research assistants who conducted the neuropsychological and EEG assessments were blinded to the rationale of the study and the group assignment. During the EEG assessm[66]. Details of the Go/No-go task are elaborated in the Measures section. The electrode impedances were maintained at ≤10 kΩ. The EEG signals were referenced to linked ears and sampled at 256 samples per second, with a high-frequency limit band pass of 30 Hz, and then fast Fourier transformed. Artifact-free EEG data were selected based on visual examination for eye movements and muscle artifacts and then captured for subsequent sLORETA analyses. The sLORETA method is a properly standardized discrete, three-dimensional (3D) distributed, linear, minimum norm inverse solution for exact and zero-error localization of the source of scalp EEG activity [55]. The baseline assessment was performed two weeks before the start of the intervention. After the baseline assessment, the two groups of children were provided with training on Nei Yang Gong and Progressive Muscle Relaxation, respectively, for one month. Two weeks after the intervention, the same set of assessments on self-control, daily behaviors and EEG activity were administered to the children and their parents. BODY.MEASURES: BODY.NEUROPSYCHOLOGICAL ASSESSMENTS ON SELF-CONTROL: The children’s ability to exercise self-control was assessed by the following three standardized neuropsychological tests, namely the Tower of London Test, the Children’s Color Trails Test and the Five Point Test: The Tower of London Test – Drexel Version (TOLDX) [67] is designed to test the well-controlled and flexible execution of goal-directed behavior. It consists of ten items that involve moving three colored beads on three vertical pegs to match a target arrangement while adhering to the testing rules. The test is suitable for use with children aged 7 years and above [67], although it has been used in studies with children aged as young as 4 [68]. The recipient’s self-control is measured by the total number of rule violations, with fewer violations indicating better self-control. The degree of impulsivity is indicated by the initiation time, which is measured in terms of seconds, with a longer duration reflecting less impulsivity. The second trial of the Children’s Color Trails Test (CCTT-T2) [69] was adopted to test inhibitory control and flexibility in shifting mental processes and motor responses. The CCTT is designed for children aged 8 to 16 years, although some normative data are available for children aged 5 to 7 years [70]. The test involves sets of duplicate numbers embedded within pink and yellow circles, and requires the child to connect the numbers in ascending order from 1 to 15 while alternating between the two colors as quickly as possible. The completion time (in seconds) is used as a measure of inhibitory and flexible control, with a shorter duration reflecting better control. The Five Point Test (FPT) [71] is a figural fluency test that was adopted to test flexible mental control, as it requires the spontaneous generation of novel designs without repetition by connecting five points with straight lines within 5 minutes. The test is suitable for children aged 6 and above [70]. The total number of unique designs generated is measured, with higher amounts indicating better self-control and greater flexibility. BODY.PARENTAL EVALUATION ON BEHAVIORAL MEASURES: The parents were interviewed on their child’s behavioral changes in daily life using standardized questionnaires. As problems with self-control have been found to be associated with typical autistic symptoms (e.g., repetitive/disinhibitory behaviors, communication and socialization problems), the parents were asked to rate their child’s autistic symptoms based on the Autism Treatment Evaluation Checklist [72] before and after the intervention. The ATEC was selected because it is a simple one-page evaluation that has been empirically found to be a sensitive measure for evaluating treatment effectiveness for individuals with ASD [73]–[75]. Moreover, some preliminary evidence suggests that it is able to monitor the progress of children with ASD with high internal consistency [76]. The ATEC consists of four subscales: (1) speech/language communication; (2) sociability; (3) sensory/cognitive awareness; and (4) health/physical behavior. The total scores of the four subscales were used for the pre-post comparison of each group. The parents were also asked to answer another questionnaire containing items about their child’s ability to exercise self-control, including the control of his/her temper tantrums and rigid thoughts/acts, and their difficulty in verbal expression. This questionnaire was used in our previous study to evaluate the effect of the Chan-based diet modification in improving the self-control of children with ASD [23]. The parents’ rating on this questionnaire was consistent with their rating on the standardized ATEC. Given that the rationale of the present study was to examine the treatment effect on self-control, it was anticipated that the children would show improvement in self-control, but not in verbal expression. The questionnaire asked the parents to evaluate the degree of change in each problem behavior after the one-month intervention on a scale of “−5” to “+5”, where “−5” indicates a “large reduction in problem behavior”, thereby indicating a large degree of improvement; “+5” indicates a “large increase in problem behavior”, thereby indicating a large decline; and “0” indicates “no change”. The mean rating obtained from each group was used for the subsequent between-group comparisons. BODY.EVENT-RELATED EEG ASSESSMENT: The event-related EEG signals for each child were collected during an inhibitory control test, namely, the Go/No-Go task. The Go/No-Go task is a computerized test that measures the respondent’s ability to flexibly respond to changing stimuli and inhibit unwanted responses. A total of 192 black balls and 48 red balls (black:red ratio = 4∶1) were randomly displayed, one at a time, for 500 ms followed by 1000 ms of blank intervals, in the center of a computer screen. The child was required to press a key as quickly as possible in response to a black ball (Go stimulus), but to inhibit their response when a red ball (No-go stimulus) appeared. The total testing time was 6 minutes. As the No-go condition required the inhibition of an unwanted response (i.e., pressing the key), the EEG data collected during this condition were selected for subsequent sLORETA analysis of the inhibition-related neural activity. The EEG data for each child was first transformed using Excel before being imported into EEGLAB software using MatLab 7.1 to capture the correct events and epochs. The epoch limits were set as 50ms as the start and 900 ms as the end. Artifacts in the epoched data were then pruned by visual inspection and the rejection method on the EEG Plot. The selected data were exported and then spectrally processed by fast Fourier transformation (FFT) for computation of the power data in the theta band (4–7.5 Hz) through the use of the NeuroGuide software. As the ACC has been reported to be one of the generators of theta activities in the human brain [77]–[79] and to play a major role in inhibitory control, sLORETA [55] was thus adopted to localize the sources of the theta activities in response to the “No-go” condition. The sources of the theta activities were expressed as the three-dimensional cortical current density according to the Montreal Neurological Institute (MNI) brain coordinates. BODY.INTERVENTION: :Nei Yang Gong vs. Progressive Muscle Relaxation The children in the experimental and control groups attended training classes at the Chinese University of Hong Kong twice per week for four weeks, with each session lasting for an hour. The two training classes were administered on the same days of a week. The children in the experimental group were taught to practice Nei Yang Gong by a clinical psychologist, with over 10 years clinical experience in training children with autism. The Nei Yang Gong set comprised five types of movement: tranquil stand, shoulder relaxation, nasal bridge massage, Qi-circulating movement, and passive Dan Tian breathing. The movements were arranged in a fixed sequence and incorporated with specific pieces of music to facilitate the children’s mastery of the technique and to keep them engaged. While practicing Nei Yang Gong, the children were guided to move calmly and relaxingly, and were encouraged to persist with the movements. To foster self-awareness and self-control, the children were also encouraged to practice some forms of Nei Yang Gong that served as self-guided massages for relaxing and calming oneself whenever they feel distressed and frustrated, e.g., rolling their hands slowly up and down between the chest and the abdomen, resting their hands on their abdomen while quietly observing their breathing. The selected Nei Yang Gong movements involved simple bodily actions (e.g., moving hands/fingers up and down, and bending the knees) and the children were only asked to perform the movements in a relaxed and natural manner. Five years of clinical observation suggested to the research team that even children with moderate grade mental retardation would be able to learn and practice Nei Yang Gong. Each child was closely monitored by the clinical psychologist during each session to ensure that all the children were able to master the technique. As the intervention progressed, the children were observed to become less agitated and to move at a slower pace when practicing Nei Yang Gong. Each round of Nei Yang Gong with music lasted for 5 minutes. The children were encouraged to practice the movements daily one to three times. The children’s practicing times and frequencies were recorded in log booklets, which indicated that 65% of the children practiced Nei Yang Gong at least 6 days per week for between 5 and 45 minutes a day (mean = 19.38; SD = 11.82). The remaining children practiced 5 to 25 minutes a day, 3 to 5 days per week (mean = 13.66; SD = 6.12). Given that the physical conditions vary among children, they were instructed to stop doing the exercise until they began sweating to avoid exhaustion. Thus, the duration of practice was not fixed. The children in the control group were taught the Progressive Muscle Relaxation (PMR) technique by another clinical psychologist with over a decade of experience in administering PMR to pediatric population. The Chinese version of PMR for children was adopted. This version was locally developed by the Clinical Psychology Division of the Hong Kong Psychological Association, and has been used in pediatric clinical health since 2004. Clinical experience and empirical evidence [50], [51] suggested that even children with mental retardation would be able to master the PMR technique. In each training session, the therapist guided the children in sequentially tensing and relaxing seven muscle groups (nose, mouth, shoulders, arms, hands, chest and feet) as instructed by a sound track. To facilitate the learning of the ASD children, during the practice, visual and verbal cues were provided on how to contract and relax each muscle group (e.g, the tensing-relaxing of the arm muscle was represented by an image of a monkey swinging from one tree to another printed on a cue card together with the voice of a monkey presented on the sound track). The therapist monitored the progress of each child and observed that all children were able to master the technique. Each round of PMR lasted about 20 minutes. In addition to the PMR technique, other behavioral-based training techniques on social skills, emotional awareness and behavioral monitoring were incorporated into the training program. After the first training session, the children were given a cue card that listed the seven steps of the PMR technique for home practice. In each subsequent training session, any difficulties the children encountered during their home practice were reviewed. Half the children practiced PMR once (i.e. 20 minutes) a day at least 6 days a week, while the other half practiced between 1 and 5 days per week. The average practice duration of the control and experimental groups was similar, t = −1.28, p = 0.21. BODY.DATA ANALYSES: The mean performance of each group on the self-control neuropsychological test measures and the mean parental rating on the ATEC at pre- and post-training were compared using repeated measures ANOVAs and then followed by post hoc paired sample t tests. The change in mean performance/rating in each group was also computed for between-group comparison using independent sample t tests. To examine the effects of the two types of training on neural activity, the regions of interest (ROI) analysis of the sLORETA method was used to localize the source of the scalp-EEG activity and to compare within and between groups using voxel-by-voxel paired sample t tests and independent sample t tests respectively. The sLORETA comparisons were computed with subject-wise normalization and log transformation for each group to compare the pre-post changes in the sources of theta activity in the ACC during the “No-go” condition. Because specific hypotheses were tested, no adjustment to the alpha level was applied for the planned comparison to avoid lowering the power of the tests. BODY.RESULTS: BODY. :Nei Yang Gong Improves the Self-Control Test Performance Repeated measures ANOVAs were performed separately for each neuropsychological test measures to compare the effects of Nei Yang Gong and PMR in enhancing the self-control of ASD children. At the baseline, the levels of self-control in both groups were comparable, as reflected by the three neuropsychological test measures, which showed t ranges from −1.49 to 1.5, and p ranges from 0.15 to 0.72. The results of the repeated measures ANOVAs showed that there were significant and marginally significant Time (Pre vs Post) by Group (Control vs Experimental) interaction effects on the two indices of the TOLDX, i.e., the frequency of rule violation, F(1,34) = 6.02, p = 0.02, and the initial time, F(1,34) = 3.25, p = 0.08. For the other two measures, there was a significant main effect of Time (CCTT-T2: F(1,33) = 13.23, p = 0.001, FPT: F(1,35) = 18.05, p = 0.00). Subsequent post hoc t tests confirmed that the four neuropsychological test measures were consistent, indicating that the autistic children in the experimental group showed better self-control than those in the control group after the one-month intervention (see Table 2). Although both groups showed improvement in reducing the frequency of rule violation in the TOLDX, the mean reduction of the experimental group (−8.53) after the intervention was about four times that of the control group (−2.82), and the group difference was statistically significant with a large effect size (0.84), t(34) = 2.45, p = 0.019. Furthermore, the experimental group, but not the control group, became less impulsive in problem-solving, as reflected by their significantly increased average initial time in attempting the TOLDX questions [experimental: t(18) = −3.65, p = 0.002, effect size = 0.86; control: t(16) = −0.58, p = 0.57, effect size = 0.14]. The reduction in impulsivity of the treatment group was greater than that of the control group, with a medium effect size (0.77), t(33) = −2.27, p = 0.03. In regard to the CCTT-T2, the children in the experimental group showed a significant reduction in completion time (mean reduction = −17.29) with a large effect size (0.83), t(17) = 3.53, p = 0.003, while the children in the control group did not show any significant improvement (mean reduction = −6.97), t(16) = 1.71, p = 0.11, effect size = 0.41. Although the between-group difference in the mean reduction of completion time did not reach statistical significance, t(33) = 1.61, p = 0.12, the effect size (0.55) is at the medium level. Similarly, children in both the control group and the experimental group generated significantly greater numbers of unique designs in the FPT after the intervention (mean increment: control group = 4.11 and experimental group = 5.11), t(17) = −2.69, p = 0.015 and t(18) = 3.50, p = 0.003, respectively. Yet, the change in the experimental group was relatively more robust (a large effect size of 0.80) than that in the control group (a medium effect size of 0.63). In sum, the children who received training in Nei Yang Gong showed more noticeable improvement in self-control than the children in the control group across all the neuropsychological tests. 10.1371/journal.pone.0068184.t002Table 2BODY.MEAN PERFORMANCE AND DIFFERENCE SCORE ACROSS THE NEUROPSYCHOLOGICAL TESTS ON SELF-CONTROL OF THE CONTROL AND EXPERIMENTAL GROUPS AT PRE- AND POST-ONE-MONTH INTERVENTION.: Control Group Effect 95% C.I. p value Experimental Group Effect 95% C.I. p value (n = 19) Size (n = 20) Size Pre Post Diff Pre Post Diff TOLDX# Rule Violation 3.88 1.06 −2.82 1.11++ 1.52–4.13 0.00** 14.37 5.84 −8.53 0.92++ 4.06–12.99 0.00** (3.30) (2.16) (2.49) (13.22) (7.07) (9.27) Initial Time 15.48 16.10 0.62 0.14 −2.89–1.65 0.57 10.73 14.92 4.19 0.86++ −6.62 – −1.77 0.00** (12.29) (9.36) (4.41) (5.36) (6.56) (4.87) CCTT-T2# Completion Time 59.38 52.41 −6.97 0.41 −1.68–15.63 0.11 76.61 59.32 −17.29 0.83++ 6.95–27.63 0.00** (24.76) (21.71) (16.83) (41.22) (34.26) (20.79) FPT Unique Designs 15.33 19.44 4.11 0.63+ −7.33 – −0.89 0.02* 14.05 19.16 5.11 0.80++ −8.17 – −2.04 0.00** (12.43) (11.67) (6.48) (8.63) (10.31) (6.37) Note. C.I. = Confidence Interval; Diff = average of difference score by subtracting pre-training score from post-training score; TOLDX = The Tower of London Test – Drexel Version; CCTT-T2 = Trial 2 of the Children’s Color Trails Test; FPT = Five Point Test. Standard deviations are in parenthesis. #Lower value indicates better performance; * p<0.05, ** p<0.01; ++large effect size, +medium effect size. BODY. :Nei Yang Gong Reduces Self-Control Related Daily Behavioral Problems While the children who learned Nei Yang Gong demonstrated better performance in the laboratory tests of self-control, we then further analyzed their ability to exercise self-control in everyday life. The parents were interviewed using the ATEC on the common autistic symptoms before and after the intervention, and were given a questionnaire rating the treatment effect in controlling temper and obsessive behaviors, and verbal expression difficulty after intervention. The results of the repeated measures ANOVAs showed that there were significant main effects of Time on the sociability subscale, F(1,37) = 14.25, p = 0.001, and the health/physical/behavior subscale, F(1,37) = 4.74, p = 0.04, and a marginally significant main effect of Time on the sensory/cognitive awareness subscale, F(1,37) = 3.20, p = 0.08. Given the significant main effects of the three measures, the post hoc tTable 3 presents the pre-post comparisons on the subscales of the ATEC. The baseline level of the mean rating across all subscales is comparable between the two groups, with t ranging from 0.68 to 1.48, and p ranging from 0.15 to 0.50. After intervention, parents of the experimental group reported significant improvement in three subscales: sociability, t(19) = 3.06, p = 0.006, effect size = 0.68; sensory/cognitive awareness, t(18) = 2.11, p = 0.049, effect size = 0.49; and health/physical/behavior, t(18) = 2.87, p = 0.01, effect size = 0.66. However, the children in the control group were rated as showing significant improvement mainly in the sociability subscale, t(18) = 2.53, p = 0.02, effect size = 0.58. As expected, as the two intervention programs aimed to improve self-control, neither groups showed any change in the speech/language/communication subscale, as revealed by the non-significant results of the F test, F(1,37) = 0.01, p = 0.92. These results suggest that the mind-body exercise had a positive effect on the cognition and health of children with ASD, which may not be achieved by conventional behavioral intervention. 10.1371/journal.pone.0068184.t003Table 3BODY.MEAN PARENTAL RATING ON THE AUTISM TREATMENT EVALUATION CHECKLIST (ATEC) OF THE CONTROL AND EXPERIMENTAL GROUPS AT PRE- AND POST-ONE-MONTH INTERVENTION.: Control Group Effect 95% C.I. p Experimental Group Effect 95% C.I. p (n = 19) Size value (n = 20) Size value Pre Post Pre Post Speech/Language/ 6.53 5.68 0.27 −0.70–2.39 0.27 5.80 5.05 0.29 −0.46–1.96 0.21 Communication (3.39) (2.43) (3.25) (2.65) Sociability 17.53 14.63 0.58+ 0.49–5.30 0.02* 15.55 13.50 0.68+ 0.65–3.45 0.01* (6.28) (6.68) (6.18) (5.91) Sensory/Cognitive 12.32 11.58 0.20 −1.06–2.53 0.40 10.63 9.21 0.49 0.01–2.83 0.05* Awareness (5.28) (5.37) (5.51) (4.91) Health/Physical/ 19.16 17.11 0.26 −1.72–5.83 0.27 15.00 12.26 0.66+ 0.73–4.74 0.01* Behavior (9.46) (11.89) (7.78) (6.29) Note. C.I. = Confidence Interval; Standard deviations are in parenthesis. Lower value indicates less severe problems; * p<0.05, ** p<0.01; +medium effect size. The results of the parents’ evaluation of the changes in their child’s temper outbursts, obsessive behaviors and verbal expression problem, are also consistent with the above-mentioned findings. With the temper outburst scale, the experimental group reported a significantly greater reduction (−1.63) than the control group (−0.07), with a large effect size (0.86), t(28) = 2.36, p = 0.026 (see Fig. 2). With the obsession scale, the experimental group reported a mean reduction of 1.43 and the control group a reduction of 0.56, while difference was only marginally significant, t(28) = 1.90, p = 0.068, it had a medium effect size (0.69). With the verbal expression problem subscale, the experimental (−0.77) and control (−0.79) groups both showed no obvious change, t(28) = 0.35, p = 0.97, effect size = 0.02. Thus, the parents’ reports are consistent with the neuropsychological assessments that indicate that the mind-body exercise appeared to have some positive effects in improving the self-control of the ASD children. As expected, the children did not show any improvement in language expression as it was not the target of the training exercises. Again, the parental reports on this specific treatment effect cohered with the neuropsychological test results and our anticipated results, suggesting that the parents’ ratings were not biased (e.g., reporting exaggerated positive effects). 10.1371/journal.pone.0068184.g002Figure 2BODY.PARENTAL RATING OF THE TREATMENT EFFECTS AFTER ONE-MONTH TRAINING.: The treatment effects as rated by the parents in controlling the children’s temper outburst and obsessive behaviors, and verbal expression problem in daily life after one-month training. A positive value indicates improvement in the corresponding problem. * p<0.05 (independent sample t test). In addition to the positive effects, no parent reported any adverse effect of the two interventions during the study. Although some children may have experienced fatigue while practicing Nei Yang Gong, they were advised to stop whenever they felt tired or discomfort, and to resume practice after taking a rest. BODY. :Nei Yang Gong Enhances Brain Activity in the Anterior Cingulate Cortex Further pre-post comparisons of the EEG activity of the groups during the Go/No-go task (a common test of inhibitory control) were performed using the sLORETA method. The No-go condition, which requires the inhibition of a behavioral response, was selected for analysis. The ACC was set as the region of interest because it has been found to mediate inhibitory control [56]–[58] and to be hypo-active in individuals with ASD [59]–[61]. The effects of Nei Yang Gong and PMR in enhancing ACC activity during the No-go condition were compared using the sLORETA voxel-by-voxel paired t statistics. At the baseline, the results of the voxel-by-voxel independent t statistics show comparable ACC activity levels between the two groups during the No-go condition, t = 0.76, p = 0.45. After one-month intervention, the experimental group showed significantly elevated activity in the rostral ACC region (Brodmann areas 24) during the No-go condition, t = 0.30, p = 0.02 (circled region in the right-side image in Fig. 3). Treatment-induced activity elevation in this ACC region has been previously found to indicate the level of hypoactivity in children with ASD as compared with normally developed children [60]. In contrast, the control group did not show any significant change in activity in the ACC, t = −0.25, p = 0.81 (left-side image in Fig. 3). The enhanced ACC activity in the experimental group indicates that the ACC may be the neural mechanism underlying the positive treatment effects of Nei Yang Gong on the self-control of ASD children. 10.1371/journal.pone.0068184.g003Figure 3BODY.NEURO-ELECTROPHYSIOLOGICAL ACTIVITY CHANGES AFTER ONE-MONTH TRAINING.: Graphical representation of the sLORETA paired t-statistics results comparing the pre- and post-one-month theta source activity of the control and experimental groups during the No-go condition of the Go/No-go task. The regions colored in red indicate significantly elevated ACC activity (in the circle) after one-month Nei Yang Gong training (experimental group) at p<0.05, which was not observed after practicing the Progressive Muscle Relaxation (control group). BODY.DISCUSSION: Self-control problems, such as emotional outburst, repetitive or obsessive thoughts or acts, and impulsivity, are common behavioral manifestations in children with ASD. These problems hinder learning and social development, and increase caregiver burden. The present findings supported the positive effects of the Chinese Chan-based mind-body exercise, Nei Yang Gong, in enhancing self-control and reducing the typical autistic symptoms and daily emotional and behavioral problems of children with ASD. A one-month Nei Yang Gong training program, totaling 8 one-hour sessions, was able to elicit robust treatment effects that were not observable for the other group receiving a conventional behavioral training technique. One encouraging finding of the present study is that this exercise can be mastered by children with limited language ability and intellectual functioning. Because of the simplicity and effectiveness of this Chinese mind-body exercise, it can be considered as an alternative or complementary intervention for improving executive control in children with ASD. The potential benefits of the mind-body exercise in promoting increased self-control found in the present study are consistent with previous empirical studies on mind-body interventions. For instance, Bahrami et al. [31] reported a 42% reduction in stereotypic behavior in ASD children after 14 weeks of Kata techniques (a form of mind-body exercise) training compared to the no-exercise control group, which was sustained at one-month follow-up. Another study [37] also reported significantly reduced behavioral problems among 24 children with ASD after 8-weeks movement-based therapy (with yoga as one of the treatment components). A recent review article in Science [80] concluded that traditional martial arts (a form of mind-body exercise) improved executive control to a greater extent than standard physical education [36] or modern martial arts [81] for children/adolescents with executive function deficits. The effectiveness of the traditional martial arts may be related to their concurrent emphasis on physical training, self-control, discipline, and character development (e.g., respect, responsibility, and perseverance), which tend to focus more on the executive function. Similarly, as the Chinese mind-body exercise adopted in the present study was developed based upon the principles of Chinese Chan and Buddhism, the children were guided to practice with a peaceful mind and to relieve anger and distress. This may explain the distinctive effects of the exercise in enhancing self-control. The underlying mechanism that may explain the change in the participants’ self-control and daily behaviors was indicated by the change in EEG activity as analyzed by the sLORETA method. The findings show that the children who practiced Nei Yang Gong for one month had significantly increased electrophysiological activity in the rostral ACC region, whereas those who practiced PMR did not show such elevation. Studies have been repeatedly reported that children with ASD show hypoactivity in the ACC during response-monitoring and inhibitory tasks (e.g., the Go/No-go task) as compared to normally developed children [59]–[61]. The enhanced ACC activity while performing an inhibitory task observed among the children who completed one-month Nei Yang Gong practice suggests that the behavioral improvement of children with ASD may be associated with increased ACC activity. In fact, the effect of Chan-based mind-body exercise on the brain was explored in our previous randomized controlled study [43]. This study found that individuals who practiced mind-body exercises were able to foster a relaxed and attentive brain state as reflected by their altered EEG indices, whereas those practicing PMR did not show any alteration in brain activity. A relaxed and attentive brain state is crucial for achieving peak performance and exercising good self-control. Nevertheless, how mind-body exercises change and increase the activity in the neural system warrants further investigation. The therapeutic effects of Nei Yang Gong on brain functions and activity observed in the present study are in line with our other clinical observations and empirical findings in recent years. For instance, two case studies, one on a child with autism and mental retardation and another on an adolescent with Asperger’s disorder, reported significantly improved inhibitory control of emotional and behavioral disturbances after DMBI, with Nei Yang Gong as one of the treatment components [22], [24]. A recent randomized controlled study of the Chan-based mind-body intervention through dietary modification on children with ASD also showed significant enhancement in inhibitory control and elevation in ACC activity after one-month treatment [23]. Other positive outcomes on emotional control, cognitive functions (e.g., attention), and frontal brain activity were also observed in three randomized controlled trials on patients with major depressive disorders and community-dwelling adults after DMBI [25]–[26], [40]. Thus, the results of the present study shed further light on the potential application of Chinese Chan-based mind-body exercise as a complementary intervention for the rehabilitation of individuals with ASD and other emotional and cognitive problems. The results of the present study indicate that the one-month Chinese mind-body exercise had positive effects in improving the self-control and alleviating the autistic symptoms of children with ASD. However, the long-term effects of the exercise and the sustainability of the treatment effects remain unknown and are worth further investigation. This study also explored the treatment effect of a single component (mind-body exercise) of the DMBI. Our previous study reported a similar positive outcome for another component (diet modification) of the DMBI in improving the self-control of children with ASD [23]. Thus, future studies could investigate whether the use of both treatment components could have additive treatment outcomes when applied to individuals with ASD. Because self-control problems are commonly observed in patients with other brain disorders (e.g., attention-deficit/hyperactivity disorder, traumatic brain injury, and dementia), it may also be worth extending the investigation of the effects of mind-body exercise to these clinical populations. Last but not least, given the increasing research interest in mind-body interventions (e.g., yoga) for improving health in Western countries, future studies could also investigate the applicability and effectiveness of this Chinese Chan-based mind-body exercise in Caucasian populations. BODY.CONCLUSIONS: In summary, this study provides evidence that one month of training in the Chinese Chan-based mind-body exercise, Nei Yang Gong, had a positive effect in enhancing the self-control of children with autism spectrum disorders. This cognitive enhancement coincided with significantly elevated brain activity in the anterior cingulate cortex of the Nei Yang Gong group participants. In contrast, this improved brain functioning and elevated brain activity was not observed in the children practicing progressive muscle relaxation. This encouraging finding confirms the potential clinical applicability of this Chinese mind-body exercise in enhancing the self-control of individuals with various brain disorders. BODY.SUPPORTING INFORMATION: Checklist S1CONSORT checklist. (DOC) Click here for additional data file. Protocol S1Trial protocol. (DOC) Click here for additional data file.

TITLE: CHADS2 and CHA2DS2-VASc score to assess risk of stroke and death in patients paced for sick sinus syndrome ABSTRACT.OBJECTIVE: The risk of stroke in patients with atrial fibrillation (AF) can be assessed by use of the CHADS2 and the CHA2DS2-VASc score system. We hypothesised that these risk scores and their individual components could also be applied to patients paced for sick sinus syndrome (SSS) to evaluate risk of stroke and death. ABSTRACT.DESIGN: Prospective cohort study. ABSTRACT.SETTINGS: All Danish pacemaker centres and selected centres in the UK and Canada. ABSTRACT.PATIENTS: Risk factors were recorded prior to pacemaker implantation in 1415 patients with SSS participating in the Danish Multicenter Randomized Trial on Single Lead Atrial Pacing versus Dual Chamber Pacing in Sick Sinus Syndrome (Danpace) trial. Development of stroke was assessed at follow-up visits and by evaluation of patient charts. Mortality was assessed from the civil registration system. ABSTRACT.INTERVENTIONS: Patients were randomised to AAIR (N=707) or DDDR pacing (N=708). ABSTRACT.MAIN OUTCOME MEASURES: Stroke and death during follow-up. ABSTRACT.RESULTS: Mean follow-up was 4.3±2.5 years. In the AAIR group 6.9% patients developed stroke versus 6.1% in the DDDR group (NS). There was a significant association between CHADS2 score and the development of stroke (HR 1.41; 95% CI 1.22 to 1.64, p<0.001). CHA2DS2-VASc score was also significantly associated with stroke (HR 1.25; CI 1.12 to 1.40, p<0.001). CHADS2 score (HR 1.46; CI 1.36 to 1.56, p<0.001) and CHA2DS2-VASc score (HR 1.39; CI 1.31 to 1.46, p<0.001) were associated with mortality. Results were still significant after adjusting for AF and anticoagulation therapy. ABSTRACT.CONCLUSIONS: CHADS2 and CHA2DS2-VASc score are associated with increased risk of stroke and death in patients paced for SSS irrespective of the presence of AF. BODY.INTRODUCTION: Stroke is one of the dominating causes of death and consumes a substantial part of the healthcare costs in the industrialised world. The predominant part (80%) of strokes is ischaemic including cases secondary to cardiac embolisms due to atrial fibrillation (AF).1 The risk of stroke in AF patients can be quantified by various scoring systems. The most commonly used scheme for stratifying risk of stroke is the CHADS2 (Congestive heart failure, Hypertension, Age≥75 years, Diabetes mellitus, previous Stroke/transient ischaemic attack (TIA) (double weight)) score which has a range 0–6. In low-risk patients recent guidelines have recommended use of the extended CHA2DS2-VASc (Vascular disease, Age 65–74 years, (female) Sex category) score which supplements the CHADS2 score by two additional items and an alternative scoring of age with doubled weight to age ≥75 years (range 0–9).2 Patients with sick sinus syndrome (SSS) and bradycardia are treated with cardiac pacing. Recently, the Danish Multicenter Randomized Trial on Single Lead Atrial Pacing versus the Dual Chamber Pacing in Sick Sinus Syndrome (the DANPACE trial) comparing AAIR and DDDR pacing in patients with SSS found no difference in mortality or occurrence of stroke between the two groups.3 Thromboembolic events occur with a higher rate in patients with SSS and AF is common in this patient population.3–6 Patients with SSS therefore may share the same risk factors for stroke as patients with known AF. Although the CHADS2 and CHA2DS2-VASc score systems were constructed to address stroke risk in AF patients these score systems may be useful in other groups of cardiac patients. We therefore hypothesised that for patients with SSS treated with pacemaker therapy, the risk of stroke and the risk of death could be assessed by applying the CHADS2 and CHA2DS2-VASc score. BODY.METHODS: BODY.STUDY DESIGN: The DANPACE trial has previously been described in detail.3 In brief, the trial randomly assigned 1415 patients with SSS to AAIR pacing or DDDR pacing. The criteria for inclusion were: symptomatic bradycardia; documented sinoatrial block or sinus-arrest with pauses >2 s or sinus bradycardia <40 bpm for more than 1 min while awake; PR interval ≥0.22 s if aged 18–70 years or PR interval ≥0.26 s if aged ≥70 years; and QRS width <0.12 s. The main exclusion criteria were: atrioventricular block; bundle branch block; long-standing persistent AF (>12 months) or permanent AF; AF with ventricular rate <40 bpm for ≥1 min or pauses >3 s; a positive test for carotid sinus hypersensitivity, planned cardiac surgery; or a life expectancy shorter than 1 year. Documented paroxysmal AF was not an exclusion criterion. Enrolment began in March 1999 and was terminated in June 2008. The trial was conducted in accordance with the Helsinki Declaration and approved by the regional Ethics Committee and the Danish Data Protection Agency. The study was registered in Clinical Trial Gov (NCT00236158). All patients gave written informed consent before inclusion. BODY.PATIENT FOLLOW-UP: Patients were clinically evaluated and pacemaker check was done after 3 months and then once every year after implantation until September 2009. In case of suspected thromboembolic events (stroke or TIA), supplementary information on hospital admissions, diagnosis of the event and degree of disability was collected from hospital files and general practitioners. Once every month, new deaths were identified by checking the study database against the Danish Civil Registration System. BODY.DEFINITION OF STROKE: Stroke was recorded in the study Case Report Form (CRF) using clinical evaluations. Stroke was defined as: sudden development of focal neurological symptoms lasting more than 24 h. Decision on diagnostic CT or MRI scans was left to the discretion of the physician treating the patient, typically general practitioners, specialists in internal medicine or neurologists. Stroke endpoints were evaluated by an independent endpoint committee. BODY.STATISTICAL ANALYSIS: The hypotheses of the current study were established prior to data analysis. Time to first stroke and time to death were analysed using Cox proportional hazards regression. Following the lines from the primary DANPACE publication a univariate analysis of each prespecified variable was performed. Furthermore, multivariate analysis including all significant univariate variables was performed. When oral anticoagulation (OAC) treatment was used as a time-dependant covariate, the latest known value for a given patient (OAC or no OAC) at a given time (as opposed to the baseline value) was used in the Cox calculations to find the model coefficients. C statistics was calculated using Harrels's C of concordance. Relative risk was expressed as HR with 95% CI. Statistical tests were two-tailed, and p<0.05 was considered statistically significant. Statistical analysis was performed using Stata V.11 (StataCorp. 2009, College Station, Texas, USA) and BMDP release V.8.1 (Statistical Solutions Ltd, Ireland). BODY.RESULTS: BODY.POPULATION: A total of 1415 patients were included in the analysis. Of these, 708 patients were randomised to the DDDR group. Baseline characteristics of patients are presented in table 1. Of the 1415 patients randomised in the DANPACE study 1392 patients were followed up at a total of 7496 follow-up visits. Mean follow-up time until stroke or censoring was 4.3 years (SD 2.5 years), that is, 6075 patient-years of follow-up. Mean follow-up time until death, or end of study was 5.4 years (SD 2.6 years) which comprises 7643 patient-years of follow-up. At the time of randomisation, 623 of the patients had a history of AF and 197 received OAC. Table 1Baseline clinical characteristics of the patients Clinical characteristicsAAIR pacing (n=707)DDDR pacing (n=708)p-ValueFemale gender, n (%) 472 (66.8) 441 (62.3) 0.08 Age, years (mean±SD) 73.5±11.2 72.4±11.4 0.05 History of atrial fibrillation, n (%) 303 (42.9) 318 (44.9) 0.44 Hypertension, n (%) 241 (34.1) 239 (33.8) 0.90 Previous myocardial infarction, n (%) 94 (13.3) 90 (12.7) 0.74 Diabetes, n (%) 68 (9.6) 72 (10.2) 0.73 Previous TIA, n (%) 35 (5.0) 37 (5.2) 0.81 Previous stroke, n (%) 61 (8.6) 53 (7.5) 0.81 Peripheral artery embolism, n (%) 11 (1.6) 16 (2.3) 0.33 LVEF reduced (<50%), n (%) 59 (10.6) 54 (9.5) 0.55 Medication at randomisation, n (%)  Oral anticoagulation 108 (15.3) 89 (12.6) 0.14  Aspirin 369 (52.2) 361 (51.1) 0.67  β-Blocker other than sotalol 159 (22.5) 132 (18.7) 0.08  Calcium-channel blocker 137 (19.4) 142 (20.1) 0.75  Digoxin 73 (10.3) 62 (8.8) 0.32  Class I–III antiarrhythmics 80 (11.3) 82 (11.6) 0.88  Angiotensin-converting-enzyme inhibitors 160 (22.6) 170 (24.0) 0.53  Diuretics 304 (43.0) 263 (37.2) 0.03 NYHA functional class, n (%) – – 0.33  I 503 (71.4) 522 (73.9)  II 172 (24.4) 158 (22.4)  III–IV 29 (4.1) 26 (3.7) The data were not complete for LVEF reduced (n=1127), NYHA functional class (n=1410). LVEF, left ventricular ejection fraction; NYHA, New York Heart Association; TIA, transient ischaemic attack. BODY.STROKE: In the analysis of CRF data immediately at study end a total of 86 strokes were reported in 73 patients. After final evaluation by the endpoint committee with review of patient charts a total of 102 strokes in 92 patients were identified and these 92 patients were analysed as end points in the present report. Forty-nine were in the AAI group and 43 in the DDD group (NS). Eighty-two strokes (80.4%) were verified by MRI or CT-imaging, 19 strokes were diagnosed clinically and for 1 stroke verification mode was unknown. Of those verified by imaging 72 were infarcts, 9 were bleedings and 1 was other. Data from univariate analysis of the total cohort of 1415 patients showed that age as a continuous variable was associated with an increased risk of stroke (HR 1.05 per one-year increase; 95% CI 1.02 to 1.07, p<0.001). Similarly, when the cohort was dichotomised by the median value an increased risk of stroke was seen in patients older than 75 years (HR 2.05; 95% CI 1.35 to 3.12, p<0.001). Previous stroke (HR 2.28; 95% CI 1.27 to 4.11, p=0.01) and previous TIA (HR 2.18; 95% CI 1.06 to 4.51, p=0.04) were also associated with increased risk of stroke. AF, OAC or aspirin treatment at baseline were not associated with development of stroke in the univariate analysis (NS). In a multivariate analysis where only baseline variables were included age as a continuous variable (HR 1.04; 95% CI 1.02 to 1.07, p<0.001) and previous stroke/TIA (HR 2.27; 95% CI 1.38 to 3.74, p=0.001) were associated with increased risk of stroke. Similarly, if the model included baseline variables and the time-dependent variable ‘OAC treatment’ an association with risk of stroke was seen with increasing age (HR 1.039; 95% CI 1.017 to 1.062, p<0.001) and previous stroke or TIA (HR 2.38; 95% CI 1.44 to 3.93, p<0.001). In this analysis ongoing OAC was associated with reduced risk of stroke (HR 0.46; 95% CI 0.24 to 0.90, p=0.02). BODY.CHADS:2, CHA2DS2-VASc and stroke Figure 1 shows cumulative stroke rate (%) during follow-up stratified by CHADS2 and CHA2DS2-VASc scores and stratified according to age and previous stroke/TIA/arterial embolism. Figure 1Cumulative stroke rate (%) during follow-up stratified according to (a) CHADS2 score, (b) age (A) and previous stroke/TIA (S2) from the CHADS2 score, (c) CHA2DS2-VASc score and (d) age (A2+A) and previous stroke/TIA/arterial embolism (S2) from the CHA2DS2-VASc score. Applying the CHADS2 score (range 0–6) a significant association between score and risk of stroke was seen (figure 2). Among the five components in the CHADS2 score, only age and previous stroke/TIA significantly affected risk of a stroke. To address any confounding factors from patients with AF, we performed a sensitivity analysis excluding all patients with a history of AF at the time of enrolment (n=621). Forty-nine of these ‘AF-free’ patients had a stroke during follow up. The CHADS2 score was still significantly associated with risk of stroke and a univariate analysis of the CHADS2 variables in the AF-free cohort (n=794) still demonstrated age and previous stroke/TIA to be the only variables significantly associated with risk of stroke (figure 2). Of the 92 patients with stroke, 10 patients received OAC at baseline. A sensitivity analysis excluding patients receiving OAC at baseline (n=197) did not change the results (please see online supplementary table S2). Figure 2HRs for CHADS2 score and its association with stroke for all patients and patients without a history of atrial fibrillation (AF) at baseline (AF-free patients). C, NYHA class at baseline >1; H, medical treatment for hypertension; A, age≥75; D, diabetes; S2, previous stroke or TIA; AS2, A and S2 combined (A+S2); *Five patients with unknown NYHA at baseline counts as 0. **S2 takes the values 0 and 2. HR corresponds to an increase in S2 by 1. Applying the CHA2DS2-VASc score (range from 0 to 9) a significant association between score and risk of stroke was seen (figure 3). If only age and previous stroke/TIA/arterial embolism (0–4 points) were included in the model, a significant association with risk of stroke was observed. Likewise, when analysing only the ‘AF-free’ patients, a univariate analysis of the CHA2DS2-VASc score proved the composite score and the variables age and stroke/TIA/arterial embolism to be significantly associated with risk of stroke (figure 3). Excluding patients with OAC at baseline did not change results (please see online supplementary table S3). Figure 3HRs for CHA2DS2-VASc score and its association with stroke for all patients and patients without a history of atrial fibrillation (AF) at baseline (AF-free patients). C, NYHA class at baseline >1; H, medical treatment for hypertension; A2, age≥75; D, diabetes; S2, previous stroke, TIA or arterial embolism; V, vascular disease; A, age 65–75; Sc, (female) sex category; A2S2A, A2, S2 and A combined (A2+S2+A); *Patients with unknown NYHA or LVEF at baseline counts as 0. **For the sum of A2 and A. ***S2 takes the values 0 and 2. HR corresponds to an increase in S2 by 1. The C statistics for predicting stroke with the CHADS2 and CHA2DS2-VASc scores were 0.62 (95% CI 0.56 to 0.68) and 0.60 (95% CI 0.54 to 0.66), respectively (see online supplementary table S1). BODY.AF, OAC AND STROKE: All 621 patients who had a history of paroxysmal AF had sinus rhythm at the time of randomisation. During the study 255 previously AF-free patients had either mode switch (as a surrogate measurement for AF, only DDDR patients) or ECG-verified AF at follow-up visits. Among patients treated with DDDR pacing AF burden (percentage of time with mode-switch; ie, a measure of time in AF) was evaluated in 650 patients. A total of 442 patients had mode switch during follow-up. Of these, only 246 had a history of AF at baseline. Of the 650 patients, 42 had stroke. Interestingly, among the 608 non-stroke patients mean percentage of time in mode switch was significantly lower than in the 42 stroke patients (mean 7.5±0.7% vs 10.8±3.6%, p=0.03). Antithrombotic treatment was used according to guidelines. At baseline 197 patients were treated with OAC and at study end a total of 345 patients were treated with OAC. In univariate analysis with time-dependent variables, patients treated with OAC had a reduced risk of stroke (HR 0.47; 95% CI 0.24 to 0.91, p=0.02). To exclude any confounding factors of AF and OAC we performed a multivariate analysis containing the most significant variables of the CHADS2 and CHA2DS2-VASc scores (age and previous stroke/TIA), presence of AF (new and old) and anticoagulation (new and old); the latter variables as time-dependent variables. Mode switch and/or ECG with AF in patients without known AF at baseline were counted as ‘new’ AF. Age (continuous) (HR 1.04; 95% CI 1.02 to 1.60, p<0.001) and previous stroke/TIA (HR 2.41; 95% CI 1.47 to 4.01, p<0.001) were still significantly associated with increased risk of stroke, while OAC was negatively associated with stroke (HR 0.41; 95% CI 0.20 to 0.80, p=0.01). AF (new or old) was not associated with risk of stroke (p=0.12). BODY.MORTALITY: In the AAIR group 209 patients (29.6%) died versus 193 (27.3%) patients in the DDDR group (unadjusted HR 1.06; 95% CI 0.88 to 1.29, p=0.53). The CHADS2 score (HR 1.46; 95% CI 1.36 to 1.56, p<0.001) and the CHA2DS2-VASc score (HR 1.39; 95% CI 1.31 to 1.46, p<0.001) were associated with mortality. When analysing the individual components of the CHADS2 score in a multivariate model age ≥75 years (HR 4.48; 95% CI 3.33 to 6.01, p<0.001), congestive heart failure (HR 2.80; 95% CI 1.88 to 4.17, p<0.001) and diabetes (HR 1.88; 95% CI 1.42 to 1.49, p<0.001) were independent factors associated with mortality. In the model there was also significant interaction between age and congestive heart failure (HR 0.52; 95% CI 0.33 to 0.83, p=0.006). Hypertension and previous stroke/TIA were not independently associated with mortality. When analysing the individual components of the CHA2DS2-VASc score in a multivariate model, age (age≥65+age≥75 years) (HR 2.79; 95% CI 2.25 to 3.46, p<0.001), congestive heart failure (HR 2.99; 95% CI 1.61 to 5.56, p=0.001), diabetes (HR 1.81; 95% CI 1.36 to 2.40, p<0.001) and arteriosclerotic heart disease (HR 1.29; 95% CI 1.05 to 1.59, p=0.015) showed independent association with mortality. Hypertension, gender and previous stroke were not independently associated with mortality. We also performed sensitivity analysis with regard to mortality first excluding patients with a history of AF at baseline (n=621) and the patients receiving OAC therapy at baseline. This did not change the results of either the CHADS2 or CHA2DS2-VASc scores (please see online supplementary tables S4 and S5). The C statistics for predicting death with the CHADS2 and CHA2DS2-VASc scores were 0.66 (95% CI 0.63 to 0.69) and 0.67 (95% CI 0.64 to 0.70), respectively (see online supplementary table S1). BODY.DISCUSSION: The present study is the first to evaluate the prognostic impact of the CHADS2 and the CHA2DS2-VASc score systems to assess risk of stroke and mortality in a large cohort of patients with SSS. Results are based on more than 6000 patient-years of follow-up. The main findings of our study were that the CHADS2 and CHA2DS2-VASc scores could be used to assess risk of new stroke and death in this population of patients paced for SSS irrespective of the presence of AF. Age and prior stroke/TIA were the most significant components of the CHADS2 and the CHA2DS2-VASc scores associated with future stroke. BODY.CHADS:2, CHA2DS2-VASc and stroke The CHADS2 and the CHA2DS2-VASc scores were originally constructed to evaluate risk of stroke in patients with AF with the purpose of clarifying the possible need of antithrombotic therapy.7 8 We found that the association between the CHADS2 and CHA2DS2-VASc scores and stroke was still significant when analysing only the patients without a history of AF at baseline in our cohort. The significance of the CHADS2 and CHA2DS2-VASc scores in a non-AF population has not been well established. In a recent study of patients with acute coronary syndrome, Poçi et al9 also found that the CHADS2 score could be used to identify non-AF patients at high risk of subsequent stroke. A retrospective study of patients screened for ischaemic heart disease (343 with AF and 2945 without) demonstrated that the CHADS22 score.10 The reason for the CHADS2 and the CHA2DS2-VASc scores being able to predict stroke risk in a non-AF population is unclear. It is well known that SSS and AF often coexist. Our results indicate that patients with SSS share many of the same risk factors as AF patients. It may be that these risk factors predict increased risk of stroke, preceded or not preceded by AF. The risk factors contained in the CHADS2 and CHA2DS2-VASc scores may in future studies prove to be associated with stroke in other patient groups and in the general population. Nonetheless, these results suggest that OAC should be considered in patients paced for SSS, irrespective of the presence of AF. This however, needs further investigation, ideally in a randomised trial testing the possible net benefits of OAC versus no OAC in SSS patients without AF (and without other indications for OAC treatment) and with a CHADS2 or CHA2DS2-VASc score of ≥1.2 BODY.AF, OAC AND STROKE: The number of strokes reported in the present study with more than 6.000 patient-years of follow-up is comparable with the observation of 90 strokes in 5664 patient-years of follow-up reported in The Mode Selection Trial (MOST) in SSS patients.6 Similar numbers were reported in small-sized studies of SSS patients.4 5 In our study a rather large proportion (44%) of the patients had a history of AF at baseline and at the end of the study 62% of the total cohort had either AF at baseline or ‘new’ AF documented by ECG or mode switch. However, this number is most likely underestimated since it was not possible to detect mode switch in the AAIR-paced group of patients. It is well established that AF increases the risk of stroke.11 However, excluding these patients from our analyses did not change the results. A possible explanation could be that all AF patients in the present study were appropriately anticoagulated and therefore had a reduced risk of stroke compared with other AF-populations. The method used for AF detection during follow-up in the present study was relatively non-sensitive, recording an ECG once per year. This may also explain why we could not confirm a recent report by Healey et al,12 indicating that short episodes of AF detected by the pacemaker also increases the risk of stroke. However, we did find a higher ‘mode-switch burden’ among the DDDR-paced patients who developed a stroke during follow-up, supporting the association between AF and stroke. OAC is known to reduce the risk of stroke in AF patients significantly.13 The number of OAC-treated patients increased from 14% to 24% during the course of the trial and the use of OAC was associated with a markedly lower risk of stroke. Nonetheless, excluding patients receiving OAC at baseline did not change the association between the CHADS2 and CHA2DS2-VASc scores and stroke, nor did adjusting for OAC in the multivariate analysis. This finding may be explained by appropriate anticoagulation of patients with AF at high risk of stroke. BODY.CHADS:2, CHA2DS2-VASc and mortality The CHADS2 and CHA2DS2-VASc scores were not primarily designed to predict mortality. Nevertheless, in recent years a few studies have tested the scores’ ability to predict death in AF and non-AF populations. Poçi et al9 found that the CHADS2 score could predict subsequent death in AF and non-AF patients hospitalised for acute coronary syndrome as well as did Crandall et al10 in 3288 AF and non-AF patients undergoing coronary angiography for suspicion of coronary artery disease. A substudy from the RE-LY (Randomized Evaluation of Long-term anticoagulant therapY) trial also found that mortality rates increased with increasing CHADS2 score in 18 112 patients with AF receiving OAC.14 Our results confirm this association in a population of patients paced for SSS, even when excluding patients with AF or patients receiving OAC. BODY.LIMITATIONS: Since follow-up in the trial was performed after 3 months and then only once a year (eg, ECG recording and registration of medication status) the number of ‘new AF’ patients in the AAIR group could be underestimated. Likewise, the sensitivity of the analysis of OAC as a time-dependant variable may also be slightly limited. Since OAC treatment strategies for AF patients have changed since the course of this trial (1999 to 2008) favouring more anticoagulation,2 this could also have influenced the number of OAC-treated patients and number of strokes in the AF group. Finally, we have no data on time spent within therapeutic range for the patients receiving OAC. BODY.CONCLUSION: This study indicates that the risk of stroke and death in patients with SSS treated with pacemaker can be evaluated by using either the CHADS2 score or the CHA2DS2-VASc score irrespective of the presence of AF. The score components age and previous stroke/TIA seem to contain the most important information about the risk of future stroke in this population. BODY.SUPPLEMENTARY MATERIAL: BODY.WEB APPENDIX:

TITLE: Heparin binding protein in patients with acute respiratory failure treated with granulocyte colony-stimulating factor (filgrastim) – a prospective, placebo-controlled, double-blind study ABSTRACT.BACKGROUND: Heparin Binding Protein (HBP) is released to blood circulation from activated neutrophils in bacterial infections. It is a potential inducer of vascular leakage and precludes the development of septic shock. Filgrastim induces the production of new neutrophils and modulates their bacterial-killing activity. We evaluated the effect of filgrastim on HBP –concentrations in critically ill patients with acute respiratory failure. ABSTRACT.METHODS: 59 critically ill patients with acute respiratory failure were included in this randomised, double-blind, placebo-controlled study of filgrastim 300 micrograms/day or corresponding placebo for 7 days. Plasma samples were drawn on baseline, day 4 and day 7. HBP –concentrations, absolute leukocyte and neutrophil counts were measured. ABSTRACT.RESULTS: The median [IQR] HBP concentrations were 23.6 ng/ml [13.9-43.0 ng/ml], 25.1 ng/ml [17.7-35.5 ng/ml] and 15.9 ng/ml [12.6-20.7 ng/ml] in patients receiving filgrastim on baseline, day 4 and day 7, respectively. The HBP concentrations in placebo group were 21.6 ng/ml [16.9-28.7 ng/ml], 13.9 ng/ml [12.0-19.5 ng/ml] and 17.8 ng/ml [13.6-20.9 ng/ml]. At day 4, the filgrastim group had significantly higher HBP –concentrations when compared to placebo group (p < 0.05). No correlation between HBP –concentrations and absolute neutrophil count or P/F –ratios was found. ABSTRACT.CONCLUSIONS: Filgrastim treatment is associated with increased circulating HBP levels compared to placebo, but the absolute neutrophil count or the degree of oxygenation failure did not correlate with the observed plasma HBP –concentrations. ABSTRACT.TRIAL REGISTRATION: Clinicaltrials.gov NCT01713309 BODY.BACKGROUND: Plasma leakage from the vasculature is an important step in the development of septic shock in patients with infections [1]. Heparin binding protein (HBP; also known as azurocidin and CAP37) is an immunomodulatory mediator released from activated neutrophils [2]. The release of HBP is stimulated e.g. when neutrophils adhere to the endothelial lining or when these cells encounter bacterial products in the circulation. The released HBP initiates rearrangement of the endothelial cell cytoskeleton, leading to openings in the endothelial barrier and increased macromolecular leakage to interstitial space [3]. This is an important mechanism of uncontrollable leakage in inflammatory conditions. In capillary leakage such as septic shock, burns or erysipelas caused by group A Streptococci, elevated levels of HBP have been documented [4-8]. The levels of HBP were elevated up to 12 h prior to the clinical manifestation of septic shock [5]. Pulmonary infections and septic infections are responsible for more than a third of acute respiratory failure leading to ICU admission [9]. In bacterial infections, neutrophils become activated leading to the release of HBP [10]. Filgrastim is a granulocyte colony stimulating factor (G-CSF) which stimulates neutrophil production in bone marrow as well as strengthens their functions, such as phagocytosis and bacterial killing [11]. Increased HBP –levels have been documented in several different bacterial infections [4-6,12]. However, no clinical trial has studied the effect of G-CSF on HBP –levels. We hypothesized that increased number of circulating neutrophils in response to G-CSF treatment would be associated with increased HBP concentrations in plasma compared to placebo. Accordingly, we analyzed the HBP concentrations in critically ill patients with acute respiratory failure who participated in a prospective, randomised, double-blind study of G-CSF vs. placebo. BODY.METHODS: This is a study of plasma HBP –levels of a previously published trial of G-CSF in critically ill patients [13]. The study was approved by the Ethics Committee of Department of Anaesthesiology in Helsinki University Central Hospital. The notice to the Finnish National Agency for Medicines was submitted 60 days before the initiation of the study as required by Finnish legislation. Informed consent was obtained from the study patients or from a close relative. BODY.STUDY PATIENTS: During a 16 –month period from February 1997 to June 1998 altogether 636 patients were admitted to the mixed 10 –bed ICU. 59 consecutive patients were included into the study. The inclusion criteria were 1. age >18 years, 2. admission <12 h before study inclusion, 3. intubation <48 h of study inclusion, 4. expected ICU stay >48 h, 5. Informed consent signed. The exclusion criteria were 1. age <18 years, 2. admission >12 h before study inclusion 3. Intubated >48 h before study inclusion, 4. expected stay in ICU <48 h, 5. no informed consent, 6. pregnant or nursing, 7. administration of filgrastim, sargramostin or other biological response modifiers within 7 days, 8. participation in another medicinal trial. BODY.STUDY DESIGN: The study was a prospective, randomised, double-blind, placebo-controlled trial of filgrastim in patients with acute respiratory failure requiring intubation. The patients were treated with subcutaneous injections of 300 micrograms of filgrastim or corresponding placebo (1 ml of 0.9% NaCl –solution) once daily for 7 days or until discharge from the ICU. If the neutrophil count exceeded 50x109/L filgrastim/placebo was administered every other day and if neutrophil count exceeded 75x109/L filgrastim/placebo was discontinued. The study personnel were blinded to study patients’ leukocyte and neutrophil counts until the data had been analyzed. Other intensive care treatments were performed according to the written standard operating procedures of the ICU. Blood samples were drawn at study entry (baseline), day 4 and day 7 afterwards. The samples were taken before administration of the study drug. The sampling was performed only during the ICU stay and if a patient was discharged by day 7, blood collection was also discontinued. The blood samples were drawn and placed immediately in ice and centrifuged at +4°C. The plasma was stored in −70°C until further analysis. Total leukocyte count and absolute neutrophil count were determined in routine analysis of Helsinki University Central Hospital laboratory. The plasma HBP was determined by enzyme-linked immunosorbent assay as described earlier [2]. The detection limit of the method was 0.25 ng/ml and CV variance was <5%. The primary endpoint of the main study were number of adverse events, the number of patients developing ARDS, disseminated intravascular coagulation or acute renal failure during days 1–28, and changes in MOD score [13]. For this substudy, the clinical data comprised of APACHE II [14], SOFA -score [15,16], admission diagnosis, and laboratory values for white blood cell (WBC), neutrophil count and inflammatory mediators: (Interleukin (IL)-6, Interleukin (IL)-10, soluble E-selectin (sE-selectin) and soluble Interleukin 2 receptor (sIL-2R) from baseline and day 3 [17]. BODY.STATISTICAL ANALYSIS: The data are presented as median and interquartile ranges (IQR) or absolute numbers and ranges, as appropriate. The normality of distribution of parameters was tested with Kolmorogov-Smirnov one-sample test. In case of non-normal distribution, the parameters were transformed to natural logarithm before analysis. The difference between groups is calculated by one-way analysis of variance with a p value <0.05 considered statistically significant. The correlations between parameters were calculated from values transformed to natural logarithm. Intraclass correlation coefficient (ICC) was used to examine the correlation between HBP -levels, P/F –ratio, SOFA –score, WBC and neutrophil count as well as inflammatory mediators. BODY.RESULTS: Altogether 59 patients were included into the study, 30 in the filgrastim group and 29 in the placebo group. Due to an error in the preparation of randomisation envelopes number 45 was omitted and only 59 patients were randomised instead of the intended 60 patients. The baseline characteristics of the patients are presented in the Table 1. Table 1Baseline characteristics of study population VariableControls (n = 29)Filgrastim (n = 30)Age (yr) 52 (28–73) 45 (20–76) Gender (F/M) 6/23 9/21 APACHE II 13 (3–28) 11 (2–19) Admission diagnosis    Bacterial infection* 14 12    Inflammatory process** 6 6    Other 9 12 SOFA 9 (3–15) 8.5 (2–15) APACHE, Acute Physiology and Chronic Health Evaluation; SOFA, Sequental Organ Failure Assessment, median (range). *Bacterial infection: pneumonia, blood culture positive sepsis, meningitis, peritonitis. **Inflammatory process: aspiration pneumonia, acute pancreatitis. The plasma HBP –concentrations, absolute neutrophil counts and white blood cell counts at baseline, day 4 and day 7 are presented in Figure 1 and Table 2. The plasma concentrations of HBP in filgrastim and placebo groups were similar at baseline, median [IQR] 23.6 ng/ml [13.9-43.0 ng/ml] vs. 21.6 ng/ml [16.9-28.7 ng/ml], respectively (p = 0.785). At day 4, the filgrastim group had significantly higher HBP –concentrations when compared to placebo group 25.1 ng/ml [17.7-35.5 ng/ml] vs. 13.9 ng/ml [12.0-19.5 ng/ml], respectively (p < 0.05). At day 7, there was no difference in HBP levels 15.9 ng/ml [12.6-20.7 ng/ml] vs. 17.8 ng/ml [13.6-20.9 ng/ml] in filgrastim and placebo groups (p = 0.774), respectively. Figure 1The concentrations (ng/ml; median, 95% CI) of HBP between patients receiving filgrastim 300 microgr/day or placebo for 7 days. Table 2Concentrations of HBP, absolute neutrophil count and WBC in patients receiving daily filgrastim 300 microgr/day or placebo  BaselineDay 4‡Day 7FilgrastimPlaceboFilgrastimPlaceboFilgrastimPlaceboHBP (ng/ml) 23.6 (13.9-43.0) 21.6 (16.9-28.7) 25.1 (17.7-35.5)* 13.9 (12.0-19.5) 15.9 (12.6-20.7) 17.8 (13.6-20.9) Neutrophil count (x109)/L 7.3 (4.0-11.1) 8.2 (3.6-12.8) 24.0 (14.6-32.8)† 9.4 (7.5-13.7) 27.1 (18.7-38.9)† 9.4 (6.1-12.0) WBC count (x109)/L 9.1 (5.6-12.3) 12.1 (4.3-17.1) 27.2 (17.6-36.0)† 12.1 (10.0-16.5) 29.7 (20.1-39.8)† 12.2 (14.9) HBP/WBC –ratio 2.9 (1.4-8.7) 2.8 (1.5-11.0) 0.9 (0.6-1.7)* 0.7 (0.5-0.9) 1.6 (1.1-2.1) † 0.7 (0.7-1.0) HBP/Neutrophil –ratio 4.0 (1.7-9.8) 3.0 (1.6-12.5) 1.2 (0.7-2.2) 1.8 (1.3-2.4) 0.7 (0.5-1.2)* 1.7 (1.3-2.1) Median (IQR), WBC white blood cell count. * p < 0.05 filgrastim vs. placebo. † p < 0.001 filgrastim vs. placebo. ‡ Neutrophil count and WBC count from day 3. The absolute neutrophil count and white blood cell counts were similar in filgrastim and placebo groups at baseline but at days 4 and 7 there were significant increase in filgrastim group (p < 0.01 for both; Table 2). The HBP/WBC –ratio was similar in both groups at baseline, but at days 4 and 7, the ratio decreased more in placebo group compared to filgrastim group. HBP/absolute neutrophil count –ratio also decreased from baseline in both groups, but the decrease was greater in filgrastim group (Table 2). In patients with septic shock vs. no septic shock, no difference in plasma HBP –concentrations could be detected in any of the time points (baseline, 4 –day or 7 –day) regardless of filgrastim treatment. The HBP –concentrations were above 15 ng/ml in 44 of the 59 patients at baseline. No correlations between HBP –concentrations and absolute neutrophil count or P/F –ratio could be demonstrated (Figure 2). The concentrations of HBP (baseline or day 4) did not correlate with SOFA –score. No correlation of HBP with inflammatory mediators IL-6 (p = 0.42), IL-10 (p = 0.44), or sIL-2R (p = 0.32) was found. A modest correlation with sE-Sel was found (rho 0.275, p < 0.01). Figure 2The correlation of HBP –concentrations to P/F –ratio (left panel) and absolute neutrophil count (right panel). BODY.DISCUSSION: Our study was the first to assess HBP –levels in critically ill patients receiving filgrastim or placebo in a controlled study. The HBP –levels were similar in both treatment groups at baseline, but differed at day 4. The difference in HBP –levels did not persist to day 7, and HBP –concentrations did not correlate with absolute neutrophil count or P/F –ratios. The concentrations of HBP were elevated in both groups already at the baseline samples. In healthy volunteers without infections and in patients with local infections only, the concentrations of HBP have been around 7 ng/ml compared to 24 ng/ml and 22 ng/ml in filgrastim and placebo groups at baseline, respectively [8]. These baseline concentrations were, however, lower than those previously described for febrile patients at hospital admission who later develop septic shock (42 ng/ml) or severe sepsis (30 ng/ml) [5]. When compared to burn patients or sepsis patients at ICU admission, the HBP –levels were similar [4,7]. In our study population, all patients had acute respiratory failure, but not all had bacterial infection. HBP is released in systemic inflammatory reactions including sepsis, and a cut-off value of 15 ng/ml is found to have the best predictive value for the development of septic shock [5]. In our study patients 44 out of 59 exceeded this level at baseline even though only 16 of the 59 patients were diagnosed as having severe sepsis or septic shock. HBP is an important mediator of bacterial infections transforming to septic shock. In septic situations, HBP is released from activated neutrophils by exocytosis of granule compartments. Filgrastim stimulates the production of new neutrophils in bone marrow and enhances the effector functions of mature neutrophils [18]. As HBP is released from activated neutrophils, the stimulation of neutrophil production and function by filgrastim could lead to increased secretion of HBP. In our study, the levels of HBP were already elevated at baseline compared to healthy volunteers or to patients with local infections only, but not compared to febrile patients who develop septic shock or severe sepsis. At day 4, there was a significant difference in the HBP –levels between filgrastim and placebo groups. This seems to be caused by a decrease of HBP –concentrations in placebo group rather than an increase in HBP –concentrations of filgrastim group. Previously, in burn patients, the levels of HBP have decreased to near-normal levels in 24–48 h after the ICU admission [4]. In our patients the high HBP –concentrations did not persist to day 7, at that day both groups had median HBP –levels lower than that at baseline. The correlation of HBP concentrations to WBC count has been studied in previous trials [4,5]. In a subset of patients with severe sepsis (with or without septic shock), a positive correlation was found whereas no correlation was found in burn patients. In our patients, no correlations between WBC and HBP –levels was found. In previous trials the correlation between HBP –levels and absolute neutrophil count has not been studied. In our patient population, however, no such correlation could be verified. The M1 –protein mediated release of neutrophil granule contents is responsible for subsequent lung injury [19]. As HBP is situated in secretory vesicles along with azurophilic/primary granule subsets, it is readily released when neutrophils become activated [2,10]. Initially filgrastim was thought to have beneficial effects on the outcome of critically ill patients with septic infections. However, no mortality benefit could be shown in critically ill patients with pneumonia and severe sepsis or septic shock in a large randomised placebo-controlled trial [20]. The worsening of pulmonary function and even the development of acute respiratory distress syndrome (ARDS) has been of concern in patients receiving filgrastim. As filgrastim treatment results in prolonged high levels of HBP, this could have deleterious effects on pulmonary capillary leakage and pulmonary function. In our patients, the plasma concentrations of HBP did not correlate with the P/F –ratios. Our study had some limitations. The number of patients was low and, accordingly, there is considerable risk to type II error, i.e. not detecting a true difference even though it is present. Due to the limited number of patients in this randomized pilot study, we were not able to analyze HBP –levels in subgroups of patients with bacterial infections, other inflammatory processes, or with non-infectious causes of acute respiratory failure. Second, the sampling was not primarily designed for measurements of plasma HBP –concentrations, but rather to show the change in organ failure score before and after the intervention. As the decline in HBP occurs within days in critically ill patients [4], more frequent sampling would have been better to detect the maximal effect of filgrastim. Thus, it is possible that filgrastim may have caused a temporary increase in HBP –levels, and we were not able to detect it. Finally, as the primary study was performed already in 1997, the blood samples have been conserved in −70° over time. This may have had effect on the HBP levels. If degradation of HBP had occurred, it can be suspected that the degradation of HBP would have occurred to the same degree in both treatment groups. BODY.CONCLUSIONS: Filgrastim treatment is associated with increased circulating HBP levels compared to placebo, but the absolute neutrophil count or the degree of oxygenation failure did not correlate with the observed plasma HBP –concentrations. BODY.KEY MESSAGES: Plasma levels of heparin-binding protein (HBP) were elevated in critically ill patients with acute respiratory failure requiring intubation at baseline. A significant decrease in HBP –levels in placebo –treated patients was observed at day 4. HBP –levels did not correlate with absolute white blood cell or neutrophil counts. HBP –levels did not correlate with worsening of oxygenation measured by P/F –ratio. BODY.ABBREVIATIONS: HBP: Heparin-binding protein; IQR: Interquartile range; G-CSF: Granulocyte colony stimulating factor; ICU: Intensive care unit; APACHE II: Acute physiology and chronic health evaluation; SOFA: Sequential organ failure assessment; CV: Coefficient of variation; ARDS: Acute respiratory distress syndrome; DIC: Disseminated intravascular coagulopathy; WBC: White blood cell; ICC: Intraclass correlation coefficient; P/F –ratio: Arterial oxygen tension to fraction of inspired oxygen –ratio. BODY.COMPETING INTERESTS: Hansa Medical AB has filed a patent application on the use of HBP as a diagnostic tool in sepsis. HH is listed as inventor. KMK, LL and VP declare no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: KMK has performed statistical analysis of the HBP data and is responsible for the draft writing and submission of the manuscript. HH and LL have contributed in the planning of the study, are responsible for the determination of HBP –concentrations, contributed on the analysis of the data and writing of the manuscript. VP is responsible for the planning, execution and reporting of the original study and contributed in HBP data analysis and manuscript preparation. All authors have read and approved the final version of the manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2334/13/51/prepub

TITLE: Epirubicin, oxaliplatin, and capecitabine with or without panitumumab for patients with previously untreated advanced oesophagogastric cancer (REAL3): a randomised, open-label phase 3 trial ABSTRACT.SUMMARY: ABSTRACT.BACKGROUND: EGFR overexpression occurs in 27–55% of oesophagogastric adenocarcinomas, and correlates with poor prognosis. We aimed to assess addition of the anti-EGFR antibody panitumumab to epirubicin, oxaliplatin, and capecitabine (EOC) in patients with advanced oesophagogastric adenocarcinoma. ABSTRACT.METHODS: In this randomised, open-label phase 3 trial (REAL3), we enrolled patients with untreated, metastatic, or locally advanced oesophagogastric adenocarcinoma at 63 centres (tertiary referral centres, teaching hospitals, and district general hospitals) in the UK. Eligible patients were randomly allocated (1:1) to receive up to eight 21-day cycles of open-label EOC (epirubicin 50 mg/m2 and oxaliplatin 130 mg/m2 on day 1 and capecitabine 1250 mg/m2 per day on days 1–21) or modified-dose EOC plus panitumumab (mEOC+P; epirubicin 50 mg/m2 and oxaliplatin 100 mg/m2 on day 1, capecitabine 1000 mg/m2 per day on days 1–21, and panitumumab 9 mg/kg on day 1). Randomisation was blocked and stratified for centre region, extent of disease, and performance status. The primary endpoint was overall survival in the intention-to-treat population. We assessed safety in all patients who received at least one dose of study drug. After a preplanned independent data monitoring committee review in October, 2011, trial recruitment was halted and panitumumab withdrawn. Data for patients on treatment were censored at this timepoint. This study is registered with ClinicalTrials.gov, number NCT00824785. ABSTRACT.FINDINGS: Between June 2, 2008, and Oct 17, 2011, we enrolled 553 eligible patients. Median overall survival in 275 patients allocated EOC was 11·3 months (95% CI 9·6–13·0) compared with 8·8 months (7·7–9·8) in 278 patients allocated mEOC+P (hazard ratio [HR] 1·37, 95% CI 1·07–1·76; p=0·013). mEOC+P was associated with increased incidence of grade 3–4 diarrhoea (48 [17%] of 276 patients allocated mEOC+P vs 29 [11%] of 266 patients allocated EOC), rash (29 [11%] vs two [1%]), mucositis (14 [5%] vs none), and hypomagnesaemia (13 [5%] vs none) but reduced incidence of haematological toxicity (grade ≥3 neutropenia 35 [13%] vs 74 [28%]). ABSTRACT.INTERPRETATION: Addition of panitumumab to EOC chemotherapy does not increase overall survival and cannot be recommended for use in an unselected population with advanced oesophagogastric adenocarcinoma. ABSTRACT.FUNDING: Amgen, UK National Institute for Health Research Biomedical Research Centre. BODY.INTRODUCTION: Gastric and oesophageal cancers are among the most common causes of cancer-related mortality, and were responsible for more than 1·1 million deaths worldwide in 2008.1 Combination chemotherapy is beneficial in perioperative and advanced disease settings, although overall survival is poor. In patients with metastatic disease, median overall survival with best supportive care is about 3 months, which can be extended to about 10 months with chemotherapy.2,3 No internationally accepted standard of care regimen exists for advanced oesophagogastric adenocarcinoma, although most centres use doublet or triplet chemotherapy combinations with a platinum-fluoropyrimidine backbone. The REAL2 non-inferiority study established epirubicin, oxaliplatin, and capecitabine (EOC) as a standard first-line regimen, and noted a median overall survival of 11·2 months.3 This result compared favourably with the alternative regimens assessed in REAL2, including a combination of epirubicin, cisplatin, and fluorouracil that had a median overall survival of 9·9 months. In the past decade, the EGFR pathway has been recognised as one of the key proliferative pathways that is dysregulated during tumorigenesis. Preclinical data confirm that transfection of EGFR into human cancer cells is associated with an aggressive phenotype,4 and several molecular aberrations within this pathway can function as potent oncogenes. In oesophagogastric adenocarcinoma, EGFR overexpression is reported in 27–55% of cases in published literature,5,6 and has been associated with reduced overall survival in some series.5,7 Amplification of EGFR, measured by fluorescence in-situ hybridisation, is also reported in a smaller subset of oesophagogastric adenocarcinoma.5,8 This pathway therefore represents a potential therapeutic target in this disease setting, and single-drug phase 2 trials have reported a small number of responses with monoclonal antibodies9 or tyrosine-kinase inhibitors10 targeting EGFR in advanced oesophagogastric adenocarcinoma. Panitumumab is a fully human immunoglobulin G2 monoclonal antibody directed against EGFR that has confirmed survival benefits in advanced colorectal cancer.11 We designed the REAL3 trial to assess the addition of panitumumab to EOC in molecularly unselected patients with advanced oesophagogastric adenocarcinoma. BODY.METHODS: BODY.STUDY DESIGN AND PARTICIPANTS: REAL3 was an open-label, multicentre, phase 3, randomised controlled trial, undertaken at 63 parti-cipating centres (tertiary referral centres, teaching hospitals, and district general hospitals) in the UK. Safety, response, and survival outcomes were compared between standard EOC and modified EOC plus panitumumab (mEOC+P). The mEOC+P schedule was established in an unplanned dose-finding study,12 which was done because excessive toxicity (primarily diarrhoea) was noted when the drugs were initially combined at full dose. As reported previously,12 eligible patients had histologically verified, untreated, metastatic or locally advanced inoperable adenocarcinoma or undifferentiated carcinoma of the oesophagus, gastro-oesophageal junction, or stomach. Patients were aged at least 18 years and had measurable disease on CT or MRI. Other eligibility criteria included WHO performance status of 0–2, and adequate cardiac, renal, liver, and bone marrow function. Cardiac function was not formally measured at baseline unless there were concerns based on previous medical history. Renal, liver, and bone marrow function were assessed via routine blood tests (full blood count and biochemistry). Exclusion criteria included previous chemotherapy (including adjuvant chemotherapy), previous anti-EGFR therapy, known brain metastases, and clinically significant cardiac disease or other significant comorbidity. Recruitment largely predated routine HER2 testing in oesophagogastric adenocarcinoma and therefore HER2 status did not affect eligibility. The study was done in accordance with good clinical practice guidelines and the declaration of Helsinki, and was approved by the North West Research Ethics Committee. All patients provided written informed consent before screening investigations. BODY.RANDOMISATION AND MASKING: We randomly allocated patients (1:1) to standard EOC chemotherapy (intravenous epirubicin 50 mg/m2 on day 1, intravenous oxaliplatin 130 mg/m2 on day 1, and oral capecitabine 1250 mg/m2 per day on days 1–21) or modified-dose EOC in combination with panitumumab (mEOC+P; intravenous epirubicin 50 mg/m2 on day 1, intravenous oxaliplatin 100 mg/m2 on day 1, oral capecitabine 1000 mg/m2 per day on day 1–21, and intravenous panitumumab 9 mg/kg on day 1) every 21 days. Randomisation was done independently at the Institute for Cancer Research Clinical Trials and Statistics Unit (ICR-CTSU) by random permuted blocks (block sizes of six and eight) and stratified by centre region (locations were divided into 11 regions), extent of disease (locally advanced vs metastatic disease), and performance status (0 vs 1 vs 2). Patients were enrolled by trials office staff at the Royal Marsden Hospital, who then faxed confirmation of the allocated treatment group to local site staff. The trial was open-label with no masking of patients or study staff to the treatment allocation. Patients received a maximum of eight cycles of treatment. Advice regarding dose modifications for toxic effects was provided in the protocol (appendix). BODY.PROCEDURES: The primary endpoint was overall survival, defined as the time from randomisation until death from any cause. Secondary endpoints were progression-free survival (PFS), defined as the time from randomisation until documented disease progression or death from any cause; response rate according to RECIST 1.0 criteria;13 toxicity graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0; patient-reported outcomes; and KRAS mutation status. CT scans were done every 12 weeks. No central imaging review was done in relation to response or PFS endpoints. Details of the translational research methods are available in the appendix. Results from patient-reported outcomes will be reported separately. Data for patients recruited at Royal Marsden Hospital were subject to source data verification by trust-appointed monitoring staff. For other UK centres, the sponsor deemed it appropriate, in keeping with good clinical practice requirements, to undertake central monitoring and provide sites with training meetings and written guidance to ensure appropriate conduct of the trial. Participating centres were required to provide evidence confirming patient eligibility, including blood test results, histopathology reports, and imaging reports to ensure appropriate randomisation and stratification at trial entry. During trial treatment, any discrepancies in data received compared with expected were queried with sites in real time. Recorded toxicities were cross-referenced against reported serious adverse events to ensure that all toxicities were captured and that all events meeting the criteria for a serious adverse event were reported as such. BODY.STATISTICAL ANALYSIS: The trial was powered to detect a 10% improvement in 1 year survival, from 45% for EOC to 55% with mEOC+P, equating to a hazard ratio (HR) of 0·749. To achieve 90% power and a two-sided α of 0·05, we needed to include 509 events (deaths from any cause) and planned a total accrual of 730 patients. We planned to do an interim survival analysis after 255 events (50%). We also did a preplanned non-comparative interim analysis of response rate with mEOC+P after the first 200 patients were assessable for response (phase 2 population). These data were reviewed by the independent data monitoring committee (IDMC), and confirmed an acceptable response rate of 52% in the mEOC+P group, which exceeded the predefined futility threshold of 45%. Throughout the trial, unmasked data were reviewed by the IDMC to examine the safety, scientific validity, and conduct of the trial. At annual review of the data in October, 2011, the IDMC noted a statistically inferior overall survival outcome in the mEOC+P group based on the occurrence of 169 events (HR 1·53, p=0·0062). In discussion with the trial management group, we decided to close the trial to further recruitment with immediate effect, withdraw panitumumab, and cross all patients over to full-dose EOC. We did our efficacy analyses in the intention-to-treat population, defined as all eligible randomised participants. Data for patients still on treatment were censored at the time of crossover to allow accurate comparison between the two trial groups. Toxicity analyses included all participants who received at least one dose of study drug. We estimated overall survival and PFS with the Kaplan-Meier method. We compared groups with the log-rank test and used Cox regression analysis to generate HRs and 95% CIs. We compared response rates between groups with logistic regression, and generated odds ratios and 95% CIs. We used the χ2 test to compare incidence of toxic effects between groups. In multivariate analysis, we used forward stepwise Cox regression to calculate corrected HRs and 95% CIs. Statistical analyses were done with SPSS version 19. This study is registered with ClinicalTrials.gov, number NCT00824785. BODY.ROLE OF THE FUNDING SOURCE: Amgen were involved in planning of the study design. The funding sources were not involved in collection, analysis, and interpretation of data, writing of the report, or the decision to submit the paper for publication. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. BODY.RESULTS: Between June 2, 2008, and Oct 17, 2011, we enrolled 575 patients, three of whom were withdrawn because they did not fulfil eligibility criteria. Additionally, 19 patients randomly allocated during the phase 1 dose-finding study were excluded from the intention-to-treat analysis.12 We included 553 eligible patients in the phase 3 intention-to-treat population, representing 76% of the planned accrual at the time of trial closure (figure 1; table 1). 494 (89%) patients had metastatic disease. Median follow-up in patients who were alive at the time of analysis was 4·6 months (IQR 1·8–10·1) in the EOC group and 5·3 months (2·6–9·5) in the mEOC+P group. Based on 251 events (EOC 110, mEOC+P 141) at the time of reporting, median overall survival was lower in the mEOC+P group than in the EOC group (HR 1·37, 95% CI 1·07–1·76, p=0·013; figure 2). Median overall survival was 8·8 months (95% CI 7·7–9·8) in the mEOC+P group compared with 11·3 months (9·6–13·0) in the EOC group (figure 2). 35 (13%) of 278 patients in the mEOC+P group and 54 (20%) of 275 patients in the EOC group were on treatment at the time of trial closure and were censored at this timepoint. 1-year overall survival was 33% (95% CI 26–41) in the mEOC+P group and 46% (38–54) in the EOC group. Subgroup analyses according to extent of disease, site of primary tumour, and KRAS mutation status were prespecified. Other subgroup analyses (age, sex, performance status, and number of metastatic sites) were exploratory. Figure 3 shows the forest plot analysis for subgroups tested, with a similar effect favouring EOC noted between all subgroups. Based on 333 events (EOC 153, mEOC+P 180), PFS did not differ between treatment groups (HR 1·22, 95% CI 0·98–1·52, p=0·068; figure 4). Median PFS was 6·0 months (95% CI 5·5–6·5) in the mEOC+P group and 7·4 months (6·3–8·5) in the EOC group. 1-year PFS was 20% (95% CI 14–26) in the mEOC+P group compared with 21% (14–27) in the EOC group. 61 patients who were still on treatment (37 in the EOC group and 24 in the mEOC+P group) and had not reached their first response assessment at the time of data censoring were excluded from the response analysis. Patients who died, progressed, or withdrew before response assessment were classed as non-responders. 116 (46%) of 254 patients in the mEOC+P group had a complete or partial response compared with 100 (42%) of 238 patients in the EOC group (odds ratio 1·16, 95% CI 0·81–1·66, p=0·42; table 2). 542 patients received at least one cycle of chemotherapy and were assessed for toxicity (table 3). Addition of panitumumab was associated with increased rates of grade 3–4 diarrhoea, mucositis, rash, and hypomagnesaemia. Patients in the EOC group had increased rates of grade 3–4 peripheral neuropathy, neutropenia, febrile neutropenia, and thrombocytopenia compared with patients in the mEOC+P group. Overall, we noted no significant difference between the two groups in terms of grade 3–5 toxicity when all toxicities were combined (p=0·21). However, when we excluded haematological and biochemical toxicities, more patients had grade 3–5 toxicities in mEOC+P group (p=0·035, table 3). Four patients died from toxicities regarded as related to mEOC+P: one each of septicaemia, neutropenic sepsis, pulmonary embolism, and upper gastrointestinal haemorrhage. Six patients died from toxicities regarded as related to EOC: one each of pneumonia, diarrhoea and dehydration, pneumonitis, and myocardial infarction, and two cases of neutropenic sepsis. For 542 patients who started treatment, the median number of cycles delivered was five (IQR three to eight) in both trial groups. 80 (30%) of 266 patients who started EOC received all eight planned cycles of chemotherapy, as did 74 (27%) of 276 patients who started mEOC+P. These findings are restricted by data censoring for the 89 patients still on treatment at the time of trial closure. Excluding these patients, the median number of cycles delivered was six (IQR three to eight) with EOC (80 [38%] of 212 patients completed eight cycles) and five (IQR three to eight) with mEOC+P (74 [31%] of 241 patients completed eight cycles). Overall, 1307 cycles of EOC were administered compared with 1375 cycles of mEOC+P. Table 4 shows the median relative dose intensity for each drug according to treatment group and the proportion of patients in each group achieving a relative intensity of at least 80%. In exploratory biomarker analyses of 276 patients treated with mEOC+P, development of any grade of rash (219 patients) was associated with significantly longer overall survival compared with patients with no rash (57 patients; figure 5). Median overall survival was 10·3 months (95% CI 8·9–11·6) and median PFS was 6·8 months (5·9–7·8) in patients with grade 1–4 rash compared with median overall survival of 4·3 months (3·3–5·4) and a median PFS of 3·7 months (2·3–5·1) in patients with grade 0 rash (p<0·0001 for overall survival and PFS). However, outcomes in the grade 0 rash population might be negatively skewed by patients with rapid disease progression who received insufficient panitumumab to develop rash. A further exploratory analysis was therefore done including only 194 patients (177 with rash, 17 with no rash) who received at least four cycles of chemotherapy. Although possibly restricted by the smaller number of patients, in this analysis overall survival did not differ between patients who developed rash (median 10·6 months [95% CI 9·0–12·2]) and those who did not (8·5 months [7·1–9·9]; HR 0·64 [95% CI 0·37–1·11], p=0·11). Few tissue biomarker analyses have been undertaken to date. Frequencies of tested biomarkers in the first 200 patients are shown in table 5 and have been reported elsewhere.14 For ten patients with known KRAS mutant tumours, we noted a potential association with benefit from mEOC+P, although this association was not significant (figure 3). In multivariate overall survival analysis incorporating performance status and disease stage, both KRAS mutation (HR 2·21, 95% CI 1·15–4·26; p=0·017) and PIK3CA mutation (HR 3·63, 95% CI 1·13–11·67, p=0·030) were negatively prognostic. No prognostic effect was associated with HER2 or PTEN status, and no BRAF mutations were noted (data not shown). These biomarker analyses are exploratory and were limited by the small numbers of mutations identified to date. Further biomarker analyses are ongoing in the full trial cohort. BODY.DISCUSSION: The REAL3 trial is one of two concurrent randomised phase 3 trials (the other being the EXPAND trial15) assessing the addition of anti-EGFR monoclonal antibodies to chemotherapy in first-line oesophagogastric cancer. Based on the findings of REAL3, use of panitumumab in combination with EOC cannot be recommended in an unselected population with advanced oesophagogastric adenocarcinoma, and was associated with inferior overall survival and PFS. Notably, this detrimental outcome in the experimental group was not predicted by the phase 2 endpoint of response rate (overall response rate 52% with mEOC+P). This trial does, however, confirm the efficacy of the EOC control group in this setting, with median overall survival and PFS results that are consistent with those previously reported in REAL2 (11·2 months for overall survival and 7·0 months for PFS).3 The poor outcome associated with mEOC+P in this trial did not seem to be attributable to increased treatment-related deaths, and therefore other potential explanations for our findings need to be considered. First, as reported previously,12 combination of panitumumab with full-dose EOC in the initial stages of the trial was associated with unacceptably high rates of grade 3 diarrhoea (four of the first five patients by cycle four). Therefore, we had to reduce the starting doses of oxaliplatin (by 23%) and capecitabine (by 20%) in the experimental group. Although these changes undoubtedly reduced the frequency of grade 3–4 diarrhoea with mEOC+P (17% in phase 3 population), they also served to reduce the dose intensity of chemotherapy, which is reflected in the reduced incidence of grade 3–4 neutropenia and peripheral neuropathy noted in the mEOC+P group. Additionally, the dose intensity data show a reduced proportion of patients achieving at least 80% of the planned capecitabine dose in the experimental group, suggesting that mEOC+P was still slightly more difficult to deliver than standard EOC. Second, a negative interaction might have occurred between panitumumab and one or more of the EOC components. Evidence in the setting of colorectal cancer suggests that the chemotherapy partner for anti-EGFR therapy might be an important determinant of treatment efficacy, with oxaliplatin-containing regimens yielding inconsistent results. The OPUS16 and PRIME11 studies provide evidence of improved outcomes with the addition of cetuximab and panitumumab respectively, whereas no benefit was associated with the addition of cetuximab in the COIN17 and NORDIC VII18 studies in the same setting. Recent cell-line data also suggest that greater synergy might exist between anti-EGFR therapy and irinotecan than with oxaliplatin.19 Additionally, the COIN trial17 results suggest that there might be a differential benefit from cetuximab dependent on the fluoropyrimidine partner, with patients receiving oxaliplatin plus fluorouracil seemingly deriving increased benefit compared with those treated with oxaliplatin plus capecitabine. At present, the significance of these potential interactions is unknown, and has not been assessed in the setting of oesophagogastric cancer. KRAS (common in colorectal cancer) and BRAF (common in malignant melanoma) are now known to be infrequent molecular events in oesophagogastric adenocarcinoma. Indeed, the 5·7% frequency of KRAS mutation in our population is in keeping with the 3–10% reported in other studies,20–22 and we did not note any BRAF mutations in 167 tumour samples tested. By contrast, gene copy number alterations (amplifications and deletions) seem to be a relatively frequent finding in oesophagogastric adenocarcinoma and are more likely to represent the key molecular alterations driving carcinogenesis. Two recent series23,24 of detailed genomic analyses in oesophagogastric adenocarcinoma reported that about 37% of tumours harbour copy number aberrations in genes that are deemed to be targetable, including KRAS, EGFR, HER2, and MET. Randomised clinical trials are therefore needed to establish whether targeting of these oncogenic signal transduction pathways can meaningfully improve outcomes for patients. In preclinical studies, cetuximab can decrease EGFR pathway signalling via reduced phosphorylation of EGFR and AKT in oesophagogastric cancer cell lines.25 In combination with chemotherapy, cetuximab results in synergistic inhibition of cell proliferation and enhanced apoptosis.25–27 In hypoxic gastric cancer cell lines the addition of anti-EGFR therapy reversed oxaliplatin resistance.26 Additionally, a synergistic antitumour effect of combined cetuximab and S-1 was apparent in gastric cancer cell lines overexpressing EGFR.25,27 In colorectal cancer, somatic mutations in codon 12, 13, or 61 of the KRAS oncogene confer resistance to panitumumab therapy.11,28 MET amplification with or without KRAS mutations might be associated with resistance to cetuximab therapy in gastric cancer cell lines;29 however, no validated predictive biomarkers for this setting exist. Unfortunately, despite preclinical data suggesting a role for anti-EGFR therapy in the treatment of oesophagogastric adenocarcinoma, the REAL3 trial findings are supported by two other phase 3 trials assessing anti-EGFR therapy in this disease setting. The EXPAND trial15 assessed the addition of cetuximab to a cisplatin-capecitabine doublet in 904 patients with previously untreated adenocarcinoma of the stomach and gastro-oesophageal junction, and did not meet its primary endpoint of improved PFS (HR 1·09, 95% CI 0·92–1·29, p=0·32).15 EXPAND also noted no improvement with the addition of cetuximab in either overall survival (HR 1·00, 95% CI 0·87–1·17, p=0·95) or overall response rate (30% in the experimental group vs 29% for controls). The COG trial30 assessed the anti-EGFR tyrosine-kinase inhibitor gefitinib compared with placebo in the second-line treatment of 450 patients with oesophageal and type I–II gastro-oesophageal junction cancers. This trial also did not meet its primary endpoint, with no improvement in overall survival (HR 0·90, p=0·285). However, improvements in PFS (HR 0·795, p=0·017) and disease control at 8 weeks (25·5% in the experimental group vs 16·0% in controls, p=0·014) were noted, suggesting some activity of gefitinib in a small undefined subset of patients. Taken together, these relatively consistent findings suggest that the EGFR pathway is unlikely to represent an important therapeutic target in most patients with oesophagogastric cancer (panel). The presented biomarker analyses accompanying the REAL3 trial are restricted by small patient numbers and low rates of tested mutations. However, this work is ongoing in the full trial dataset and these translational analyses represent a unique opportunity to further assess the molecular biology of advanced oesophagogastric adenocarcinoma within a randomised trial setting. Techniques such as gene-expression profiling and next-generation sequencing might help to provide further information regarding the driver genetic events in this complex disease. Furthermore, the evaluation of genetic aberrations in pathways linked to EGFR signalling could still offer the prospect of identification of a low-frequency biomarker that identifies a subpopulation of patients benefiting from anti-EGFR targeted therapy in this setting. This online publication has been corrected. The corrected version first appeared at thelancet.com/oncology on May 28, 2013

TITLE: Effects of trimetazidine on periprocedural microRNA-21 expression by CD4+ T lymphocytes in patients with unstable angina pectoris ABSTRACT.OBJECTIVE: Post-percutaneous coronary intervention (PCI) myocardial injury is related to the CD4+ T lymphocyte-mediated inflammatory response. microRNA-21 expression is associated with CD4+ T lymphocyte activation. The pre-PCI use of trimetazidine prevents periprocedural myocardial injury and reduces inflammatory cytokine levels. This study aimed to assess the effects of trimetazidine on periprocedural microRNA-21 expression by CD4+ T lymphocytes in patients with unstable angina pectoris. ABSTRACT.METHODS: A total of 252 patients with unstable angina pectoris were randomized to the trimetazidine (60 mg/d, administered 3 days before PCI, n=128) and control (no trimetazidine, n=124) groups. Serum CK-MB, cTnI, and hs-CRP levels were tested pre-PCI and 16-24 h post-PCI. Peripheral blood CD4+ T lymphocytes were isolated by magnetic activated cell sorting. Quantitative polymerase chain reaction was used to assess microRNA-21 and PDCD4 mRNA expression levels in CD4+ T lymphocytes, and western blot was used to evaluate PDCD4 protein expression. Enzyme-linked immunosorbent assay was used to assess serum TNF-α and IL-10 levels. ABSTRACT.RESULTS: Compared with the control group, the trimetazidine group had a lower frequency of patients with post-PCI serum CK-MB and cTnI levels higher than normal values; the trimetazidine group had also significantly lower serum hs-CRP and TNF-α levels, and higher IL-10 levels post-PCI. Finally, the trimetazidine group had significantly lower PDCD4 expression and higher microRNA-21 levels in CD4+ T lymphocytes post-PCI. ABSTRACT.CONCLUSIONS: Trimetazidine reduces the incidence of periprocedural myocardial injury, possibly by increasing microRNA-21 levels in CD4+ T lymphocytes and inhibiting PDCD4-mediated inflammatory response. BODY.INTRODUCTION: Periprocedural myocardial injury (PMI) is a frequent complication of percutaneous coronary intervention (PCI), especially among high-risk patients with acute coronary syndrome (ACS). Previous studies demonstrated that post-PCI activation of local CD4+ T lymphocytes and the subsequent myocardial inflammatory response probably play a role in PMI [1]. MicroRNAs (miRNAs) are endogenous non-coding small RNA molecules of 18-25 nucleotides. They are important post-transcriptional regulatory genes widely found in the non-coding regions of the genome, regulating cell proliferation, cell differentiation, inflammation, and apoptosis [2, 3]. Lu et al. [4] reported that miRNA-21 downregulation in CD4+ T lymphocytes enhanced Th1 cell-mediated inflammatory response and reduced Th2 cell-secreted anti-inflammatory cytokines, indicating that miRNA-21 upregulation may inhibit the inflammatory response mediated by over-activated Th1 cells. miRNA-21 inhibits its target gene (programmed cell death 4 (PDCD4) protein) and the corresponding signaling pathway, controlling the differentiation, activation, and functionalization of Th1 cells [4]. In addition, miRNA-21 upregulation is likely to reduce the secretion of the proinflammatory cytokine tumor necrosis factor (TNF)-α and promote that of the anti-inflammatory cytokine interleukin (IL)-10, suppressing Th1 cell-mediated inflammatory response [5, 6]. We hypothesized that post-PCI myocardial injury may be alleviated by miRNA-21 upregulation in CD4+ T lymphocytes, improving patient prognosis. Trimetazidine (TMZ), or 1-(2,3,4-trimethoxybenzyl) piperazine dihydrochloride, is a piprazine derivative that selectively inhibits fatty acid oxidation and stimulates glucose oxidation. TMZ improves myocardial blood supply by directly altering cardiac metabolic substrates and optimizing myocardial metabolism [7]. Labrou et al. [8] reported that the pre-PCI oral administration of TMZ significantly decreased post-PCI cTnI levels, alleviated PMI, and improved left ventricular function. Our preliminary studies demonstrated that pre-PCI TMZ is effective in lowering serum hs-CRP levels and reducing PMI, although the underlying mechanisms remain unclear. Recently, TMZ was found to upregulate miRNA-21 in cardiomyocytes and mitigate myocardial ischemia/reperfusion injury [9]. We inferred that the same mechanisms may play a role in the protective effects of TMZ in PMI. In this study, patients with unstable angina pectoris (UAP) were administered TMZ before PCI. Post-PCI expression levels of miRNA-21 and the target gene PDCD4 were assessed in CD4+ T lymphocytes. Serum levels of cytokines (including IL-10, TNF-α, and high-sensitivity C-reactive protein (hs-CRP)) and cardiac markers of myocardial necrosis (including creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI)) were also evaluated. This study may help clarify the cardioprotective mechanisms of TMZ during the periprocedural period, and provide a new approach for preventing and treating coronary artery diseases as well as PMI. BODY.RESULTS: BODY.BASELINE CHARACTERISTICS OF THE PATIENTS: The TMZ group was not significantly different from the control group in terms of age, sex, smoking status, diabetes mellitus, dyslipidemia, hypertension, history of cardiovascular disease, and medication use (all P>0.05). The two groups were comparable in terms of coronary lesion location, stenosis severity, stenosis number, and stent number (all P>0.05) (Tables 1 and 2). Table 1BODY.CLINICAL CHARACTERISTICS OF PATIENTS: ParametersTrimetazidine (n = 128)Control (n = 124)PMale 100 (78) 93 (75) 0.331 Age, years 65.18±10.97 63.87±10.13 0.326 BMI, kg/m2 23.8 ±2.7 24.1 ±3.2 0.565 Hypertension 59 (46) 52 (42) 0.295 Diabetes mellitus 29 (23) 26 (21) 0.432 Dyslipidemia 45 (35) 37 (30) 0.222 Previous AP 27 (21) 21 (17) 0.248 Current smoker 52 (41) 46 (37) 0.328 Systolic BP, mmHg 138.02±17.11 135.24±15.01 0.192 Diastolic BP, mmHg 82.98±11.67 78.19±10.88 0.483 LVEF, % 52.26±10.74 53.41±11.33 0.563 Treatment received Aspirin 128 (100) 124 (100) NS Clopidogrel sulfate 128 (100) 124 (100) NS Statins 128 (100) 124 (100) NS ACEIs/ARBs 105 (82) 93 (75) 0.114 Beta-blockers 74 (58) 67 (54) 0.317 Calcium antagonists 31 (24) 26 (21) 0.321 Nitrates 55 (43) 46 (37) 0.205 GPIIb/IIIa inhibitors 7 (5.5) 6 (4.8) 0.106 Values are numbers of patients with percentages in parentheses, or mean ± SD. BMI = Body mass index; AP = angina pectoris; BP = blood pressure; LVEF = left ventricular ejection fraction; ACEIs = angiotensin-converting enzyme inhibitors; ARBs = angiotensin-receptor blockers; GPIIb/IIIa = glycoprotein IIb/IIIa; NS = Not significant. Table 2BODY.TREATMENT CHARACTERISTICS OF PATIENTS: ParametersTrimetazidine (n = 128)Control (n = 124)PVessel treated  Left anterior descending 97 (76) 98 (79) 0.647  Left main 14 (11) 16 (13) 0.312  Left circumflex 54 (42) 47 (38) 0.341  Right coronary artery 70 (55) 61 (49) 0.127 One-vessel Intervention 73 (57) 73 (59) 0.402 Two-vessel Intervention 42 (33) 35 (28) 0.262 Multi-vessel Intervention 13 (10) 16 (13) 0.387 Stents per patient 1.72 ± 0.84 1.56 ± 0.79 0.622 Total stent length 25.17 ± 7.91 24.08 ± 8.13 0.193 Stent diameter 3.00 ± 0.44 3.00 ± 0.51 0.927 Stent deployment pressure 13.16 ± 2.68 12.63 ± 2.47 0.152 Drug-eluting stent 128 (100) 124(100) NS Dissection 0 (0) 0 (0) NS TIMI flow 3 before PCI 113 (88.3) 113 (91.1) 0.551 TIMI flow 3 after PCI 124 (96.9) 117 (94.4) 0.738 Values are number of patients with percentage in parentheses, or mean ± SD; TIMI = thrombolysis in myocardial infarction; NS = Not significant. BODY.SERUM CARDIAC MARKERS AND HS-CRP: Baseline serum CK-MB and cTnI levels were normal and not significantly different between the TMZ and control groups. Compared with the control group, the TMZ group had a significantly lower proportion of patients with post-PCI cTnI (38.2% vs. 70.4%, P<0.05) and CK-MB (18.7% vs. 44.2%, P<0.05) levels higher than the reference values at 16-24 h. Baseline serum hs-CRP levels were not significantly different between the TMZ and control groups (1.99±0.82 mg/l vs. 1.63±0.67 mg/l, P>0.05). Both groups had significantly elevated hs-CRP levels post-PCI compared with baseline values, with significantly higher levels found in the control group (5.85±1.32 mg/l vs. 9.63±0.67 mg/l, P<0.05). BODY.SERUM TNF-Α AND IL-10 LEVELS: Baseline serum TNF-α levels were not significantly different between the TMZ and control groups (7.14±0.77 pg/ml vs. 7.51±0.82 pg/ml, P>0.05). TNF-α levels were significantly increased in both groups at 16-24 h post-PCI, with higher amounts detected in the control group (13.18±2.62 pg/ml vs. 19.84±2.91 pg/ml, P<0.05). Baseline serum IL-10 levels were not significantly different between the TMZ and control groups (6.27±0.93 pg/ml vs. 6.41±0.86 pg/ml, P>0.05). IL-10 levels were significantly increased at 16-24 h post-PCI, with higher values detected in the TMZ group (15.13±2.36 pg/ml vs. 11.87±1.95 pg/ml, P<0.05). BODY.CHANGES IN MIRNA-21 LEVELS AND PDCD4 MRNA EXPRESSION IN CD4+ T LYMPHOCYTES: CD4+ T lymphocytes were sorted using magnetic beads and had >90% purity (Figure 1). Baseline miRNA-21 levels and PDCD4 mRNA expression were not significantly different between the two groups (P>0.05). The TMZ group had significantly reduced PDCD4 mRNA expression and increased miRNA-21 expression post-PCI (P<0.05). The control group had significantly higher PDCD4 mRNA levels post-PCI (P<0.05) (Figure 2), but miRNA-21 expression was not significantly changed (P>0.05) (Figure 3). Figure 1(A) Isolation of CD4+ T cells isolated from mononuclear cells. Pure CD4+ T cells are gated in LR. (B) Purity and viability of CD4+ T cells. Pure CD4+ T cells are gated in LR. UL = Upper left; UR = upper right; LL = lower left; LR = lower right. Figure 2BODY.FLUORESCENCE-BASED RT-PCR WAS USED TO DETECT PDCD4 GENE EXPRESSION: #P<0.05 vs. the control group (n=124, pre-PCI) as assessed by the paired t test. *P<0.05 vs. the trimetazidine group (n=128, pre-PCI) as assessed by the paired t test. Figure 3BODY.FLUORESCENCE-BASED RT-PCR WAS USED TO ASSESS MIRNA-21 LEVELS: *P<0.05 vs. the trimetazidine group (n=128, pre-PCI) as assessed by paired t test. BODY.CHANGES IN PDCD4 PROTEIN LEVELS IN CD4+ T LYMPHOCYTES: Baseline relative PDCD4 protein levels were not significantly different between the two groups (P>0.05). The TMZ group had significantly lower relative PDCD4 protein amounts post-PCI (P<0.05). The control group had significantly higher relative PDCD4 protein levels post-PCI (P<0.05) (Figure 4). Figure 4BODY.WESTERN BLOT ANALYSIS OF PDCD4 PROTEIN EXPRESSION: Relative expression levels of PDCD4 were determined by western blot. #P<0.05 vs. the control group (n=124, pre-PCI); *P<0.05 vs. the trimetazidine group (n=128, pre-PCI) as assessed by the paired t test. BODY.CORRELATIONS OF CD4+ T LYMPHOCYTE PDCD4 LEVELS WITH INFLAMMATION MARKERS AND MIRMA-21: PDCD4 levels were positively correlated with hs-CRP (r=0.714, P<0.01) and TNF-α (r=0.634, P<0.01) levels, and negatively correlated with miRNA-21 (r=-0.679, P<0.01) and IL-10 (r=-0.525, P<0.01) levels. BODY.DISCUSSION: PMI is a common complication of elective PCI, occurring in 30-70% of cases, as indicated by elevated CK-MB or cTnI levels [10]. Previous studies demonstrated that PMI significantly increases long- and short-term cardiovascular adverse events and leads to unfavorable prognosis. Meanwhile, the severity of PCI-induced myocardial injury is highly correlated with the incidence of long-term cardiovascular adverse events [11, 12]. As shown above, serum CK-MB and cTnI levels were elevated post-PCI, accompanied by changes of hs-CRP, TNF-α, and IL-10 amounts, indicating that the inflammatory response likely plays a role in PMI. Pre-PCI oral administration of TMZ may significantly improve PMI by reducing serum hs-CRP and TNF-α levels and elevating IL-10 amounts. TNF-α is an important pro-inflammatory cytokine that suppresses cardiac contractility and activates neutrophils. It stimulates neutrophils and endothelial cells to excessively express adhesion molecules, leading to tissue neutrophil accumulation and subsequent tissue impairment [13]. IL-10 is an anti-inflammatory cytokine secreted by Th2 cells. Derkacz et al. [14] reported that elevated serum IL-10 levels is associated with reduced incidence of PMI. Our results demonstrated that TMZ may alleviate PMI through its anti-inflammatory effects. PMI results from multiple factors such as microvascular embolization, coronary artery spasm, microcirculation hypoperfusion, and ischemia-reperfusion injury. Activation of cardiac CD4+ T lymphocytes and the subsequent myocardial inflammation play crucial roles in PMI. We previously reported that the PDCD4 protein is involved in post-PCI CD4+ T lymphocyte-mediated inflammatory response and subsequent PMI by increasing serum TNF-α levels and reducing IL-10 levels [15]. Recent studies have shown that miRNAs regulate the development of the immune system and immune response, participating in immunity-mediated diseases. miRNAs exert their functions mainly through the post-transcriptional regulation of protein synthesis [16–18]. Since PDCD4 is one of the key target genes of miRNA-21, it is possible to regulate PDCD4 protein synthesis by controlling miRNA-21 expression. Garchow et al. [19] showed that miRNA-21 downregulation increases PDCD4 expression levels in CD4+ T lymphocytes, indicating that miRNA-21 negatively regulates PDCD4 expression and reduces the inflammatory response. Therefore, we inferred that a drug likely to affect miRNA-21 expression would also likely decrease CD4+ T lymphocyte-mediated inflammatory response post-PCI and result in reduced PMI incidence. Recently, the cardioprotective effects of TMZ have been attributed to miRNA-21 regulation. Ma et al. [9] reported that TMZ improves myocardial ischemia-reperfusion injury by upregulating miRNA-21 and modulating the Akt and Bcl-2/Bax pathways. Yang et al. [20] showed that TMZ exerts its cardioprotective effects by suppressing hypoxia-reperfusion cardiomyocyte apoptosis and upregulating miRNA-21. As shown above, pre-PCI administration of TMZ significantly upregulated miRNA-21, effectively reduced PDCD4 secretion by CD4+ T lymphocytes, lowered serum TNF-α levels, and increased IL-10 levels, resulting in decreased PMI incidence. Interestingly, miRNA-21 correlated with PDCD4, which was also correlated with TNF-α and IL-10, as shown above. These findings confirmed the notion that TMZ exerts cardioprotective effects by regulating periprocedural miRNA-21 expression to reduce PMI. Limitations: Clinically important endpoints, such as death, myocardial infarction, and revascularization rate, were not recorded. In future studies, more clinically relevant endpoints will be considered. PDCD4 may be a downstream target of other factors (e.g., TGF-β, IL-2, IL-15, IL-12, etc.) [21, 22], but the present study did not examine all these factors that may be associated with periprocedural myocardial injury. On the other hand, miRNA-21 levels were only increased in the TMZ group and this suggests that the increase in miRNA-21 could be induced by TMZ. Additional studies are necessary to examine these factors. Furthermore, no mechanistic studies could be made in the present preliminary study. Finally, large-scale randomized controlled trials are warranted to validate these findings. BODY.MATERIALS AND METHODS: BODY.STUDY DESIGN AND PATIENTS: From February 2011 to July 2015, 252 patients with UAP (193 males and 59 females) were randomized to the TMZ (20 mg tid TMZ, for 3 days before PCI, n=128) and control (no TMZ, n=124) groups, according to a random number table. All patients with UAP had coronary stenosis confirmed by coronary angiography, and had indications for PCI. Cardiac makers (CK-MB and cTnI) were negative in these patients at baseline. No patients had contraindications to statins. All patients had either new-onset angina, worsening effort angina, or resting angina. The exclusion criteria were: 1) severe infections or tumors; 2) severe liver or renal dysfunction; 3) allergy to TMZ; 4) stroke; 5) left ventricular ejection fraction <30%; or 6) emergency PCI. The study protocol complied with the human trial regulations developed by the Institutional Review Board of Guangxi Medical University. Written informed consent was obtained from all participants. BODY.SAMPLE COLLECTION: Peripheral venous blood (20 ml) was drawn before PCI and at 16-24 h after PCI (mean of 20 h in both groups, P>0.05). One ml of blood was kept at room temperature for 20 min and centrifuged at 2000 rpm for 10 min; the resulting serum was assessed for CK-MB, cTnI, and hs-CRP levels. Serum TNF-α and IL-10 levels were measured with specific ELISA detection kits (eBioscience, USA), according to the manufacturer's instructions. The remaining 19 ml of peripheral venous blood were added with heparin for cell isolation. BODY.CELL ISOLATION: Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation. PBMCs were then resuspended in 1 ml of RPMI 1640 culture medium (Hyclone, USA), and 10 μl of PBMC suspension were added to 90 μl of phosphate-buffered saline for cell counting. The remaining PBMCs were sorted for CD4+ T lymphocytes, according to the instructions of the Dynabeads FlowComp Human CD4 kit (Dynal, Norway). Briefly, the beads were first washed according to the manufacturer's protocol. Then, 500 μL of cells were added with 25 μL of the FlowComp Human CD4 antibody, mixed well, and incubated 10 min at 4°C. The cells were washed with 2 mL of isolation buffer and centrifuged at 350 g. The supernatant was discarded and 75 μL of FlowComp Dynabeads were added. The cells were incubated at room temperature for 15 min under gentle agitation. One mL of isolation buffer was added, mixed for 2-3 s, and the tube was placed in a magnet for 2 min. The supernatant was discarded with the tube still in the magnet. Washing was repeated twice. The cells were released using the release buffer and the beads were caught using the magnet. The supernatant containing the bead-free cells was transferred to a new tube. Stained cells were analyzed on a FACScan flow cytometer (BD Biosciences, USA) with a 488-nm argon laser and Cell Quest 3.1 software (BD Biosciences, USA). Lymphocytes were gated by forward scatter (FSC) and side scatter (SSC). The sorted lymphocytes were counted. Cell viability was evaluated by 0.4% Trypan blue staining. CD4+ T lymphocytes with cell viability >90% were used for the subsequent experiments. BODY.MIRNA-21 LEVELS AND PDCD4 MRNA EXPRESSION BY QUANTITATIVE FLUORESCENCE POLYMERASE CHAIN REACTION (Q-PCR) IN CD4+ T LYMPHOCYTES: Total RNA was extracted from CD4+ T lymphocytes using TRIzol (KeyGen, China) according to the manufacturer's instructions, and quantitated using a Nanodrop system. RNA integrity was detected by 1% agarose gel electrophoresis. After adjusting the RNA concentration to 1 μg/μl, cDNA was synthesized using the Reverse Transcription Kit (Fermentas, Lithuania). PCR products were assessed by the SYBR Green I method, in a total reaction volume of 20 μl. Primers for miRNA-21 and the internal control U6 were provided by GeneCopeia (Maryland, USA; primers ID: hsmq-0057 and hsnRNAU6). The following primers (Sangon Biotech, China) were used: PDCD4, forward 5′-AAC TGT GCC AAC CAG TCC AA-3′ and reverse 5′-TCT TCT CAA ATG CCC TTT CAT C-3′; GAPDH forward 5′-GAG TCA ACG GAT TTG GTC GT-3′ and reverse 5′- GAC AAG CTT CCC GTT CTC AG-3′. The reaction system and parameters were based on the manufacturer's instructions. Each specimen was detected in multiple wells, and each reaction system included negative control wells. PCR products were sequenced. The final results were analyzed by the 2−ΔΔCT method. BODY.WESTERN BLOT FOR PDCD4 PROTEIN LEVEL ASSESSMENT: Sorted CD4+ T lymphocytes were treated with 100 μl of protein lysis buffer and centrifuged at 12,000 r/min for 20 min at 4°C. Supernatants were transferred to clean centrifuge tubes and tested for protein concentration using the bicinchoninic acid assay, with GAPDH as internal control (Beyotime Institute of Biotechnology, China). Equal amounts of protein were separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with 5% stacking gel, and transferred onto PVDF membranes by the semi-dry method for 40 min. The membranes were blocked in Tris-buffered saline-Tween containing 5% skimmed milk for 1 h, and incubated with the primary antibody (PDCD4, Abcam, USA; 1:2500) at 4°C overnight. After five washes of 5 min with TBST, the samples were incubated with 1:5000 infrared fluorescent secondary antibody (LI-COR, USA) at room temperature for 2 h. The membrane was then scanned on an Odyssey double-color infrared laser imaging system. BODY.STATISTICAL ANALYSIS: Data were analyzed with SPSS 23.0 (IBM Corporation, Armonk, USA). Continuous data are presented as mean ± standard deviation. Inter-group comparison was performed by paired t test. Multiple group comparison was performed by one-way analysis of variance (ANOVA) with the Fisher's least significant difference post hoc test. Categorical data were expressed as frequency and constituent ratio, and compared by the chi-square test. P<0.05 was statistically significant.

TITLE: Comparing use of lidocaine periprostatic nerve block and diclofenac suppository alone for patients undergoing transrectal ultrasound guided prostate biopsy ABSTRACT.INTRODUCTION: The objective of this study is to make a comparison between the effectiveness of the diclo- fenac suppository alone and periprostatic local anaesthesia infiltration alone to reduce the pain during a transrectal ultrasound-guided prostate biopsy procedure. ABSTRACT.MATERIAL AND METHODS: Between January 2014 and December 2015, 100 patients from two centres who were scheduled for transrectal ultrasound guided prostate biopsy (TRUS Bx) were included in the study. Patients were divided into two groups; diclofenac suppository group (Group 1) and Lidocaine group (Group 2). After the prostate biopsy, patients were requested to fill out a visual analogue scale in order to evaluate their pain level during the procedure. ABSTRACT.RESULTS: Since each group had been examined for VAS score, statistical differences were detected for VAS 0 and VAS 1. VAS 0 score was stated in 38 (71%) patients in Group 2, and in 25 (50%) patients in Group 1 (p = 0.040). VAS 1 score was stated in 9 (17%) patients in group 2 and 19 (38%) patients in Group 1 (p = 0.030). ABSTRACT.CONCLUSIONS: Although statistical difference was detected at lower VAS scores (0 and 1) in favor of the lidocaine group during transrectal ultrasound guided prostate biopsies, there was not a significant difference in higher (>2) VAS scores; which was made with 100 mg of diclofenac suppository. Therefore, diclofenac suppository can be used as an alternative to periprostatic nerve block made with lidocaine. BODY.INTRODUCTION: Transrectal prostate biopsy is the existing diagnostic procedure for the identification of prostate cancer. In the beginning, this procedure was carried on with the transperineal method. Later, the transrectal method was preferred. In 1989, core biopsies having at least 10 pieces replaced the sextant biopsy concept: which was identified primarily by Hodge et al. [1]. Despite developing technology, pain control during prostate biopsy procedure is usually required. The pain during the procedure originates from the movement of the transrectal ultrasound probe towards the prostate capsule and rectal wall, and from the tissues while the sample from the prostate is being removed. This procedure is generally performed in an outpatient clinic. To reduce the pain, different methods of anaesthesia are applied, varying from regional anaesthesia to systematic analgesia. Although every method has specific advantages, there is no consensus on which analgesia method is the best. However, there is a considerable number of patients complaining about pain during the procedure. Despite the application of local anaesthesia, 65–90% of patients complain about varying degrees of discomfort, from uneasiness to intense pain [2]. It is reported that, pain during the prostate biopsy is derived from the needle stimulating sensory nerves in the prostate capsule and the parenchyma in the course of its insertion into the prostate capsule through the rectum [3]. The most common preferred anaesthetic methods are the utilization of intrarectal gel application orperiprostatic local anaesthesia infiltration alone, or the combination of these two methods. In addition to these, suppository painkillers are also used separately or in combination with other methods. Nevertheless, there is no such study which compares the applications of periprostatic local anaesthesia and diclofenac suppository alone to reduce pain during prostate biopsy. The aim of this study is to make a comparison between the effectiveness of the utilization of diclofenac suppository alone and periprostatic local anaesthesia infiltration to reduce pain during prostate biopsy procedure which is accompanied by transrectal ultrasound guidance. BODY.MATERIAL AND METHODS: Between January 2014 – December 2015, due to PSA increase and/or abnormal rectal examination, 100 patients from two centres who were scheduled fortransrectal ultrasound guided prostate biopsy (TRUSBx) were included in the study. Detailed medical history was collected from all patients and physical examinations were done. Patients who had abnormal rectal examination and PSA value higher than 2.5 ng/ml were included in the study. Patients were informed about the procedure and their written permission was taken. Patients who were allergic to lidocaine or non-steroidal anti-inflammatory drugs, who had a history of acute prostatitis, gastrointestinal ulcer or anorectal surgery, and those who had second TRUS biopsies because of their high or rising PSA, were excluded from the study. On the day before the prostate biopsy procedure, patients were started on 500 mg of ciprofloxacin orally twice a day for antibiotic prophylaxis, and it was continued for two more days after the procedure. A rectal enema was used one hour before the biopsy for rectal cleaning. Patients were divided in two groups, Group 1 (diclofenac suppository) and Group 2 (periprostatic lidocaine infiltration). Group 1 had a 100 mg Diclofenac suppository introduced one hour before the biopsy. Group 2 was given10 cc of 1% Lidocaine with an 18 g Chiba needle to both periprostatic areas in the lateral decubitus position, with the guidance of an ultrasound probe. One hour after the application of either drug, a 12 piece core biopsy, six specimens from each lobe, were taken from the base, apex and middle zones of the prostate using disposable prostate needles. After the prostate biopsy, patients were requested to fill the visual analogue scale to evaluate the pain level during the procedure. The visual analogue scale represents the severity of pain on a 10-cm line, with zero expressing no pain at all, and 10 being pain so severe that it cannot withstood. All patients were asked to wait for at least two hours after the procedure, and were examined for any kind of acute complications. Patients without any complaints and who were urinating normally were sent home. In case of necessity, patients were recommended to use analgesic and anti-inflammatory drugs. All patients included in the study were invited back to the hospital two weeks after the procedure to discuss biopsy results and were simultaneous revaluated with regard to late complications, such as, hematospermy and persistent hematuria. BODY.STATISTICAL ANALYSIS: The data was analysed with SPSS (Version 22.0, SPSS Inc., Chicago, IL, USA). Descriptive statistics with a normal distribution were presented as a mean ±standard deviation; those with a non-normal distribution were presented as median; and ordinal variables were presented as number of cases and percentage (%). Normality distributions of the groups were evaluated with Shapiro-Wilk normality tests. To evaluate the homogeneity of variance, we used Levene's test. The significances of the difference between the two independent groups were evaluated by using an independent sample T-test in case of normally distributed data and Mann-Whitney U Test in case of data that was not normally distributed. We also used chi-square test to compare the proportion of visual analogue scale (VAS) scores. P value <0.05 was considered statistically significant. BODY.RESULTS: 103 patients, between the ages of 49 and 82, underwent transrectal ultrasound guided prostate biopsy. The average age was found 64.14 ±6.7 in the Diclofenac suppository group (Group 1) and 64.98 ±7.6 in the Lidocaine group (Group 2). There was no statistically significant difference between the two groups regarding age (p = 0.557>0.05). The average PSA value was calculated as 16.16 ±31.7 for Group 1 and 30.92 ±35 for Group 2. Statistical difference regarding PSA values were detected between the two groups (p = 0.027<0.05). The average prostate volume was calculated as 51.34 ±19.15 for Group 1 and 56.1 ±18.67 for Group 2 (p = 0.081>0.05). There is no significant difference regarding prostate volume between the two groups. Table 1. Descriptive statistics of Group 1 and Group 2 Since two groups had been examined in terms of VAS score, statistical difference was detected for VAS 0 and VAS 1. A VAS 0 value was stated by 38 (71%) patients in Group 2, and by 25 (50%) patients in Group 1 (p = 0.040). A VAS score of 1 was stated by 9 (17%) patients in Group 2 and by 19 (38%) patients in Group 1 (p = 0.030). When all patients were evaluated, the maximum VAS score identified was 5 (Tables 2 and 3). Table 1Descriptive statistics of Group 1 (diclofenac – D) and Group 2 (lidocaine – L) Diclofenac-lidocaineN (D/L)Minimum (D/L)Maximum (D/L)Mean (D/L)Std. Deviation (D/L)Age 50 / 53 49 / 50 78 / 82 64.14 / 64.98 6.77 / 7.640 PSA 50 / 53 3.10 / 3.30 200.00 / 144.0 16.16 / 30.92 31.70 / 35.07 Prostate Volume 50 / 53 20 /30 110 / 145 51.34 / 56.10 19.15 / 18.67 Table 2Visual Analogue Scale Group Cross tabulation GroupplidocainediclofenacVAS 0 Count 38 25 0.040* % within Group 71.7% 50.0% 1 Count 9 19 0.030* % within Group 17.0% 38.0% 2 Count 5 0 – % within Group 9.4% 0.0% 3 Count 1 4 % within Group 1.9% 8.0% 5 Count 0 2 – % within Group 0.0% 4.0% Total Count 53 50 % within Group 100.0% 100.0% Table 3Visual analogue scale for diclofenac (D) and lidocaine (L) Frequency (D/L)Percent (D/L)Valid Percent (D/L)Cumulative Percent (D/L)VAS 0 25/38 50.0/71.7 50.0/71.7 50.0/71.7 VAS 1 19/9 38.0/17.0 28.0/17.0 88.0/88.7 VAS 2 0/5 0/9.4 0/9.4 0/98.1 VAS 3 4/1 8.0/1.9 8.0/1.9 96.0/100.0 VAS 5 2/0 4.0/0 4.0/0 100.0/0 Total 50/53 100.0/100.0 100.0/100.0 BODY.DISCUSSION: For prostate cancer diagnosis, the current standard method is taking a core biopsy from prostate tissue by transrectal ultrasound guidance. During this procedure, which can be very painful, many topical analgesics, such as lignocaine gel, glyceryl trinitrate and analgesia methods, such as an eutectic mixture of local anaesthetics (EMLA) cream, intravenous or inhalation anaesthesia, periprostatic nerve block, and oral or intrarectal analgesics are used [4, 5]. In a study made by Irani J et al., it is reported that 19% of patients are not willing to undergo a repeat biopsy in cases where no analgesia is applied [6]. Generally during a biopsy, a periprostatic nerve block is applied with local anaesthetic agents to control the pain. Also, intravenous sedation or diclofenac suppository in addition to a periprostatic nerve block is used, depending on the clinician's choice. The autonomic nerve fibres of the prostate originate from the inferior hypogastric plexus. The nerves innervating the prostate arrive to the prostatic tissue passing from the prostate capsule and outside of the denonvillier's fascia. The prostatic capsule is surrounded with many nerve fibres and ganglions. This is named plexus prostaticus or plexus periprostaticus [7]. The pain felt during a prostate biopsy is due to the stimulation of the sensory nerves in the prostate capsule and the parenchyma caused by the movement of the needle passing through the rectal wall and penetrating the prostate capsule during the procedure [3]. All anaesthesia methods aimed at reducing the pain during prostate biopsy are based on desensitization of these nerve fibres. Therefore, because the prostate tissue includes a visceral nerve source, systematic anti-inflammatory drugs and various forms of diclofenac can be useful for these patients [8]. Although there is not a consensus on the amount of core that should be taken for diagnosis during a prostate biopsy, the most widely accepted view is to take at least eight core biopsies. Taking fewer cores may cause insufficient diagnosis and taking more may cause increase in the complication ratio after the procedure. In this study, we compared the effectiveness of using a diclofenac suppository alone and a periprostatic nerve block using lidocaine as a local anaesthetic agent, during transrectal ultrasound guided prostate biopsy. Since diclofenac is a cyclooxigenase inhibitor, it can be used as a systemic or local agent. Oral suppository and injection forms are available. Analgesic, antipyretic and anti-inflammatory effects demonstrate themselves by inhibiting prostaglandin synthesis [9]. Peak concentrations are reached in about 30 to 60 minutes after the introduction of the diclofenac suppository in adults, but this could take longer with oral administration [9]. Therefore, utilization of the suppository form is more effective and convenient to prevent and eliminate the pain acutely. In our study, we applied the diclofenac suppository to our patients intrarectally one hour before the procedure. We did not encounter any problems during the application of the diclofenac suppository and there was no feedback reporting that the patients in the diclofenac suppository group did not want to use the drug. During TRUS guided prostate biopsy, utilization of diclofenac suppository intrarectally in addition to a prostatic nerve block which is applied with local anaesthetic agents is evaluated in various studies. Published studies on this issue show that this combination is reliable and effective in decreasing the pain during the prostate biopsy. There was no difference detected between the two groups regarding complications [8, 10]. In a study made by Adiyat et al., it was reported that diclofenac suppository or diclofenac patches could be used as complementary treatment [11]. Another study made by Ooi et al. states that this combination does not provide any specific reduction in pain nor it does improve the tolerability of the procedure [4]. In a study made by Batch T and Tauber R, in which the utilization of diclofenac suppository in addition to intrarectal lidocaine gel was compared to placebo, regarding their effectiveness to prevent pain, it was stated that the patients in the diclofenac suppository group showed lower pain scores than the ones in the placebo group [12]. In another study, in which patients were given a ±100 mg diclofenac suppository in addition to 10 ml 2% lidocaine gel application one hour before the procedure, it was reported that the pain score was statistically lower than the patients in the control group who were given intrarectal glycerine (3.4 ±1.3 versus 5.9 ±1.7 p<0.0001). They also remarked that intrarectal diclofenac suppository application is a simple, safe and reliable method in reducing pain and developing patient tolerance during a transrectal prostate biopsy [13]. In a randomized study made by Haq et al., which compared diclofenac suppository and placebo during prostate biopsy, it was remarked that the diclofenac suppository, which had been applied one hour before the procedure, provided more relief as compared to the placebo and had was a VAS score of 2.8 [14]. We also did not detect any difference regarding pain levels between the two groups. As it can be seen in the table, even the ratio of the patients having VAS scores of 3 or less in the group of periprostatic nerve block with lidocaine was 98.1%, while the ratio in the Diclofenac suppository group was 96%. No differences regarding side effects such as hematuria, hematospermy and rectal bleeding were detected between the two groups. Three patients were catheterized due to globe vesical, which developed after the procedure. Two of these patients were in the diclofenac suppository group and the other was in the periprostatic nerve group. All three patients were discharged after conservative treatment. One patient, who received a periprostatic nerve block, had been hospitalized because of high fever. He recovered well after treatment. No life threatening infective complications were encountered. After periprostatic nerve block, haemorrhagic and infectious complications rarely occur [15]. In a study made by Turgut et al., it was reported that the complication incidence related to local anaesthesia infiltration is 2% [16]. In a study made by Song et al., it was stated that patients had syncope with a ratio of 1% after the periprostatic nerve block [17]. Another study from Sen et al. reported that syncope developed in only one patient (1.6%) [15]. Although statistical differences were detected at lower VAS scores (0 and 1) in favor of the lidocaine group during transrectal ultrasound guided prostate biopsies, there was no significant difference in higher (>2) VAS scores between two groups. Therefore, we are under the opinion that a diclofenac suppository can be used as an alternative to a periprostatic nerve block made with lidocaine. BODY.CONCLUSIONS: In transrectal ultrasound guided prostate biopsy, a 100 mg intrarectal diclofenac suppository showed similar analgesic effects as a periprostatic nerve block which with 10 cc of 1% lidocaine. Introduction of a 100 mg intrarectal diclofenac suppository was well tolerated by patients and no side effects of the drug were encountered. BODY.CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

TITLE: Effects of fenoldopam on kidney function parameters and its therapeutic efficacy in the management of acute kidney injury in dogs with heatstrokeSEGEV et al. ABSTRACT.BACKGROUND: Acute kidney injury (AKI) is common in dogs, but evidence of efficacy of its treatment is lacking. ABSTRACT.OBJECTIVE: To evaluate the efficacy of fenoldopam in the management of AKI. ABSTRACT.ANIMALS: Forty dogs with naturally occurring heatstroke. ABSTRACT.METHODS: Dogs were prospectively enrolled and divided into treatment and the placebo groups (fenoldopam, constant rate infusion [CRI] of 0.1 µg/kg/min or saline, respectively). Urine production (UP) was measured using a closed system. Urinary clearances were performed at 4, 12, and 24 hours after presentation to estimate the effect of fenoldopam on UP, glomerular filtration rate (GFR) and sodium fractional excretion (NaFE). ABSTRACT.RESULTS: At presentation, severity of heatstroke, based on a previously developed scoring system, was similar between the study groups, but was significantly worse in nonsurvivors compared with survivors. Fenoldopam administration was not associated with hypotension. Overt AKI was diagnosed, based on the International Renal Interest Society guidelines in 22/40 (55%) of the dogs. Overall, 14/40 dogs (35%) died, with no significant (P = .507) mortality rate difference between the fenoldopam (6/20 dogs; 30%) and placebo (8/20; 40%) groups. The proportion of dogs with AKI did not differ between the fenoldopam and the placebo groups (9/20; 45% versus 13/20; 65%, respectively; P = .204). There were no differences in UP, GFR, and NaFE between the fenoldopam and the placebo groups. ABSTRACT.CONCLUSION AND CLINICAL IMPORTANCE: Fenoldopam CRI at 0.1 µg/kg/min did not have a clinically relevant effect on kidney function parameters in dogs with severe heatstroke‐associated AKI. BODY.ABBREVIATIONS: ABParterial blood pressure AKIacute kidney injury CRIconstant rate infusion DA‐1dopamine‐1–receptor DAPdiastolic ABP GFRglomerular filtration rate ICUintensive care unit IRISInternational Renal Interest Society MAPmean ABP NaFEsodium fractional excretion PPpostpresentation SAPsystolic arterial blood pressure sCrserum creatinine SDstandard deviation UPurine production VTHveterinary teaching hospital 1BODY.INTRODUCTION: Acute kidney injury (AKI) in dogs is characterized by a sudden decrease in glomerular filtration rate (GFR), decreased urine production (UP) and alterations in solute excretion.1 Acute kidney injury leads to high morbidity and mortality, which are influenced mainly by the underlying etiology and therapeutic options.2, 3, 4, 5, 6, 7, 8, 9 The therapeutic approach for AKI generally is supportive, aimed at eliminating the underlying cause and correcting the hemodynamic and biochemical consequences of the attendant uremia.10 When medical management fails to control clinical and laboratory abnormalities, more advanced therapy, such as hemodialysis, can be applied, if available.9 Low‐dose dopamine therapy has been suggested as a treatment for AKI in human patients. Although past studies yielded conflicting results, recent evidence suggests that low‐dose dopamine has no role in prevention and management of AKI.11, 12, 13, 14 Fenoldopam is a selective dopamine‐1–receptor (DA‐1) agonist, inducing vasodilatation, and selectively increasing both renal cortical and outer medullary blood flow and GFR, similar to dopamine.15, 16, 17 Unlike dopamine, fenoldopam is not associated with adverse effects resulting from α‐ and β‐adrenergic receptor activation. A recent large‐scale study evaluating fenoldopam for prevention of contrast‐induced nephropathy in human patients failed to show an advantage of its use.18 Conversely, a study of AKI in human patients in intensive care units (ICU) demonstrated that fenoldopam (CRI of 0.09 µg/kg/min IV) led to a significant decrease in the occurrence of AKI.19 Yet another study failed to show similar effects.20 Finally, a recent meta‐analysis, including studies of fenoldopam in human patients in ICU and those undergoing major surgery concluded that fenoldopam significantly decreased the risk for AKI, the need for renal replacement therapy, and the mortality rate.21 The latter study, however, excluded contrast‐induced nephropathy, and included only a few placebo‐controlled trials, in which fenoldopam doses varied, and thus was limited. A recent review of the current knowledge on prevention of AKI has concluded that the role of fenoldopam in preventing AKI warrants further exploration, because there is still a lack of controlled clinical studies evaluating its clinical efficacy, and its use for preventing AKI in human patients at risk for AKI remains controversial.22 The efficacy of fenoldopam as preventive therapy for AKI in animals at high risk for AKI has not been evaluated in a controlled study, and evidence regarding its efficacy in animals with AKI is limited. In a retrospective study of dogs and cats with AKI, fenoldopam had no beneficial effects, but its effects on UP, GFR, and solute excretion were not evaluated.23 Heatstroke in dogs results from exposure to a hot environment or strenuous physical exercise, leading to high body core temperature (>41°C) and central nervous system dysfunction.6 Regardless of the insult, heatstroke leads to severe systemic inflammatory response syndrome, multiple organ dysfunction, and often death.6 Acute kidney injury is a common complication of heatstroke, in both dogs and humans,6, 24, 25, 26 probably resulting from hemodynamic instability.25, 27 Necropsy results of dogs sustaining fatal heatstroke and measurement of sensitive markers of kidney damage in dogs with heatstroke show that AKI invariably is present in affected animals,28 and is a risk factor for death.6 For these reasons, heatstroke is an excellent model for naturally occurring AKI in dogs, and can be used to test a variety of therapeutic and preventive interventions in AKI. We hypothesized that fenoldopam would increase GFR, UP, and sodium fractional excretion (NaFE) in dogs with naturally occurring heatstroke. The aim of this prospective, placebo‐controlled study was to investigate the efficacy of fenoldopam for decreasing the occurrence and severity of heatstroke‐associated AKI in dogs, and potentially preventing heatstroke‐induced AKI. Specifically, we sought: (1) to assess the influence of fenoldopam on sequential changes in GFR, UP, and NaFE; (2) to assess the proportion of AKI in dogs receiving fenoldopam versus placebo; and (3) to assess if fenoldopam would decrease the death rate in dogs naturally sustaining heatstroke. 2BODY.MATERIALS AND METHODS: 2.1BODY.STUDY DESIGN AND RANDOMIZATION: This prospective, randomized, double‐blinded, placebo‐controlled clinical trial was approved by our Institutional Animal Care and Use Committee (KSVM‐VTH/13–2009). Dogs presented to the Hebrew University Veterinary Teaching Hospital (VTH) between 2009 and 2015 and diagnosed with heatstroke were considered eligible for the study, depending on their owners' consent. Randomization was performed by drawing slip from an envelope indicating the treatment regimen. Envelopes were prepared by the VTH chief pharmacist, and investigators, attending clinicians, staff, and dog owners were blinded to the treatment regimen until the end of the study. Fenoldopam (Fenoldopam, Corlopam, 20 mg/2 mL, Teva, Milano) or saline (as placebo) were dispensed in identical syringes. 2.2BODY.CASE SELECTION: Dogs presented to the Emergency Service of the VTH and diagnosed with heatstroke were consecutively enrolled. Heatstroke was diagnosed based on a history of exposure to a hot environment, strenuous activity, or both, and presence of appropriate clinical signs, which had developed acutely only after the heat insult, including neurological dysfunction, collapse, and tachypnea. Dogs with coexisting medical conditions, and those that died or were euthanized within 4 hours of hospitalization, were excluded. 2.3BODY.DEFINITIONS: The diagnosis of AKI was made according to International Renal Interest Society (IRIS) guidelines.29 Dogs that died or were euthanized because of grave prognosis, despite ongoing intensive therapy, were defined as nonsurvivors. 2.4BODY.TREATMENT PROTOCOL AND MONITORING: Dogs enrolled in the study were treated and managed according to a previously described protocol for heatstroke.6 Upon admission, the urinary bladder was catheterized, and the catheter connected to a closed urinary system. Urine production was monitored and recorded q4h during the first 24 hours after presentation. Dogs were treated initially with crystalloid fluids as an IV bolus, at 30 mL/kg and, as needed to correct hypovolemia and shock, if present, and thereafter, as needed to restore all fluid deficits over a period of 4 hours. Once hypovolemia and dehydration were corrected, IV fluid therapy was continued initially at 5 mL/kg/h, and thereafter adjusted to meet ongoing fluid loss (eg, diarrhea and vomiting), and based on UP, periodic physical assessment of hydration status, and body weight measurement, to maintain normal hydration throughout hospitalization. Dogs received mannitol (20% Osmitrol, Baxter, Deerfield, Illinois; IV bolus, 0.5 g/kg over 20 min) q4h over the first 12 hours from admission to prevent and treat potential brain edema, a common complication of heatstroke.6, 25 Dogs also were given ampicillin (Penibrin, Sandoz GmbH, Kundl Austria; 20 mg/kg IV q8h) and enrofloxacin (Baytril, 5%, Bayer Animals Health Leverkusen; 15 mg/kg slow IV q24h) to prevent and treat gastrointestinal bacterial translocation, and famotidine (Famotidine, West‐Ward, Eatontown, New Jersey; 0.5 mg/kg IV q24h) and metoclopramide (Metoclopramide. Pramin 10MG/2ML Rafa Laboratories, Jerusalem; 0.04 mg/kg/h IV by CRI) to prevent and treat gastric ulceration and vomiting, respectively. Fenoldopam (study group) or saline (placebo group) was initiated 4 hours post‐presentation (PP), after verifying that systolic arterial blood pressure (SAP) was >120 mm Hg. Fenoldopam and saline placebo were diluted in 100 mL of saline. Fenoldopam was administered IV at a CRI of 0.1 µg/kg/min, whereas the placebo group was given saline at identical dilution and rate. Urine volume was measured gravimetrically q4h. Urine samples were obtained q4h and stored at −80°C pending analysis. Measurements included body weight, UP, serum, and urine chemistry, GFR (estimated by endogenous creatinine clearance) and urinary NaFE. Arterial blood pressure (ABP) was measured using oscillometry (Cardell, Midmark, Tampa, FL) q4h. A previously described scoring system for heatstroke was used.30 2.5BODY.URINARY CLEARANCES: Urine and blood samples were collected to calculate NaFE and creatinine clearance at 4, 12, and 24 hours PP, to estimate GFR and NaFE, as previously described.31, 32, 33 Briefly, 2 sequential 30‐minute quantitative urine collections were performed each time, to assess GFR and NaFE. Blood was collected in plain tubes with gel separators when the first urine sample was collected, and when the second urine sample collection ended. The urinary bladder was emptied and irrigated 3 times with 10 mL of sterile water, at the start and end of each urine collection. The urinary bladder rinse volume, at the end of each urine collection period, was added to the urine volume collected. The urine volume of each urine collection was measured gravimetrically. Urine sample aliquots were stored at −80°C pending analysis. Blood samples were allowed to clot, centrifuged, and serum chemistry analysis (Cobas Integra 400 Plus, Roche, Mannheim, Germany; at 37°C) was performed within 60 minutes from collection, including urea, creatinine (sCr), and sodium. The results for the two individual 30‐minute clearances were averaged to provide an estimate of the renal clearance of solutes. The renal clearance (C), and FE of solute (x) were calculated conventionally,1 where Cx = Ux V/Px and FEx = Cx/Ccr, where Cx = clearance of solute x; Ccr = clearance of creatinine (mL/min); V = urine volume (mL/min); Ux = urine solute (x) concentration; Px = serum solute (x) concentration. 2.6BODY.STATISTICAL ANALYSIS: Descriptive statistics were used to document sequential changes in kidney function tests and solute FE throughout the study period. The distribution pattern of continuous variables was examined using the Shapiro‐Wilk test. Continuous variables (eg, UP and GFR) were reported as mean ± SD or median and range, and compared between groups using the Student t‐test and the Mann Whitney U‐test, for normally and non‐normally distributed data, respectively. Categorical variables (eg, proportions) were compared between groups using χ2 or Fisher Exact tests. The Friedman test was used to assess changes in continuous variables over the first 24 hours PP. All tests were 2‐tailed, and P < .05 was considered significant. Statistical analysis was performed using a statistical software package (SPSS 22.0 software, Chicago, Illinois). 3BODY.RESULTS: 3.1BODY.DOGS: The study included 40 dogs, 20 in each treatment group. There were 33 males (neutered, 7) and 7 females (neutered, 4). There was no mean age difference (P = .747) between the fenoldopam (4.6 ± 2.7 years) and placebo (4.4 ± 2.3 years) groups. The study included 13 brachycephalic dogs (32.5%), including boxer (6 dogs), French bulldog (4), Pug (2), and English bulldog (1). There were 37 dolichocephalic dogs (67.5%), including mixed breed (6 dogs), golden retriever (4), Belgian Malinois (4), Labrador retriever (3), Samoyed, German shepherd and Siberian husky (2 each) and Dogo Argentino and Caucasian shepherd (1 each). There was no mean body weight difference (P = .569) between the fenoldopam (30.7 ± 12.5 kg) and placebo (32.5 ± 10.7 kg) groups. 3.2BODY.SEVERITY OF HEATSTROKE: The median rectal temperature at presentation to the VTH was 38.6°C (range 35.0–42.8°C). The median rectal temperature measured by the referring veterinarians (n = 11) before cooling was 42°C (range, 40.0–43.0°C). When a previously established severity scoring system for dogs with heatstroke was applied,30 the average score was 39.3 ± 14.9, with no difference (P = .939) between the fenoldopam (39.2 ± 3.3) and placebo (39.5 ± 3.4) groups. The severity score at presentation was significantly (P = .024) higher in the nonsurvivors (46.5 ± 12.5) as compared with the survivors (35.5 ± 15.0). Overall, 14/40 dogs (35%) died, with no significant (P = .507) mortality rate difference between the fenoldopam (6/20 dogs; 30%) and placebo (8/20; 40%) groups. 3.3BODY.ARTERIAL BLOOD PRESSURE: The overall means of systolic ABP (SAP), diastolic ABP (DAP) and mean ABP (MAP) at presentation were 130 ± 27, 75 ± 23, and 106 ± 23 mm Hg, respectively, with no treatment group differences (P = .601, P = .261, and P = .094, respectively). There were no differences between mean SAP, MAP, and DAP before and 4 hours after initiation of fenoldopam therapy (143 ± 26 versus 146 ± 35 mm Hg, P = .563; 106 ± 26 versus 116 ± 25 mm Hg, P = .220; and, 90 ± 27 versus 96 ± 29 mm Hg, P = .229, respectively). Systolic arterial hypotension (SAP < 100 mm Hg) was documented only on 2 occasions at presentation and after fenoldopam initiation, and MAP was >60 mm Hg in all dogs after initiation of fenoldopam therapy. 3.4BODY.ACUTE KIDNEY INJURY: Kidney function parameters are presented in Table 1. Based on IRIS guidelines, 22/40 (55%) of the dogs had AKI (Grade I, 1/22; Grade II, 5/22; Grade III, 11/22, and Grade IV, 5/22). Of these, 6 dogs developed overt AKI during hospitalization, whereas the rest already had presented with overt AKI. No significant difference was observed in the proportion of dogs that developed overt AKI during hospitalization between the fenoldopam and placebo groups (3 of each group, P = 1.00) Table 1Kidney function parameters at presentation Fenoldopam (n = 20)Placebo (n = 20)AnalyteMedian (range)Median (Range) P valueSerum creatinine (mg/dL) 1.6 (0.5‐4.7) 1.7 (0.7‐2.6) .583 Urine production (mL/kg/h) 6 (0.4–28) 4.3 (0.9–19) .295 Glomerular filtration rate (mL/min/kg) 0.92 (0.0–3.7) 0.6 (0.0–3.1) .201 Sodium fractional excretion 0.07 (0.01‐0.35) 0.07 (0.01‐0.41) .678 Serum creatinine was measured at presentation and urine production, sodium fractional excretion, and GFR were measured 4 hours from presentation. The proportion of dogs with AKI was not significantly different (P = .204) between the fenoldopam (9/20; 45%) and the placebo (13/20; 65%) groups. The mortality rate of dogs diagnosed with AKI (11/22 dogs; 50%) was significantly (P = 0.046) higher than in those in which overt AKI was absent (3/18; 17%). 3.5BODY.UP, GFR, AND NA FE: Urine production was high in both the study and placebo groups (Figure 1). Oliguria (<1 mL/kg/h), based on UP once fluid deficits had been corrected (ie, 4 hours PP), was documented in only 2 dogs (1 of each group). A significant UP change occurred over time in the fenoldopam group (P = .044), but not in the placebo group (P = .683; Figure 1), but no significant UP differences were identified between groups at any time point during the first 24 hours PP (Figure 1). Figure 1A, urine production (mean ± SD) over time in dogs sustaining heatstroke, receiving fenoldopam (n = 20) or placebo (n = 20). B, glomerular filtration rate (mean ± SD) over time in dogs sustaining heatstroke, receiving fenoldopam (n = 20) or placebo (n = 20). C, sodium fractional excretion (mean ± SD) over time in dogs sustaining heatstroke, receiving fenoldopam (n = 20) or placebo (n =20) No change in GFR was documented during the first 24 hours PP in the fenoldopam group (P = .155), but a significant change was noted in the placebo group (P = .019), with no significant GFR group differences at any time point (Figure 1). The NaFE was high in both groups at presentation (Figure 1). There was no significant change NaFE over time, nor was there a difference, at any time point, between the treatment groups during first 24 hours PP (Figure 1). 4BODY.DISCUSSION: We evaluated the effect of fenoldopam on sequential changes in GFR, UP, and NaFE during the first 24 hours of hospitalization in dogs with naturally occurring heatstroke‐induced AKI. Our results do not support the use of fenoldopam at a dosage of 0.1 µg/kg/min for managing heatstroke‐associated AKI. Heatstroke often is complicated by AKI, which adversely affects mortality in dogs.6 Although, based on IRIS criteria, only 55% of the dogs in our study had AKI, previous studies using biomarkers with higher sensitivity compared with sCr concentration (which is a functional marker) or by histopathology of renal tissue in fatal cases of heatstroke in dogs both suggest that AKI is invariably present in these animals.25, 28 Fenoldopam, a DA‐1 receptor agonist, induces vasodilatation of the peripheral arteries. It has been studied as a substitute for dopamine, which failed to show beneficial effects in managing AKI in human patients.11, 13 In the renal proximal tubule, DA‐1 receptor activation results in vasodilatation of the renal arteries, promoting natriuresis and diuresis, increasing renal blood flow, and therefore, fenoldopam is considered part of the management of AKI in human patients.34, 35, 36, 37 Fenoldopam may increase UP by increasing both natriuresis and GFR.38 In our study, UP was high at presentation (Figure 1), with no significant difference between the study and the placebo groups, suggesting that the effect of fenoldopam on UP was mild at best. This lack of apparent group difference might have resulted from relatively aggressive IV fluid therapy and the use of mannitol, which were administered early PP. Many dogs with AKI, and typically those with heatstroke, which invariably sustain kidney damage,28 are not oliguric when initially presented,3 a finding that appears to be associated with a better prognosis compared to oliguric AKI.1, 9, 39 Therefore, the current treatment protocol in our hospital includes aggressive IV crystalloid therapy and mannitol to prevent oliguria or anuria, resulting in relatively high UP. This high, albeit variable, UP among the dogs might have negated the differences between the fenoldopam and placebo groups. The relatively low GFR documented here in dogs sustaining heatstroke further exemplifies the insensitivity of sCr as a kidney function marker, and further supports previous results, indicating that kidney injury is present dogs with heatstroke, even if not reflected by increased sCr at presentation.28 Because dogs sustaining heatstroke usually are presented for veterinary care relatively promptly after the acute and severe clinical signs of this syndrome have begun,6 sCr concentration at presentation does not reflect the severity of kidney injury, because a steady state has not been reached. Glomerular filtration rate in our study increased only mildly and insignificantly after fenoldopam administration, and no significant GFR differences were noted between treatment groups. Information regarding the effect of fenoldopam on GFR in the veterinary literature is limited and inconsistent. In cats, GFR initially decreased, but had increased by 6 hours after initiation of fenoldopam therapy.40 In healthy dogs, fenoldopam has led to a 0.78 mL/min/kg increase in the GFR, as measured by iohexol clearance, but the increase was inconsistent among the study dogs, with some showing only minimal to no GFR change. In humans, fenoldopam administered to healthy subjects at dosages ranging from 0.03 to 0.3 µg/kg/min led to significant GFR changes.41 The inconsistency of the present and previous results might be related to differences between the species studied, the dosage used (0.1‐0.8 µg/kg/min),20, 38, 41 and the health status of the patients (ie, healthy animals versus those with AKI). Nonetheless, the currently used fenoldopam dosage did not increase GFR in a clinically relevant manner. In our study, NaFE also was not significantly different between groups. In a previous study of healthy dogs, NaFE increased significantly after fenoldopam administration,38 but, the increase was inconsistent and relatively mild (0.016). Possibly, the higher fenoldopam dosages used in that previous study, as well as the higher variability that exists among dogs with kidney injury, render the changes in NaFE insignificant. In our study, the relatively high NaFE noted 4 hours PP reflects the extensive disruption of tubular resorptive function in the injured kidneys, consistent with a previous study.1 Fractional sodium excretion might be influenced by the sodium‐containing fluids administered, which can increase NaFE, possibly negating any treatment group difference. Fenoldopam is used in human patients to manage hypertension and prevent and manage AKI.42, 43, 44 In our study, fenoldopam administration did not decrease the extent of azotemia or the occurrence of kidney injury (based on IRIS guidelines29) compared to placebo, in agreement with a previous study using fenoldopam (0.05 µg/kg/min IV by CRI) in human patients.20 Consistent with previous studies, presence of overt AKI was significantly associated with death.6 The lack of efficacy of vasodilators in clinical trials of AKI may result from their relatively late administration in the disease course. We therefore have elected to study the effect of fenoldopam in a naturally occurring model, where dogs are mostly presented relatively soon after the insult. Nonetheless, in most dogs (73%) the diagnosis of AKI was already made by presentation. Therefore, the effect of fenoldopam on the progression of kidney injury cannot be assessed in these animals. Fenoldopam, a dompaminergic agonist, may promote vasodilatation and hypotension.42, 43 To prevent hypotension, fenoldopam administration was initiated 4 hours PP, after hydration status and hypovolemia had been corrected. Hypotension episodes after fenoldopam initiation were uncommon, and with aggressive IV fluid administration, it is quite safe at the currently administered dosage, in agreement with a previous pharmacokinetic study of fenoldopam in dogs,34 and others, in which fenoldopam administration was uncommonly (7%) associated with hypotension in dogs.23, 38 In a previous study, a scoring system for dogs sustaining heatstroke at presentation for care was developed.30 Using that scoring system in our cohort showed that the median scores were similar in both treatment groups, suggesting that the 2 groups were comparable at presentation, but was significantly higher in the nonsurvivors. This scoring system originally was developed and tested on the same cohort, which was a limitation of that study.30 The present cohort is relatively small for the independent validation of this scoring system. Nevertheless, the score was significantly higher in the nonsurvivors compared with the survivors, potentially supporting its validity, but this suggestion warrants further evaluation in larger cohorts. Our study had several limitations. First, the limited cohort size might have rendered our study underpowered, and thereby prone to type‐II error. For the occurrence of AKI documented in our study to become statistically significant, a substantially higher number of dogs should ideally have been included (assuming an alpha of 5% and power of 80%). Therefore, larger scale studies are warranted to evaluate the effect of fenoldopam on the occurrence of AKI in dogs at risk before concluding that fenoldopam does not decrease that risk. Second, 45% of the dogs did not fulfill the IRIS criteria for AKI, limiting our conclusion as to the potential beneficial effect of fenoldopam, and therefore it should be tested in dogs with overt AKI. Third, the fenoldopam dosage used in our study was relatively low (although comparable to that used in previous studies in humans). When our study protocol was originally designed, veterinary information regarding the effect of fenoldopam on kidney function parameters was unavailable, and the dosage used here was based on the human medicial literature. Since then, fenoldopam has been evaluated in veterinary patients, using higher dosages (0.5–0.8 µg/kg/min). The relatively low fenoldopam dosage used in our study might have resulted in a lack of detectable beneficial effects of the drug. Additional studies using higher fenoldopam dosages in dogs are warranted. In conclusion, administration of fenoldopam at a dosage of 0.1 μg/kg/min IV by CRI in dogs with heatstroke was not associated with any observable adverse effects. However, it had no beneficial effects on UP, GFR, NaFE, or on the occurrence and severity of AKI. BODY.CONFLICT OF INTEREST DECLARATION: The authors declare that they have no conflict of interest with the content of this article. BODY.INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION: Authors declare no IACUC or other approval was needed. BODY.OFF‐LABEL ANTIMICROBIAL DECLARATION: Authors declare no off‐label use of antimicrobials.

TITLE: The effect of iodine supplementation in pregnancy on early childhood neurodevelopment and clinical outcomes: results of an aborted randomised placebo-controlled trial ABSTRACT.BACKGROUND: Concern that mild iodine deficiency in pregnancy may adversely affect neurodevelopment of offspring has led to recommendations for iodine supplementation in the absence of evidence from randomised controlled trials. The primary objective of the study was to investigate the effect of iodine supplementation during pregnancy on childhood neurodevelopment. Secondary outcomes included pregnancy outcomes, maternal thyroid function and general health. ABSTRACT.METHODS: Women with a singleton pregnancy of fewer than 20 weeks were randomly assigned to iodine (150 μg/d) or placebo from trial entry to birth. Childhood neurodevelopment was assessed at 18 months by using Bayley Scales of Infant and Toddler Development (Bayley-III). Iodine status and thyroid function were assessed at baseline and at 36 weeks’ gestation. Pregnancy outcomes were collected from medical records. ABSTRACT.RESULTS: The trial was stopped after 59 women were randomly assigned following withdrawal of support by the funding body. There were no differences in childhood neurodevelopmental scores between the iodine treated and placebo groups. The mean cognitive, language and motor scores on the Bayley-III (iodine versus placebo, respectively) were 99.4 ± 12.2 versus 101.7 ± 8.2 (mean difference (MD) −2.3, 95 % confidence interval (CI) −7.8, 3.2; P = 0.42), 97.2 ± 12.2 versus 97.9 ± 11.5 (MD −0.7, 95 % CI −7.0, 5.6; P = 0.83) and 93.9 ± 10.8 versus 92.4 ± 9.7 (MD 1.4, 95 % CI −4.0, 6.9; P = 0.61), respectively. No differences were identified between groups in any secondary outcomes. ABSTRACT.CONCLUSIONS: Iodine supplementation in pregnancy did not result in better childhood neurodevelopment in this small trial. Adequately powered randomised controlled trials are needed to provide conclusive evidence regarding the effect of iodine supplementation in pregnancy. ABSTRACT.TRIALS REGISTRATION: The trial was registered with the Australian New Zealand Clinical Trials Registry at http://www.anzctr.org.au. The registration number of this trial is ACTRN12610000411044. The trial was registered on 21 May 2010. BODY.BACKGROUND: Iodine is essential for the production of thyroid hormones. Iodine deficiency encompasses a spectrum of disorders, including impaired growth and neurodevelopment [1]. Pregnant women have a higher risk of iodine deficiency because of their increased iodine requirement [2]. Severe iodine deficiency during pregnancy causes cretinism and irreversible brain damage in the offspring [1]. There is increasing concern that mild to moderate iodine deficiency during pregnancy—which has emerged as a public health issue in a number of developed countries, including Australia and the UK—may lead to cognitive deficits and learning disability in children. A recent systematic review of randomised controlled trials (RCTs) highlighted that the effect of iodine supplementation in pregnancy in regions with mild to moderate iodine deficiency is unclear because none of the RCTs conducted in those regions assessed developmental outcomes of children [3]. There is some evidence from non-randomised intervention studies suggesting that iodine supplementation in pregnancy in regions of mild to moderate iodine deficiency may improve cognitive function in children [4]. Conversely, adverse effects on child development in relation to iodine supplementation in pregnancy have also been reported from cohort studies [5]. These emerging data have been differentially interpreted by expert groups and government authorities worldwide, resulting in various approaches to address this public health issue. The American Thyroid Association and the European Thyroid Association have recommended routine iodine supplementation in pregnancy [6, 7], whereas the recommendation for iodine supplements in pregnancy by the World Health Organization (WHO) is dependent on the iodised salt coverage and the iodine status of the population [8]. There are no specific recommendations from the government authorities in the UK or USA. In Australia and New Zealand, mandatory iodine fortification of bread was implemented in 2009. In addition, the Australian National Health and Medical Research Council (NHMRC) recommended that all pregnant women take an iodine supplement of 150 μg/d [9] because of concerns that the mandatory iodine fortification may not be adequate to prevent iodine deficiency in pregnant women [10]. The present study was designed as a double-blind placebo-controlled multi-centre RCT in Australia and New Zealand. The aim of the study was to assess the effect of iodine supplementation in pregnancy over and above the mandatory iodine fortification on childhood neurodevelopment and other clinical outcomes, including pregnancy outcomes, maternal thyroid function, mental health and general well-being. BODY.METHODS: BODY.PARTICIPANTS AND RECRUITMENT: Pregnant women were approached to enter the trial at their first antenatal visit. They were eligible if they were less than 20 weeks’ gestation with a singleton pregnancy. Women were excluded if they were taking a supplement containing iodine, had a history of thyroid disease or drug or alcohol abuse, their fetus had a known major abnormality, or if English was not the main language spoken at home. Ethical approval was obtained from the Human Research Ethics Committee at each participating centre (Children, Youth & Women’s Health Service Research Ethics Committee and the Flinders Clinical Research Ethics Committee), and written informed consent was obtained from each participant. The trial was registered on the Australian and New Zealand Clinical Trials Registry (#ACTRN12610000411044). BODY.RANDOMISATION AND TREATMENT: Women were randomly assigned to iodine or placebo in the ratio of 1:1 through a web-based randomisation service. The randomisation schedule was generated independently with balanced, variable-sized blocks and was stratified by centre, parity (0 versus ≥1), and gestational age at randomisation (≤16 weeks’ versus >16 weeks’). Neither the women nor the research staff were aware of the women’s group allocation. The iodine supplements contained 150 μg of iodine per tablet as potassium iodide, whereas the placebo tablets contained no iodine. Women were asked to take one trial tablet daily from randomisation to the end of their pregnancy. The trial tablets were manufactured and donated by Blackmores (Warriewood, Australia). All tablets were similar in size, shape, smell and colour. Women were supplied with excess tablets and were asked to return any unused tablets at the end of the pregnancy as a measure of compliance. Regular telephone calls during the intervention period were made to monitor adverse side effects and encourage compliance. BODY.OUTCOME ASSESSMENTS: BODY.CHILDHOOD NEURODEVELOPMENT: The primary outcome of childhood neurodevelopment was assessed at 18 months of age by using the cognitive, language and motor composites of the Bayley Scales of Infant and Toddler Development, third edition (Bayley-III) [11]. Composite scores are age-standardised with a normative mean of 100 and a standard deviation of 15. The standardised scores were also classified into the categories of any developmental delay (<85) and moderate/severe developmental delay (<70). The social-emotional behaviours and adaptive behaviours scales of the Bayley-III were also administered. Bayley-III was used to assess childhood development as it is the most widely used objective measure of early development. It is used extensively in research including neonatal trials and has a moderate association with later intelligence quotient (IQ) [12]. BODY.IODINE STATUS AND THYROID FUNCTION: At study entry and 36 weeks’ gestation, women were asked to collect a spot urine sample to assess urinary iodine concentration (UIC). UIC was determined by using the modified WHO Method A [13]. A blood sample was also collected at baseline by venepuncture to assess thyroid hormone concentrations, including thyroid-stimulating hormone (TSH), thyroglobulin (Tg), free triiodothyronine (fT3) and free thyroxine (fT4). Thyroid function of newborns was also assessed from cord blood (TSH, fT3, fT4 and Tg) and from newborn screening (TSH only). A breast milk sample 6 weeks after birth was collected, where possible, to assess breast milk iodine concentration by inductively coupled plasma mass spectrometry method [14]. BODY.PREGNANCY AND OTHER CLINICAL OUTCOMES: Pregnancy and birth outcome data were collected by blinded review of medical records. Small and large for gestational age were defined as birth weight below the 10th and above the 90th percentile, respectively, for gestational age and sex [15]. Preterm birth was defined as gestational age at birth of fewer than 37 completed weeks. Gestational age was estimated on the basis of a composite of the last menstrual period and a dating ultrasound early in pregnancy, where available. General health and well-being of women were assessed by using validated questionnaires, including the 36-Item Short Form Health Survey (SF-36) [16] and the Depression Anxiety Stress Scale (DASS) [17] at study entry, 36 weeks’ gestation and 6 weeks’ post-partum. BODY.OTHER ASSESSMENTS: Demographic characteristics were recorded at study entry. Safety of the intervention was assessed via telephone calls to women at 2 weeks after randomisation, and at 20, 28 and 36 weeks’ gestation, to assess potential side effects, including the frequency of sweating and palpitation, gastrointestinal side effects, including nausea, diarrhoea and constipation, as well as any serious adverse events defined as death or intensive care admission of either mother or baby. BODY.SAMPLE SIZE AND STATISTICAL ANALYSIS: A sample size of 542 women per group was required to detect a minimum clinically meaningful difference in the Bayley-III composites of 4 points between the treatment groups with 90 % power, using a Bonferroni adjusted α = 0.017 for each of the three primary Bayley-III composites and allowing for adjustment for potential confounders and loss to follow-up. A 4-point difference was considered important in the context of other nutritional deficiencies and environmental exposures that have resulted in major public health campaigns [18, 19]. The primary analysis was based on the intention-to-treat principle by comparing the outcomes between the randomised treatment groups. Continuous outcomes were analysed by using t tests, or Kruskal-Wallis tests for non-normally distributed outcomes. Binary outcomes were analysed by using chi-squared tests, or Fisher’s exact tests for rare outcomes. Owing to a substantial sex imbalance between groups, a post hoc analysis was performed with adjustment for infant sex for the Bayley-III composites and anthropometric measurements at birth. No adjusted analysis was performed for other outcomes, because of the small sample size. BODY.RESULTS: Of the 645 women approached, 205 met the eligibility criteria. A total of 59 out of 205 (29 %) women were enrolled in the study from the Women’s & Children’s Hospital and the Flinders Medical Centre in Adelaide, Australia, between June 2010 and October 2010 (Fig. 1). Twenty-nine were randomly allocated to iodine and 30 to placebo (Fig. 1). The baseline demographic characteristics of the participants are listed in Table 1. The trial was stopped early, before recruitment began in other Australian centres and New Zealand, because the funding body (NHMRC) withdrew its support for the trial. The NHMRC considered a placebo-controlled trial inconsistent with its recommendation for iodine supplementation in pregnancy. The ethics committees did not withdraw approval for the trial, but in view of the funding body’s position it supported the trial management committee’s decision to unblind the study and to follow all randomly assigned women as planned to monitor safety. Women were informed of their treatment group allocation and were provided with a copy of the NHMRC recommendation for iodine supplementation in pregnancy [9]. All women except two (one from each group) consented to continue with the follow-up after unblinding. The mean gestational age at unblinding was 33 ± 7 weeks. The mean duration of intervention before unblinding was 16 weeks (range 2–23 weeks). Nine (31 %) women in the iodine group and 4 (13 %) in the placebo group gave birth before unblinding. The decision regarding whether to continue taking trial supplements or to take commercially available iodine supplements was at the women’s discretion. Five women in the iodine group and 18 in the placebo group stopped taking trial supplements. Only one woman in the placebo group commenced iodine supplements after unblinding.Fig. 1Participant flowchart Table 1Baseline demographic characteristics Iodine (n = 29)Placebo (n = 30)Age at trial entry, yearsa 29.1 (5.7) 29.8 (5.1) Gestational age at trial entry, weeksa 15.2 (2.6) 14.9 (2.4) Primiparousb 13 (44.8) 13 (43.3) Completed secondary educationb 20 (69.0) 22 (73.3) Completed further educationb 22 (75.9) 24 (80.0) Smoke at trial entry or leading up to pregnancyb 7 (24.1) 5 (16.7) Miscarriage in previous pregnancyb 8 (27.6) 12 (40.0) Previous or current depressionb 4 (13.8) 7 (23.3) BMI at trial entrya 25.3 (5.9) 23.6 (3.9) Pre-pregnancy BMIa 23.6 (5.5) 22.2 (4.3) Infant sex: maleb 20 (71.4) 9 (31.0) BMI body mass index aData are presented as mean (standard deviation) bData are presented as number (percentage) BODY.NEURODEVELOPMENT OF CHILDREN: The mean composite score of the children did not differ between the iodine and the placebo groups in cognitive (99.4 ± 12.2 versus 101.7 ± 8.2, mean difference (MD): −2.3; 95 % confidence interval (CI) −7.8, 3.2; P = 0.42), language (97.2 ± 12.2 versus 97.9 ± 11.5, MD −0.7; 95 % CI −7.0, 5.6; P = 0.83) or motor (93.9 ± 10.8 versus 92.4 ± 9.7, MD 1.4; 95 % CI −4.0, 6.9; P = 0.61) development (Table 2). Adjustment for sex of the children did not change the outcome (data not shown). There were no differences in the percentage of children with any or moderate/severe developmental delay or in the parent-reported social-emotional behaviours and adaptive behaviour scores between the groups (Table 2).Table 2Developmental outcomes from the Bayley Scales of Infant and Toddler Development (Bayley-III) OutcomeIodine (n = 27)Placebo (n = 26)Treatment effect (95 % CI) P valueCognitive Standardised Scorea 99.4 (12.2) 101.7 (8.2) −2.3 (−7.8, 3.2) 0.42 Language Standardised Scorea 97.2 (12.2) 97.9 (11.5) −0.7 (−7.0, 5.6) 0.83 Motor Standardised Scorea 93.9 (10.8) 92.4 (9.7) 1.4 (−4.0, 6.9) 0.61 Social-Emotional Standardised Scorea 105.8 (15.9) 105.4 (16.2) 0.4 (−8.5, 9.3) 0.93 Adaptive Behaviour Standardised Scorea 105.2 (15.2) 103.5 (14.9) 1.8 (−6.4, 10.0) 0.67 Cognitive scoreb <85 1 (3.7) 0 (0.0) N/A >0.99 Cognitive scoreb <70 1 (3.7) 0 (0.0) N/A >0.99 Language scoreb <85 3 (11.1) 3 (11.5) N/A >0.99 Language scoreb <70 0 (0.0) 0 (0.0) N/A N/A Motor scoreb <85 2 (7.4) 5 (19.2) N/A 0.25 Motor scoreb <70 1 (3.7) 0 (0.0) N/A >0.99 CI confidence interval, N/A not applicable aThe data are presented as mean (standard deviation), and the treatment effect is the difference in means bThe data are presented as number (percentage) BODY.IODINE STATUS AND THYROID FUNCTION OF MOTHERS AND INFANTS: The median (interquartile range) UICs of women at baseline (15 weeks’ gestation) and at 36 weeks’ gestation are shown in Fig. 2. The UIC increased from baseline to 36 weeks in the iodine group (median change (interquartile range) from baseline was 87 (−1 to 134) μg/l, P = 0.001) but not the placebo group (−2 (−76 to 37) μg/l, P = 0.71). The median (interquartile range) breast milk iodine concentration at 6 weeks after birth was 107 (79–147) μg/l overall, and there was no difference in breast milk concentration between the iodine and the placebo group (Table 3). Similarly there were no differences in cord blood fT3, fT4, TSH and Tg concentration between the groups (Table 3). Mean TSH of newborn or percentage of newborn with TSH of more than 5 mU/l from the routine newborn screen test also did not differ between the groups.Fig. 2Median urinary iodine concentration of women. GA gestational age Table 3Biomarkers of iodine status IodinePlaceboEffect (95 % CI) P valueCord blood n = 19 n = 22 Free triiodothyronine, pmol/la 2.3 (0.4) 2.3 (0.6) 0.0 (−0.4, 0.3) 0.81 Free thyroxine, pmol/la 14.4 (2.1) 13.8 (2.3) 0.6 (−0.9, 2.2) 0.40 Thyroid-stimulating hormone, mIU/lb 8.2 (5.9–13.5) 6.6 (4.5–9.6) N/A 0.25 Thyroglobulin, μg/lb 73.0 (48.0–100.0) 64.0 (44.0–121.0) N/A 0.66 (n = 28) (n = 29) Newborn TSH, mIU/la 2.1 (1.0) 2.2 (1.2) −0.1 (−0.7, 0.5) 0.79 Newborn TSH > 5c 0 (0.0) 0 (0.0) N/A N/A (n = 20) (n = 25) Breast milk iodine at 6 weeks’ post-partum, μg/lb 106.0 (84.0–146.0) 124.0 (76.0–155.0) N/A 0.74 CI confidence interval, N/A not applicable, TSH thyroid-stimulating hormone aData are presented as mean (standard deviation), and the treatment effect is the difference in means bData are presented as median (interquartile range) cData are presented as number (percentage) BODY.PREGNANCY AND OTHER CLINICAL OUTCOMES: The mean birth weight, length, head circumference and gestational age at birth did not differ between the groups (Table 4). Adjustment for infant sex did not change the outcome (data not shown). The percentage of infants classified as low birth weight (<2500 g) or small for gestational age or large for gestational age, or with a neonatal complication or major congenital abnormality, did not differ between the treatment and placebo groups (Table 4). Other pregnancy outcomes, including rate of preterm birth, miscarriage, still birth and antenatal hospital admission, were also not different between the groups (Table 4).Table 4Pregnancy and neonatal outcomes IodinePlaceboTreatment effect (95 % CI) P valuePregnancy outcome n = 29 n = 29 Miscarriagea 1 (3.5) 0 (0.0) N/A >0.99 Stillbirtha 0 (0.0) 0 (0.0) N/A N/A Caesarean sectiona 9 (32.1) 5 (17.2) N/A 0.19 Post-term inductiona 3 (10.7) 4 (13.8) N/A >0.99 Gestational diabetesa 1 (3.6) 2 (6.9) N/A >0.99 Pregnancy-induced hypertensiona 0 (0.0) 0 (0.0) N/A N/A Pre-eclampsiaa 0 (0.0) 0 (0.0) N/A N/A Antenatal hospital admissiona 6 (22.2) 7 (24.1) N/A 0.87 Neonatal outcome n = 28 n = 29 GA at birth, weeksb 39.3 (37.8 – 40.4) 39.7 (39.3 – 40.3) N/A 0.23 Preterm birth, GA < 37 weeksa 5 (17.9) 4 (13.8) N/A 0.73 Birth weight, gc 3325.4 (474.7) 3204.3 (689.4) 121.1 (−194.2, 436.3) 0.45 Birth length, cmc 49.4 (2.3) 48.7 (3.3) 0.7 (−0.9, 2.2) 0.37 Birth head circumference, cmc 34.6 (1.3) 33.9 (2.2) 0.7 (−0.3, 1.7) 0.15 Placental weight, gc 533.5 (136.2) 514.4 (129.5) 19.1 (−114.9, 153.2) 0.77 Low birth weight, <2500 ga 1 (3.6) 3 (10.3) N/A 0.61 SGAa 3 (10.7) 2 (6.9) N/A 0.67 LGAa 2 (7.1) 4 (13.8) N/A 0.67 Major congenital abnormalitya 0 (0.0) 0 (0.0) N/A N/A Neonatal complicationa 5 (17.9) 5 (17.2) N/A >0.99 Admission to NICUa 0 (0.0) 2 (6.9) N/A 0.49 Neonatal deatha 0 (0.0) 0 (0.0) N/A N/A CI confidence interval, N/A not applicable, GA gestational age, SGA small for gestational age, LGA large for gestational age, NICU neonatal intensive care unit aData are presented as number (percentage) bData are presented as median (interquartile range) cData are presented as mean (standard deviation) No women had medically diagnosed depression in pregnancy, and one woman in the iodine group had post-natal depression. There were no differences in the SF-36 or DASS outcomes or the frequency of sweating and palpitation, gastrointestinal side effects or any serious adverse events between the treatment groups (data not shown). BODY.DISCUSSION: Our study is the first randomised, double-blind, placebo-controlled trial conducted in an industrialised country to assess the effect of routine iodine supplementation in pregnancy on childhood development. Although the unforeseeable early cessation of the trial resulted in a shorter duration of intervention and a significant reduction in the sample size, which may bias our results toward a null finding, we found no consistent trend of a higher or lower mean score in the neurodevelopmental outcomes between the iodine-supplemented and the placebo groups. Based on a recent national health survey of school-age children and non-pregnant adults, including childbearing aged women, Australia is no longer iodine-deficient after mandatory iodine fortification [20]. The question of whether iodine supplementation in pregnancy is required over and above the mandatory iodine fortification in Australia remains unanswered. Despite the small sample size, our findings are consistent with the only two published RCTs conducted in regions of severe iodine deficiency more than three decades ago [21, 22], which showed no difference in IQ or cognitive development of children between the iodine-supplemented and the control groups in the absence of overt iodine deficiency (i.e., cretinism). Concerns that mild iodine deficiency may lead to cognitive impairment were based largely on two non-randomised intervention studies which showed that children whose mothers commenced iodine supplements in the first trimester had better neurodevelopment than children whose mothers took iodine supplements in the third trimester [23] or no supplements in pregnancy [4]. However, both studies have major methodological limitations and small sample sizes, with only 13 % of the original cohort selected for developmental assessment in one of the studies [23]. Thus, the results are likely to be subject to bias. Currently there is a lack of evidence from RCTs investigating the effect of iodine supplementation in pregnancy on childhood development in populations of mild to moderate iodine deficiency. Findings from cohort studies investigating the relationship between mild iodine deficiency in pregnancy, defined as maternal UIC of less than 150 μg/l, and neurodevelopmental outcomes of children are inconsistent. Whereas two cohort studies showed that mild to moderate maternal iodine deficiency in early pregnancy was associated with lower IQ [24] and reduced educational outcomes [25], other cohort studies showed no difference in the developmental outcomes of children between mothers who had mild to moderate iodine deficiency or iodine sufficiency [5, 26]. The mean cognitive and language scores of the children in our study are comparable to a large sample of children (the DOMInO study [27]) prior to mandatory iodine fortification of bread in Australia. The mean composite motor score of children in our study is approximately half a standard deviation below the population mean and is considerably lower than the children in the DOMInO study [27]. Our study sample is unlikely to be representative of the general population, and this may partly explain the lower motor score, although the effect of mandatory iodine fortification in Australia on child development is unknown. A recent larger Spanish cohort study (>1500 mother-and-child pairs) in regions of iodine sufficiency showed that maternal intake of multivitamin supplements containing at least 150 μg of iodine per day was associated with an increased risk of Bayley motor score of less than 85 in children at 1 year of age compared with iodine supplements containing less than 100 μg per day [5]. This suggests that potential adverse effects of iodine supplementation in pregnancy at the recommended dose of 150 μg/d in regions of iodine sufficiency like Australia post-mandatory iodine fortification cannot be excluded in the absence of quality RCTs. We observed no differences in markers of thyroid function in cord blood or newborns between the groups, and this is consistent with findings from systematic reviews of RCTs [3, 28] in regions of mild to moderate iodine deficiency where the majority of the trials found no differences in thyroid hormone concentration between the iodine-supplemented and the control groups. This is in contrast to RCTs in the regions of severe iodine deficiency and suggests that pregnant women in regions of mild to moderate iodine deficiency are able to maintain adequate thyroid hormone production to meet increased requirements in pregnancy, and this may partly explain the lack of benefit of iodine supplementation in pregnancy on child development observed in our study. BODY.CONCLUSIONS: There are widespread recommendations for routine iodine supplementation in pregnancy, yet the efficacy and safety of routine iodine supplementation in pregnancy in a population with mild iodine deficiency or iodine sufficiency remain unclear. Although placebo-controlled randomised trials in such populations are viewed by some as unethical, conversely recommendations made in the absence of quality evidence also raise issues of ethical responsibility for clinicians and may result in lower compliance with such recommendations. A definitive RCT with an adequate sample size is warranted to provide the rigorous evidence necessary to inform clinical practice and public health policy in order to provide the best care for pregnant women and optimal growth and development of their children.

TITLE: The Glubran 2 glue for mesh fixation in Lichtenstein's hernia repair: a double-blind randomized study ABSTRACT.INTRODUCTION: With an average incidence rate of 11%, chronic pain is considered the most serious complication of inguinal hernioplasty after surgical site infection. One of the proposed solutions to this problem is to use tissue adhesive for mesh fixation, which helps prevent nerve and tissue damage. ABSTRACT.AIM: The goal of this study was to compare mesh fixation with the use of sutures vs. adhesive in Lichtenstein's inguinal hernia repair in a randomized, double-blind one-center study. ABSTRACT.MATERIAL AND METHODS: The study group consisted of 41 males with primary inguinal hernia undergoing Lichtenstein's repair (20 – adhesive; 21 – suture) and remaining in follow-up from July 2008 to November 2010. Randomization took place during the operation. The follow-up was performed by one surgeon (blinded) according to a pre-agreed schedule; the end-of-study unblinding was performed during the last follow-up visit, usually 16 months postoperatively. ABSTRACT.RESULTS: In 1 patient from the “adhesive” group, a recurrence was observed one year after the initial repair. The early postoperative pain was less intense in this group. In later postoperative periods the method of mesh fixation had no influence on the pain experienced by the patient. Other complications were not correlated with the method of mesh fixation. ABSTRACT.CONCLUSIONS: In this randomized, one-center double-blind clinical study of males with primary inguinal hernia it has been show during follow-up that the use of Glubran 2 cyanoacrylate adhesive for mesh implant fixation yields similar recurrence and chronic pain rates as the classical suture technique. In the early postoperative period, the pain reported by these patients was relatively weaker; patients undergoing adhesive mesh fixation experienced a quicker return to daily household activities. BODY.INTRODUCTION: One of the most crucial criteria for successful modern inguinal hernia repair is short recovery time combined with postoperative pain as minor as possible. Despite the evident advancement in the domain, these goals are still unachievable. The occurrence of postoperative chronic pain is especially disturbing as it is felt by 0-43% of patients, 11% on average [1]. Postoperative pain is the consequence of tissue dissection and postoperative local inflammation. As a result, the surrounding nerves become damaged and nociceptive stimulation occurs. Surgeon's experience, local complications, synthetic material implantation are considered to be dominant causative factors. Inflammation appears with the ligation of the hernia sack, neurectomy, when a nerve gets captured into a suture or scar around the implant and with ischemic testicle. Sutures applied behind the periosteum of the pubic tubercle lead to chronic periostitis, another source of the chronic pain. Surgical access and the way implant is fixed are irrelevant [2, 3]. Matyja et al. showed that tension-free technique does not influence the incidence of the postoperative pain [4]. Also the comparison of Lichtenstein and Desard technique did not reveal any significant differences in the early postoperative painful sensation [5]. The traditional method of penetrating mesh fixation in the groin has its flaws. Except for bleeding after femoral or epigastric vessels damage caused by a needle, sutures may cause tissue ischemia, muscle contracture and nerve damage. The change may be the source of postoperative and chronic pain. Remission of the chronic ailments after sutures are removed in the evidence of cause and effect reliance [6]. Attempts have been made to replace non-absorbable with absorbable sutures, but clinical trials did not show any significant differences [7, 8]. Similarly, staples or tackers caused new dangers, but did not relieve pain [9]. There are numerous articles on the prevention and pain occurrence after inguinal hernia repair in the literature. Reports concerning chronic pain after hernia repair are especially disturbing. Pain is diversely defined. In the following report, the British Pain Society definition was accepted: chronic pain is continuous pain, lasting over 12 weeks or – in case of surgical procedures or damages – it is pain occurring after tissues have healed. According to the International Association for the Study of Pain (IASP), chronic pain is recognized when it occurs after the surgery and lasts minimum 2 months. This definition is criticized as it does not take into account changes occurring later on when the pain starts to decrease gradually e.g. due to the termination of inflammation. Such sequence of events may be expected in hernioplasty: after tissue structures have been strengthened by synthetic material. Those issues are recapped in the newest reports [10]. Apposition of tissues without excessive tension is a relatively new factor mentioned, which corresponds to the experience of the surgical team. Too energetic surgical maneuvers (the strength applied here amounts up to 20 N) causes micro damages of the surrounding tissue which activates painful sensations occurring regardless of other prophylactic treatment. Two groups of patients should be acknowledged in pain assessment: young people, professionally and physically active and middle-aged or older patients with limited physical activity. The risk of chronic pain occurrence in the 2nd group is significantly lower [11]. Recently, the methods of implant fixation have been discussed during meetings. Penetrating (staplers/tackers) and adhesive (sealants) methods of fixation are compared and situations when an implant does not have to be fixed to the surrounding tissues are defined. The first indexed report dates back to 1984. Russian doctors, Shapkina et al. used cyanoacrylic sealant in surgery in children. One hundred and eight hernioplasties were performed with good results [12]. The next trials were performed a decade later by Farouk et al. who modified Lichtenstein method by fixing the mesh with cyanoacrylic adhesive in grown-up patients. No recurrences were observed in the 1st year after the surgery [13]. The results achieved using a fibrin sealant, created by mixing fibrinogen component with a thrombin solution, were presented for the first time by Canonico et al. The method was employed in patients with coagulation disturbances (1999), and next in healthy patients and professional athletes [14–16]. To determine a valid conclusion different methods of mesh fixation have to pass through all the phases of clinical trials. Having considered the importance of the issue and its practical implications on the one hand, and lack of reliable clinical information with regard to the usage of cyanoacrylic sealant (3 indexed randomised control trials – RCTs), on the other hand, we started our own, maintaining the highest possible methodological standard prospective, randomized, double-blind clinical trial (both a physician and patient). BODY.MATERIAL AND METHODS: Fourty-one male patients with primary inguinal hernia, operated on using Lichtenstein technique from July 2008 to November 2010 were included in the trial in the Department of General and Endocrine Surgery, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz. The inclusion criteria were as follows: age ≥ 21 years old, male sex, primary inguinal hernia (in case of contralateral hernia, one side was selected for the study). Patients aged below 21 years, with a recurrent or incarcerated hernia, after attempted reduction of a hernia (with hospital stay), with a postoperative scar in the area of the planned procedure, cryptorchism, varices of the spermatic cord (clinical trial – no such cases were observed), degenerative spine diseases or other pathologies causing pain radiating to the abdomen and groin were excluded from the study. Patients who did not fully understand the nature of the study or did not give their consent to participate were also excluded. The trial was planned according to the following scheme:patient selection (verification of diagnosis, assessment of inclusion and exclusion criteria), acquainting the patient with the schedule and goals of the clinical trial, patient's consent to treatment supported by patient's signature on an informed consent form, routine preparation for the procedure; fractionated heparin only in patients previously receiving anticoagulant treatment, without antibiotic prophylaxis, the procedure performed using Lichtenstein method – the course of the procedure identical in all patients until mesh fixation, intraoperative randomization, mesh implantation; soaking the mesh in 80 ml gentamicin/250 ml physiological saline solution before placement [17], finalizing the procedure and registering surgical variables, follow-up appointments on the 2nd (hospital discharge), 7th (suture removal), 30th, 60th, 180th and 360th postoperative day. Randomization was prepared with the use of software available at http://www.randomization.com/ (1st generator was employed). Information concerning mesh fixation was available in sealed envelopes in the operating room. During mesh fixation stage, randomly chosen envelope was opened and the information was discreetly passed to the surgeon so that the conscious patient would not be aware of the surgeon's proceedings. Data concerning mesh fixation ware gathered in a separate database, but were excluded from the surgical protocol, patient's history and release form. The way nerves were handled in the surgical field was described in each case (ilioinguinal nerve, hypogastric nerve and genital branch of genitofemoral nerve were always identified). Whether the nerve was left intact or excised was recorded in the database. Prolene (Ethicon) polypropylene mesh sized 15 cm × 7-10 cm was employed (its transverse size adapted to patient's anatomy). Glubran sealant (N-butyl -2-cyanoacrylate; GEM S.r.l., Viareggio, Italy) was used according to the manufacturer's instructions. Points of sealing approximated typical areas where sutures were placed, 1 mm was used each time. In the “suture” series, the mesh was fixed to the inguinal ligament with the continuous 2-0 polypropylene suture, and to the aponeurosis of the internal oblique muscle with single 2-0 PDS sutures. Closing off the inguinal canal, sutures for the subcutaneous tissue and suturing the skin were identical in both groups. The procedures were performed by 4 surgeons (26, 10, 4 and 1 procedure). The method of anesthesia was not standardized. The patient decided in agreement with the anesthesiologist on the basis of the clinical situation. The procedures were performed under general, subarachnoid or local anesthesia (50: 50 mixture of 1% lidocaine and 0.25% bupivacaine). In the postoperative period, ketoprofen was administered and in case of contraindications – tramadol. Doses of analgesics used were recorded. Patients were evaluated during follow-up appointments by a surgeon who did not know the method used and had no access to documentation. Patients had scheduled checkups, but in practice, with time they became less self-disciplined and the appointments got postponed. The initial tests took place 1 day before the procedure (they were marked as –1), the first checkup on the 2nd postoperative day. The actual median values for the subsequent follow-up appointments were: 10th, 32th, 78th, 202nd and 401st postoperative day (Table I). In the description below, the following designations were used: –1st, 2nd, 7th, 30th, 80th, 200th and 400th postoperative day. Table IPlanned and actual follow-up periods Scheduled appointment (post-operative day)Actual date of the appointment (days after procedure)MedianMin-max1st-3rd quartile–1 –1 – – 2 2 1-6 2-3 7 10 7-14 9-11 30 32 25-63 31-36 60 78 54-107 65-95 180 202 120-532 187-234 360 401 330-849 370-591 During the follow-up appointments, all complications, sensation disturbance, foreign body sensation and sexual dysfunction were notified. Return to driving, typical house chores, work and sport was recorded. Visual Analog Scale (VAS) scale was used to assess pain. On a 10 cm scale the patient marked the point corresponding to the pain intensity (0 – no pain whatsoever, 10 – agonizing pain). The distance from the beginning of the scale was measured in mm translating VAS into verbal assessment. It was assumed after Jensen et al. that 0-4 mm mean no pain, 5-44 mm mean mild pain, 45-74 mm moderate pain and over 75 sever pain [18]. In some cases patients were unable to unambiguously determine the intensity of pain sensation. Unblinding the patient and physician took place during the last follow-up appointment – on average 16.7 months after the procedure. The above described trial was accepted by the Nicolaus Copernicus University's Bioethical Commission (201/2008). BODY.STATISTICAL ANALYSIS: Normality distribution of variables was assessed and adequate parametric and non-parametric tests were employed. For a normal distribution, average value and standard deviation were used, for non-parametric distribution – median and 1st and 3rd quartile [always in square brackets]. VAS determination, repeated several times, was analyzed by ANOVA (Analysis of Variance) Friedman test. Differences between groups were tested by Mann-Whitney U test and by Pearson's χ2 statistics with Yates correction, correlation of variables – with Spearman Rank correlation test. Values of p < 0.05 were accepted as statistically significant. BODY.RESULTS: Randomized, clinical, double-blind trial (patient, physician) was carried out in 41 patients. It assessed the results of surgical treatment in patients with primary inguinal hernia using the polypropylene mesh fixed with sutures or Glubran 2 adhesive. Twenty patients had mesh fixed with sealant (sealant group) and 21 – traditionally with sutures (suture group). Demographic and surgical variables did not fundamentally differ in both groups (Table II). Table IIDemographic data and details of the surgery in both groups VariableSealant group (n = 20)Suture group (n = 21)Value of p Average age ± standard deviation 47.4 ±13.4 45.4 ±14.8 0.67 Manual work/headwork/no work 10/5/5 10/7/4 0.31 VAS before procedure 35.0 [15; 40] 23.0 [12; 30] 0.23 Hernia right side/left 10/10 11/10 0.88 Local/subarachnoid/general anesthesia 16/1/3 17/4/0 0.91 Hernia type oblique/straight/both 12/8/0 15/5/1 0.35 Width of hernia ring 1/2/3 fingers 8/8/4 11/8/2 0.26 Dissected hernia sac/reduced 2/18 5/16 0.40 Inguinal nerves* dissected/intact 16/4 16/5 0.87 Length of hospital stay 2 [2-4] 3 [2-6] 0.06 *At least one of 3 nerves In the early postoperative period, in 4 cases, hematoma was observed in the body area where the procedure was performed, in 2 cases leakage of serum content from the wound, and in 1 – inflammatory infiltration (similarly in both groups). All the complications were treated using conservative methods. The patients operated on with sealant stayed in hospital a bit shorter (median: 2 vs. 3 days; p = 0.06). Until hospital discharge, both groups took similar amounts of analgesics. In 1 patient who had mesh fixed with sealant, hernia recurred 1 year after the procedure – it was confirmed by USG (sealant group vs. suture group, p = 0.767) BODY.POSTOPERATIVE PAIN: Before the procedure pain intensity on a VAS scale ranged 0-80 mm, on average 28 ±19 mm (Table I). Pain intensity measurements according to Jensen classification in the intraoperative and later period are presented in Table III [18]. Before the surgery, the number of patients without pain was equaled by the number of those with moderate and severe pain (n = 7). The most numerous group consisted of patients with mild pain (n = 27). In the postoperative period, pain intensity changed. ANOVA results for all the patients and both subgroups were statistically significant (p < 0.001) until the 30th postoperative day. Later on, VAS measures did not differ; pain intensity was similar. Translating VAS into subjective ailments, it could be observed that on the 2nd and 7th postoperative day every patient felt pain; on the 2nd day as many as 22 patients felt pain of moderate intensity. Only on the 7th day mild pain was predominating (n = 30). During the next follow-up appointments, pain intensity in practice did not change; between the 80th and the 400th day the same people felt constant pain of similar, mild intensity. Table IIIPain severity on individual days of the postoperative period Pre/postoperative dayPain intensityNo painMildModerateSevere–1 7 27 6 1 2 0 13 22 1 7 0 30 4 1 30 12 19 1 0 80 18 9 1 0 200 17 9 2 0 400 24 7 0 0 Classification according to [18] No relations between pain intensity and the method of mesh fixation were observed on the 30th or later postoperative day (only patients with mild and moderate pain, numbers bolded in Table III). When comparing the postoperative course in both groups, different significant values of pain intensity were observed on the 2nd postoperative day (Figure 1). In the sealant group, pain was relatively less intense than in the suture group (VAS, sealant group vs. suture group, 43 mm vs. 50 mm respectively, p < 0.05). In Figure 1, also on the 7th day, the disparity of VAS values is visible (median differs almost by 10 points), but the big interquartile range in the suture group precludes grasping the significant difference. Figure 1A summary of VAS pain scores upon subsequent follow-up visits Smaller painful ailments after using sealant in the early postoperative period are confirmed by significantly shorter time necessary for return to daily activities (Table IV). Return to professional activity depended on the kind of job done; the time was significantly longer in the group of manual workers (median: 45 vs. 30 days; p < 0.05). Return to daily activities, sport and driving was similar in both groups. Table IVReturn to normal activity levels in both groups Return to activitiesSealant group(n = 20)Suture group (n = 21)Value of p Median [1st-3rd quartile], daysDaily 3 [2-4] 5 [4-7] 0.003 Professional 40 [30-90] 40 [30-60] 0.31 Sport 30 [30-40] 35 [30-45] 0.27 Driving 7 [5-14] 7 [7-14] 0.74 In patients with minimum of 2 nerves in the groin cut, the time necessary to return to work was shorter (median: 30 vs. 45 days, pnd and 7th postoperative day and the return to everyday activities (both cases R = 0.4, p < 0.05). Pain intensity correlated positively on the 2nd and 7th and 200th and 400th postoperative day (in both cases R = 0.45, p < 0.05). There was no correlation between preoperative pain and its postoperative values (VAS scale). BODY.DISCUSSION: The randomized, single-center, double-blind clinical trial including male patients with primary inguinal hernia operated on using Lichtenstein method and observed from 11 months to 28 months (median: 16.4 months) showed that polypropylene mesh fixation with Glubran 2 cyanoacrylic sealant allows achieving similar results in the case of chronic pain recurrence to standard mesh fixation with sutures. In the early postoperative period in patients with sealant used, painful ailments were relatively less intense (VAS scale, 2nd day); patients operated on in such way came back earlier to everyday activities. The differences appeared also on the 7th postoperative day, but they were statistically insignificant. Although, it is believed that uncomplicated inguinal hernia do not cause painful ailments, it is not supported by studies. In our own observations, patients felt pain of an average VAS value of 28 ±19 mm, which corresponds to mild pain. These values are higher than quoted by Page et al. [19]; in their trial fewer patients with moderate and severe preoperative pain were included. In a multicenter study by Paajanen et al., in the identical group of patients, preoperative pain was of higher intensity – it corresponded to 4.0 ±2.4 on a VAS scale [20]. EHS congress in 2011 confirmed increasing interest in non-penetrating mesh fixation. The results of such procedures in inguinal and abdominal hernia repair were discussed. Nowadays, in medicine numerous tissue adhesives are used: biological, synthetic and genetically engineered proteinaceous ones (Table V). Table VCommercially available tissue glues, including their composition and brand names OriginContentsCommercial namesChoiceBiological Biological or fibrin adhesive Tissucol Hematogenous medicinal product Briplast Tachosil Quixil Fibrin autologous preparation Vivostat Medicinal product class III Magellan Bovine gelatin and thrombin Floseal Porcine gelatin Surgiflo Semisynthetic Glutaraldehyde Bioglue Bovine albumin Formaldehyde GRF Glutaraldehyde Porcine gelatin Resorcinum Synthetic Polyethylene glycol Coseal Polyethylene glycol Cyanoacrylate Glubran Ifabond Omnex Dermabond Medicinal product class IIa Histoakryl Indermil Cyanoacrylic adhesives are synthetic sealants firstly employed in medicine. They were synthesized in 1942 by Harry Coover in Kodak laboratories when he was searching for a super clean yielding material for rifle datives. After a few unsuccessful applications, Kodak created “Eastman #910” glue out of the product. In 1964, as a result of Kodak and Ethicon's studies, it was submitted to Food and Drug Administration (FDA) as an agent for closing wounds. Since 1966 the sealant was used in the Vietnam War. Used at the battle ground in the form of spray, it stopped bleeding very successfully. According to veterans of this war, it saved many lives. Otherwise, soldiers would have bled to death. It is ironic, but FDA forbade the use of the adhesive. It did not prevent its use during the war, though [21]. FDA did not approve of the sealant because of skin irritation it caused and its reaction with water which released cyanoacetate and formaldehyde. Only 2-octyl-cyanoacrylate met the expectations; it caused less skin irritations and it bound the tissues better than previously synthesized compounds. In 1998, FDA approved this compound for wound closing, and in 2001 – as antibacterial barrier. The product was introduced to the market as Dermabond. There are many cyanoacrylic adhesives available for different uses. The majority of them are non-absorbable, characterized by cytotoxicity and polymerization at high temperature. Some cyanoacrylic adhesives are used in the medical industry: N-butyl-1,2- cyanoacrylate (Histoacryl, B. Braun), 2-octyl-cyanoacrylate (Dermabond, Ethicon), N-butyl-2-cyanoacrylate (Indermil, Covidien) and N-hexyl-cyanoacrylate (Glubran 2, Ifabond, Fimed, France). Glubran 2 sealant used in the trial is characterized by fast polymerization, and the accompanying chemical reaction does not cause the temperature rise above 45°C. Its low viscosity facilitates application through a catheter. It is gradually absorbed, without an excessive foreign-body reaction. It is very effective; 1 ml is enough for bilateral hernia. Experimental investigations provide contradictory information about Glubran adhesive safety. Fortelny et al. revealed that tissue integration with a mesh was impaired in the area where the sealant was used. The adhesive induced local, excessive inflammation and its remains stiffened tissues – the abdominal wall of a rat [22]. Losi et al. on the same experimental model observed mild inflammation with a small number of macrophage and well supplied with blood connective tissue around the adhesive and mesh filaments. Sealant remains were visible in the whole 5-month observation period [23]. The newest, still awaiting publication, results of Kukleta's research showed that after a year Optline LP mesh fixed in the rabbit's abdominal wall with histoacryl was faster integrated than one fixed with MonoPlus 3-0 sutures. After a year, the adhesive was absorbed completely and its local tolerance was assessed as good [24]. The clinical literature concerning cyanoacrylic adhesive in open hernia surgeries in grownups is rather scarce. There are 7 indexed publications altogether, and two of them pertain to laparoscopy [25, 26]. The first prospective, randomized report comparing cyanoacrylic sealant with sutures was presented by Helbling et al. [8]. No evident differences in pain sensation were observed after 3 months with a slight tendency towards decrease in ailments in the adhesive group. Testini using Rytkow technique in 156 patients observed that chronic pain occurred only in 3.4% of the operated on with sutures; in the series with sealant (fibrin or cyanoacrylic) it did not occur at all [27]. In a Nowobilski et al. trial no fundamental differences were observed in the postoperative course within 4.7 months in patients operated on using Lichtenstein technique with sealant (Indermin, Loctite) and sutures [28]. The value of the report is decreased by the fact that authors do not write about randomization or control in patients. In a short report concerning the same series of patients (however with different numerical amount) there is information about 1 recurrent hernia in a sealant group [29]. A report by Paajanen et al. may be used to benchmark our own results, as they randomized Glubran (n = 151) and Dexon 3-0 (n = 151) fixation in a multicenter study. Procedures were performed on an outpatient basis, Optilene mesh was implanted, oblique hernia sac was dissected. During 1 year observation, hernia recurred twice in each group (1.4%). Chronic pain (VAS ≥ 2, over 3 months) in patients from both groups occurred with the similar frequency (sealant vs. sutures, 20% vs. 15% respectively). It was demonstrated that chronic pain depended on preoperative pain occurrence, patient's age and the length of the procedure; the method of mesh fixation was irrelevant [20]. The results of clinical assessment did not reveal fundamental differences between a sealant and sutures. With a corresponding definition of the chronic pain patient, our own results do not diverge from the quoted observations (22% of patients with chronic pain). Penetrating and adhesive fixation was compared more often in laparoscopic TAPP than in open surgeries. Three RCT and 5 controlled clinical trials were published. It was observed that adhesive fixation reduces recurrence (0.4% vs. 0.6%) and painful sensations (in 4 out 5 trials). For example, Olmi et al. compared different techniques of fixation in a large series of patients. They showed the superiority of fibrin sealant over penetrating fixation [30]. Kukleta's clinical experience is also convincing. He performed 1337 transabdominal pre-peritoneal (TAPP) procedures using only Glubran 2 adhesive fixation [31]. Results of previous trials (however, mostly concerning TAPP) reveal that sealant fixation reduces the risk of chronic pain in comparison to staplers/tackers (grade of recommendation 1B). It is not without significance that biological and synthetic sealants are less expensive than devices for penetrating fixation. Our own research, carried out in a series of patients operated on through an anterior approach, did not reveal fundamental differences between adhesive and penetrating fixation. With the similar results of 16-month observation, it was demonstrated that adhesive mesh fixation may have positive influence on the early postoperative period.

TITLE: A phase I pharmacokinetic study of ursolic acid nanoliposomes in healthy volunteers and patients with advanced solid tumors ABSTRACT.BACKGROUND: Ursolic acid is a promising anticancer agent. The current study aims to evaluate the single- and multiple-dose pharmacokinetics (PK) as well as the safety of ursolic acid nanoliposomes (UANL) in healthy volunteers and in patients with advanced solid tumors. ABSTRACT.METHODS: Twenty-four healthy volunteers in the single-dose PK study were divided into three different groups, which received 37, 74, and 98 mg/m2 of UANL. Eight patients in the multiple-dose PK study were administered with 74 mg/m2 of UANL daily for 14 days. The UA plasma concentrations were determined using ultra-performance liquid chromatograph-tandem mass spectrometry. ABSTRACT.RESULTS: The plasma concentration profiles of all subjects were characterized by a biexponential decline after infusion. The mean peak plasma concentration (Cmax) increased linearly as a function of the dose (r = 0.999). The mean area under the plasma concentration-time curve (AUC) from 0 to 16 hours also increased proportionally with dose escalation (r = 0.998). However, the clearance was constant over the specific dose interval. In the multiple-dose PK study, the trough and average concentrations remained low. The mean AUC, half-life, Cmax, time to Cmax, and the volume of distribution on the first day were similar to those on the last day. All subjects tolerated the treatments well. Most UANL-associated adverse events varied from mild to moderate. ABSTRACT.CONCLUSIONS: UANL exhibits relatively linear PK behavior with dose levels from 37 mg/m2 to 98 mg/m2. No drug accumulation was observed with repeated doses of UANL. The intravenous infusion of UANL was well tolerated by healthy volunteers and patients with advanced tumors. BODY.INTRODUCTION: Ursolic acid (UA; 3β-hydroxy-urs-12-en-28-oic acid) is a naturally derived pentacyclic triterpene acid that is widely present in food, medicinal herbs, and other plants.1 Its extensive pharmacological effects include hepatoprotective, antioxidant, anti-inflammatory, antiviral, and cytotoxic activities. In recent years, UA has become a major focus in cancer research because of its activities at various stages of tumor development and its low toxicity. Although the concrete mechanisms of its anticancer effects are poorly understood, several studies have found that UA can inhibit proliferation and induce apoptosis of many tumor cell lines.2 UA-induced apoptosis occurs through multiple pathways such as the inhibition of DNA replication,3 induction of Ca2+ release,4 activation of caspases5,6 and c-Jun N-terminal kinase,7,8 phosphorylation of glycogen synthase kinase 3-β, downregulation of antiapoptotic genes,9 inhibition of cyclooxygenase-2 and inducible nitric oxide synthase,10,11 suppression of matrixmetallopeptidase-9,12 and the inhibition of protein tyrosine kinase,13 phosphatidylinositol-3-kinase,14 single transducer and activator of transcription 3,15 adenosine 5′-monophosphate-activated protein kinase,16 and nuclear factor κ-light-chain-enhancer of activated B cells17 pathways. Furthermore, UA can inhibit the differentiation, angiogenesis, invasion, and metastasis of tumor cells as well as interfere with numerous enzymes such as those directly involved with DNA synthesis and repair.2,18–20 However, because of its poor solubility in water, the oral administration of UA often causes its low bioavailability.21 The literature has limited data on the mechanism of absorption and pharmacokinetics (PKs) of UA. A previous study using a Caco-2 cell monolayer model suggested that the main mechanism involved in the absorption of UA was passive diffusion and P-glycoprotein transporter mediated active transport. Liao et al performed a PK study of UA in rats after administration of the traditional Chinese medicinal preparation, Lu-Ying extract.22 The results showed that the absorption of UA was rapid, with the peak concentration occurring 1 hour after oral administration of Lu-Ying extract. Although the oral administration dose of Lu-Ying extract contained UA 80.32 mg/kg, its concentrations in rat plasma were extremely low. Recently, a PK study of UA in rats showed that the time to peak concentration of UA in plasma was about 30 minutes, indicating that UA was rapidly absorbed. The half-life of UA in plasma was less than 1 hour, indicating that UA was rapidly eliminated.23 Thus, the therapeutic development of UA was limited by its low bioavailability and the enhancement of UA solubility is a real challenge for its development. The nanocarrier-mediated drug delivery system has been used to improve the therapeutic activity and safety of drugs for years. The nanoliposomal formulation is a promising approach for poorly water-soluble drugs.24 Thus, UA nanoliposomes (UANL) were developed for the first time in the People’s Republic of China. The intravenous (IV) administration of UANL is considered to improve bioavailability because the nanoparticles bypass the stomach. To date, a wide range of preclinical studies in China have been completed (Li, unpublished data, 2005). These studies revealed that the nanoliposome-encapsulated UA inhibited the growth of various human cancer cells and nude mice xenografts. A further unpublished study has shown that UANL induces minimal toxic effects even with long-term application. Tissue distribution experiments in mice suggested that UANL first enters the stomach and intestines, and then its concentrations decline rapidly. Conversely, the hepatic UA concentration increased rapidly, and exceeded the UA concentrations in the stomach and intestines at 4 hours after IV injection. The results demonstrated that UANL delivers UA to the liver, where it accumulates. Consequently, the drug disposition behavior changes in vivo, and the toxic and side effects of UA on other tissues is decreased (unpublished data). The antitumor activities of UA and the minimal toxic effects that were observed in preclinical studies promoted the human clinical trials of UANL. Thus, a PK study on UANL was started by the Tianjin Medical University Cancer Institute and Hospital. To date, no PK studies of UANL in humans have been reported. This study is the first report of a PK investigation of UANL in humans. Thus, this study aimed to characterize the PK profiles of UANL in healthy Chinese volunteers and in Chinese patients with advanced solid tumors, as well as to monitor the UANL toxicities after IV administration. BODY.PATIENTS AND METHODS: BODY.SELECTION OF HEALTHY VOLUNTEERS AND PATIENTS: This present study was performed in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of Tianjin Cancer Hospital. All subjects were informed about the study program and signed informed consent forms before participating in the study. The healthy volunteers in the single-dose PK study were chosen based on the following criteria: aged 18 years to 75 years; with normal organ function throughout the body; no drug administration for 2 weeks prior to participation in the study; good living habits; willingness to consume no cigarettes or alcohol during the entire treatment period; and good compliance with the study program requirements. Patients with histologically confirmed solid tumors that had proven intractable to standard therapy or had received no standard therapy were eligible for the multiple-dose PK study. One inclusion criterion for the multiple-dose PK study is that the patients had not undergone any operation, chemotherapy, biotherapy, or radiotherapy for 4 weeks before participating in the study. Other inclusion criteria included: aged 18 years to 75 years; Eastern Cooperative Oncology Group (ECOG) status of 0 to 2; survival expectancy of over 3 months; adequate bone marrow function, as defined by a white blood cell count of ≥4.0 × 109/L, an absolute neutrophil count of ≥2.0 × 109/L, a platelet count of ≥100.0 × 109/L, and a hemoglobin level of ≥100.0 g/L; adequate renal function, as defined by serum creatinine ≤ 1.5 times the upper limit of normal (ULN); adequate hepatic function, as defined by alanine aminotransferase (ALT) level ≤ 1.5 × ULN, aspartate aminotransferase (AST) level ≤ 1.5 × ULN, and a normal total bilirubin level; and no obvious cardiac, pulmonary, or psychiatric disorders. Patients with an active infection and serious brain tumors or metastases were excluded from the study. All patients with probable chances of pregnancy were required to use adequate contraception during the entire study. BODY.STUDY DESIGN: Injectable UANL was supplied by Liyuanheng Pharmaceutical Co, Ltd (Wuhan, China). The UANL was manufactured as a freeze-dried powder, with 3 mg of UANL in each vial. The present study has an open-label, dose-escalation study design. Based on previous pharmacological, toxicological, and tolerability data,25 the initial UANL dose for the single-dose PK study was 37 mg/m2. The dosage was sequentially increased to 74 and 98 mg/m2 in the other corresponding dose-assigned groups. Twenty-four patients in the single-dose PK study received 37, 74, and 98 mg/m2 of UANL via a 4-hour IV infusion. Eight patients who were enrolled in the multiple-dose PK study received 74 mg/m2 of UANL daily for 2 consecutive weeks. BODY.PK SAMPLE COLLECTION: Single-dose PK 4 mL blood samples were collected at the following time points: before treatment, at 0.5, 1, and 2 hours during the infusion, at the end of the 4-hour infusion, and finally, at 5, 15, and 30 minutes as well as at 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after the infusion. On the 1st and 14th days of the multiple-dose PK study, 4 mL blood samples were collected before treatment, at 30 minutes as well as at 1 and 2 hours during the infusion, at the end of the 4-hour infusion, and finally, at 5, 15, and 30 minutes as well as at 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after the infusion. On the 6th, 7th, and 8th days of the study, 4 mL blood specimens were collected before treatment and at the end of the 4-hour infusion. The samples of plasma were prepared by centrifugation (10 minutes at 600× g), and were subsequently stored at −80°C until further analysis. BODY.ANALYTICAL ASSAYS: Ursolic acid (>99.5% purity) and glycyrrhetinic acid (internal standard, >99.5% purity) were obtained from the National Institute for Food and Drug Control (Beijing, China). High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were purchased from Fisher Scientific Inc (Fairlawn, NJ, USA). HPLC-grade ethyl acetate was purchased from Concord Technology Co, Ltd (Tianjin, China). HPLC-grade water was prepared using a Milli-Q system (EMD Millipore, Billerica, MA, USA). Analytical grade ammonium formate was purchased from Runjie Chemical Reagent Co, Ltd (Shanghai, China). The chromatographic system consisted of an ACQUITY™ ultra-performance liquid chromatography (UPLC) system coupled to an ACQUITY™ triple quadrupole mass spectrometry system and a BEH C8 column (100 mm × 2.1 mm; inner diameter, 1.7 μm; Waters Corp, Milford, MA, USA). Data were processed using the MassLynx version 4.1 software (Waters Corp). The determination of UA in human plasma was performed by UPLC/tandem mass spectrometry (MS/MS) method as previously described.26 The lower limit of quantitation (LLOQ) for UANL was 10 ng/mL, and the assays were linear from 10 ng/mL to 5000 ng/mL. The mean extraction recovery of UANL was 73.2%, and the matrix ion suppression ranged from −11.4% to −5.6%. The intra- and interday precisions were less than 7.0% and 7.2%, respectively. The accuracy of UANL was within ±2.0%. BODY.PK DATA ANALYSIS AND STATISTICAL ANALYSIS: The plasma concentration vs time data were analyzed using noncompartmental methods. The DAS 2.1 PK analysis system (Anhui, China) was used to assess the PK parameters. The peak plasma concentration (Cmax) and the time-to-peak plasma concentration (Tmax) were obtained via the experimental observations. The elimination half-life (t1/2) was calculated as 0.693/λz (λz is the slope of the terminal phase). The area under the curve (AUC) of the plasma concentration versus time from zero to infinity (AUC0–∞) was equivalent to the sum of the areas from time T0 to the time of the last measured concentration. Its value was calculated using the linear trapezoidal method (until Cmax), the log-trapezoidal method (until the last measurable concentration), and the extrapolated area. The extrapolated area was determined by dividing the final measured concentration by the slope of the terminal log-linear phase. Trough values on the 6th, 7th, and 8th days were averaged for each dose level. All statistical tests were two-tailed, and significance was set at the 0.05 level. Differences in the mean values of the physical examinations and in the PK parameters among the three groups were compared by analyses of variance or the Kruskal–Wallis test using the Statistical Package for the Social Sciences (SPSS) software (version 16.0; IBM, Armonk, NY, USA). The t-test or Wilcoxon’s test were used to investigate the differences between the two groups. BODY.RESULTS: BODY.SUBJECT CHARACTERISTICS: Thirty-two eligible and consenting subjects were recruited for this study. Twenty-four healthy volunteers were enrolled in the single-dose PK study, whereas there were eight patients in the multiple-dose study. The characteristics of the healthy volunteers and patients are shown in Table 1. All healthy subjects had normal results for the physical and laboratory examinations. No statistically significant differences were found in the age, height, weight, body surface area, and body mass index among the three healthy groups. In the multiple-dose PK study, the mean age of the eight patients was 49 years. There were six male and two female patients, all of whom had ECOG performance status score of 0 to 1. Four patients had non-Hodgkin’s lymphoma, two had hepatoma, one had Hodgkin’s lymphoma, and one had gastric cancer. BODY.SINGLE-DOSE PK STUDY IN HEALTHY VOLUNTEERS: The total UA plasma concentration-versus-time profiles of the healthy volunteers who received 37 to 98 mg/m2 of UANL are shown in Figure 1. The plasma concentration profiles of all subjects were characterized by a biexponential decline after infusion. The major PK parameters at each dose are summarized in Table 2. The respective mean Cmax for the subjects in the UANL groups were 1835, 2865, and 3457 ng/mL, in order of increasing UANL dose. The respective mean AUC0–16 h were 4203, 7175, and 9696 ng · hour/mL. The mean Cmax increased linearly as a function of dose (r = 0.999). Similarly, the mean area under the plasma concentration-time curve from 0 to 16 hours (designated as AUC0–16h) increased proportionally with dose escalation (r = 0.998). However, the mean t1/2, volume of distribution (Vd) and clearance (CL) were not significantly different among the three doses (P > 0.05), suggesting that clearance was constant over that specific dose interval. These results show that UANL displays linear PKs after IV administration. No correlation was found between the observed PK profile (in terms of the AUC) and the patient characteristics (age, height, weight, body mass index, or body surface area). BODY.MULTIPLE-DOSE PK STUDY IN PATIENTS WITH ADVANCED SOLID TUMORS: The mean plasma concentration versus time plot for the eight patients after receiving 74 mg/m2 of UANL during the 14-day period via continuous IV infusion is illustrated in Figure 2. The UANL PK parameters on the first day and the last day for the patients are summarized in Table 3. The mean steady-state Cmax, trough concentration (Cmin), and average concentration (Cav) were 1211, 7.31, and 196 ng/mL, respectively. No accumulation was observed with repeated doses of UANL. Comparison of the PK parameters at 74 mg/m2 between the healthy volunteers and patients showed that Tmax, t1/2, and Vd were not significantly different between the two subject groups (P > 0.05). However, the mean AUC, Cmax, and CL were significantly lower in the patients than in the healthy volunteers (P < 0.05). BODY.SAFETY: The National Cancer Institute’s Common Terminology Criteria for Adverse Events (version 3.0) was used to grade adverse events (AEs). Table 4 presents a summary of the toxicities associated with the administration of UANL in healthy volunteers and patients. The IV infusions of UANL were well tolerated by the healthy volunteers and the patients with advanced tumors. All subjects were evaluated for tolerability. No AE was observed in patients who receiving 37 mg/m2 of UANL. The incidence of UANL-associated AEs did not show an increasing trend after the provision of the larger doses. Nausea, diarrhea, and abdominal distention were the common AEs that were observed. Most UANL-associated AEs were either grade 1 or 2. Only one patient developed grade 3 AEs in the form of elevated AST and ALT levels with diarrhea at the same time after receiving 74 mg/m2 of UANL. BODY.DISCUSSION: Medicinal plants are often investigated as sources of new drugs for treating cancer. UA is one of the most promising chemopreventive agents for cancer.27 The water solubility of UA is limited, which consequently limits its bioavailability in the body. The nanoliposome-encapsulation of UA was expected to increase its bioavailability and therapeutic efficacy. The formulated nanoliposomes prepared in this study were relatively uniform in size. The UANL average diameter was 200 nm, and the average entrapment efficiency determined by dialysis method was 95%. Conventional UA was found to be mainly distributed in tissues with extensive vascular supply such as the lung, spleen, and liver at 1 hour after oral administration.23 However, based on the unpublished preclinical studies on nanoliposomal UA in rats, UANL probably had a lower Vd and higher hepatic drug concentration over conventional UA. These UANL characteristics may contribute towards eliminating its drug-related side effects and nonspecific drug accumulation. In the present study, we determined the total UA concentration in human plasma by UPLC-MS/MS.26 The LLOQ under the present conditions was 10 ng/mL. The results indicated that the method was specific, sensitive, and convenient for the determination of the UA concentration in biological samples. Subsequently, we investigated the PKs and safety of UANL in healthy volunteers and patients with advanced solid tumors. Following a single dose via IV administration, the total UA plasma concentration of all subjects followed a biexponential decline. After the end of the IV infusion, the UA plasma concentration rapidly decreased to a concentration that was approximately ten times less than the peak concentration within 2 hours. The PK characteristics of UANL were linear and dose-proportional in the range of 37 mg/m2 to 98 mg/m2. These results showed that the plasma concentration of the total UA decreased with constant clearance. The preclinical studies of UANL in rats showed a similar trend of the PK profiles (Li, unpublished data, 2005). In the multiple-dose PK study, the steady-state Cmin and Cav remained low. The mean AUC, t1/2, Cmax, Tmax, and Vd on the first day were similar to those on the last day, thereby indicating that the PKs of UANL had no apparent changes after repeated administration. These data showed that no drug accumulation was observed with continuous administration of UANL. The mean AUC, Cmax, and CL in the patients on the first day were significantly lower than those in the healthy volunteer group. Several possible reasons may account for this difference. First, the small sample sizes in the present study may contribute to this large difference. Then, a difference in the hepatic functional reserve of the healthy volunteers and patients may explain their different levels of response to the UANL. Whether this difference can be ascribed to the hepatic metabolic enzymes and/or other catabolic enzymes is unclear. All subjects in this study tolerated the treatments well. Most UANL-associated AEs varied from mild to moderate and were not dose-related. Only one healthy volunteer developed grade 3 AEs, such as AST and ALT elevation as well as diarrhea at the same time, after receiving 74 mg/m2 of UANL. The most frequently observed AEs included abdominal distention, nausea, and diarrhea. The UANL side effects after the 14-day continuous infusion included grade 1 skin pruritus, arthralgia, and triglyceride elevation. A previous tolerability study in humans had similar results, thereby suggesting that UANL had minimal toxic effects.25 The dose-limiting toxicity of UANL was hepatotoxicity. Thus, particular attention should be given to any clinical and laboratory evidence of hepatotoxicity in future clinical studies of UANL. Correlation analyses were conducted to determine if a relationship could be established between the observed PK profiles and UANL toxicities. No correlation was found, possibly because of the small sample size. BODY.CONCLUSION: For the first time, UANL was developed in the People’s Republic of China. This study is the first report of a PK investigation of UANL in humans. In the present study, UANL is safe and presents apparent linear PK behavior for dose levels within the range of 37 mg/m2 to 98 mg/m2. No drug accumulation was observed with repeated UANL administration even with 14 days of continuous IV infusion. The IV infusion of UANL was well tolerated by healthy volunteers and patients with advanced tumors. Further Phase II studies of UANL should be conducted to extensively observe its clinical efficacy and safety.

TITLE: Comparing Inpatient Satisfaction Collected via a Web-Based Questionnaire Self-Completion and Through a Telephone Interview: An Ancillary Study of the SENTIPAT Randomized Controlled Trial ABSTRACT.BACKGROUND: Assessing the satisfaction of patients about the health care they have received is relatively common nowadays. In France, the satisfaction questionnaire, I-Satis, is deployed in each institution admitting inpatients. Internet self-completion and telephone interview are the two modes of administration for collecting inpatient satisfaction that have never been compared in a multicenter randomized experiment involving a substantial number of patients. ABSTRACT.OBJECTIVE: The objective of this study was to compare two modes of survey administration for collecting inpatient satisfaction: Internet self-completion and telephone interview. ABSTRACT.METHODS: In the multicenter SENTIPAT (acronym for the concept of sentinel patients, ie, patients who would voluntarily report their health evolution on a dedicated website) randomized controlled trial, patients who were discharged from the hospital to home and had an Internet connection at home were enrolled between February 2013 and September 2014. They were randomized to either self-complete a set of questionnaires using a dedicated website or to provide answers to the same questionnaires administered during a telephone interview. As recommended by French authorities, the analysis of I-Satis satisfaction questionnaire involved all inpatients with a length of stay (LOS), including at least two nights. Participation rates, questionnaire consistency (measured using Cronbach alpha coefficient), and satisfaction scores were compared in the two groups. ABSTRACT.RESULTS: A total of 1680 eligible patients were randomized to the Internet group (n=840) or the telephone group (n=840). The analysis of I-Satis concerned 392 and 389 patients fulfilling the minimum LOS required in the Internet and telephone group, respectively. There were 39.3% (154/392) and 88.4% (344/389) responders in the Internet and telephone group, respectively (P<.001), with similar baseline variables. Internal consistency of the global satisfaction score was higher (P=.03) in the Internet group (Cronbach alpha estimate=.89; 95% CI 0.86-0.91) than in the telephone group (Cronbach alpha estimate=.84; 95% CI 0.79-0.87). The mean global satisfaction score was lower (P=.03) in the Internet group (68.9; 95% CI 66.4-71.4) than in the telephone group (72.1; 95% CI 70.4-74.6), with a corresponding effect size of the difference at −0.253. ABSTRACT.CONCLUSIONS: The lower response rate issued from Internet administration should be balanced with a likely improved quality in satisfaction estimates, when compared with telephone administration, for which an interviewer effect cannot be excluded. ABSTRACT.TRIAL REGISTRATION: Clinicaltrials.gov NCT01769261 ; http://clinicaltrials.gov/ct2/show/NCT01769261 (Archived by WebCite at http://www.webcitation.org/6ZDF5lA41) BODY.INTRODUCTION: Numerous questionnaires have been developed since the 70s for assessing patient satisfaction with regard to hospital health care delivery. These include the Patient Satisfaction Questionnaire [1], the Client Satisfaction Questionnaire [2], the Service Quality instrument [3-5], the Hospital Consumer Assessment of Health Providers and Systems (HCAHPS) Survey [6,7], the Short Form HK Inpatient Experience Questionnaire [8,9], and the NHS National Adult Inpatient Survey [10]. In France, inpatient hospital satisfaction has been systematically measured since 2015 with the I-Satis questionnaire [11]. These numerous questionnaires attest a worldwide concern for enhancing the central role of the patient in the health care organization. Patient satisfaction assessment is also related to the technical performance and safety of hospital care [12] and is considered as a tool contributing to hospital care evaluation, although controversial [13]. Inpatient hospital satisfaction surveys are usually either self-administrated by pen and paper or conducted by telephone, with telephone interview being a common mode of questionnaire administration. However, the development of Internet has resulted in a widespread use of Web-based questionnaires, with corresponding survey costs lower than those of telephone surveys [14-17]. Moreover, the use of Internet has increased over time, with 78% of people with Internet access at home in France in 2013 [18], thereby suggesting that this mode of administration might result in a satisfactory response rate. Nevertheless, several studies have reported a lower response rate of Internet-based surveys, as compared with other modes of administration [19-21]. On the other hand, Internet self-completion has intrinsic favorable qualities such as the avoidance of any potential bias of responses related to an interviewer effect [22], and patients are more likely to freely express their opinions [23] on websites covering anonymity than through telephone. The way in which the modes of administration of patient satisfaction survey influence response rates and the issued scores remains an important issue. Several teams studied differences between pen-and-paper and Web-based questionnaires in the field of inpatient satisfaction and quality of life [21,24,25]; however, only a few investigated the differences with surveys administered through telephone [20,26], which remains a common mode of administration for inpatient hospital satisfaction surveys [27,28]. In this context, to our knowledge, this study—which is based on the multicenter SENTIPAT (sentinel patients) randomized trial [26,29]—is the first multicenter randomized trial to date comparing inpatient satisfaction collected via the Internet or through a telephone survey. Our objective was to assess whether response rates and satisfaction scores differed between these two modes of investigation of the patients' satisfaction. BODY.METHODS: This research was an ancillary study of the multicenter, randomized SENTIPAT trial [29]. We took advantage of the trial to analyze patients’ satisfaction with their hospital stay. BODY.GENERAL DESCRIPTION OF THE SENTIPAT TRIAL: The SENTIPAT multicenter (five adult acute care units in a Parisian teaching hospital participated voluntarily: departments of digestive and general surgery; gastroenterology; hepatology; infectious diseases and tropical medicine; and internal medicine) randomized trial focused on the evolution of patients’ health on returning home post hospitalization (follow-up duration of 6 weeks). The general objective was to determine whether the information on patient’s health evolution shared by volunteer patients after returning home directly via a dedicated website was comparable with that obtained via telephone interviews. The randomization of 2050 patients (410 from each unit, 205 randomized in the Internet group and 205 randomized in the telephone group) was initially planned. The study was conducted in accordance with French regulation on ethics requirements in biomedical research. Consecutive inpatients with Internet access at home were eligible for inclusion. Inclusion criteria also required inpatients who were not cognitively impaired and did not have a behavioral disorder, who spoke and wrote French, and were returning home after an acute care hospitalization, regardless of the type of stay—standard hospitalization (scheduled or not) on weekdays only (maximum Monday to Friday or any combination thereof) or outpatient hospitalization (1 day). Inpatients were enrolled on the day of hospital discharge by a clinical research technician of the trial. At that time, patients were informed about the study. Eligible patients not opposed to participate in the study were randomized into two parallel groups: Internet or telephone follow-up (inherently resulting in an open-label trial) at a ratio of 1:1. On the basis of a centralized randomization that allocated the eligible patient either to the Internet or to the telephone group through a website and using an underlying permutation block randomization stratified by service, the computer-generated list of permutation was established by a statistician independent from the study. At the time of patient inclusion, the technician also collected baseline variables (length of stay [LOS], sex, age, relationship status, level of education, activity, and type of insurance). Patient was then informed and discharged with documents explaining corresponding questionnaire administration. BODY.CHARACTERISTICS OF THE STUDY: BODY.PATIENTS: The French authorities have made available the instructions for analyzing I-Satis questionnaire [30], and according to these recommendations, the study was restricted to patients whose LOS included at least two consecutive nights. BODY.QUESTIONNAIRE STRUCTURE: The detailed I-Satis questionnaire used in this study (all questions and corresponding proposed answers) is directly accessible via the Internet [11]. The I-Satis questionnaire comprises 32 items exploring six dimensions: global care (Q1, Q2, Q4, Q13, Q14, and Q15), information to patient (Q16, Q18, Q27, Q28, Q29, and Q30), communication with health care providers (Q3, Q5, Q6, Q17, and Q20), behavior of health care providers (Q7, Q8, Q9, Q10, and Q11), hospital room convenience (Q21, Q22, Q23, and Q24), and hospital catering (Q25 and Q26). The recommendations of the French authorities for I-Satis analysis [30] indicate that 4 questions (Q12, Q19, Q31, and Q32) are not involved in score calculations. BODY.QUESTIONNAIRE ADMINISTRATION: All patients were informed that their opinions were kept anonymous. For the patients who had been randomized in the telephone group, the I-Satis questionnaire was administered during a telephone interview with a clinical research technician 7 days after discharge (the appointment was scheduled on the day of discharge), with a maximum of three attempts to contact them. For the patients who had been randomized in the Internet group, the same questionnaire was available on the dedicated website on the day of discharge (D0) and was completed directly online by the patient, who had been given oral and written instructions (information sheet) to connect for the first time 7 days post discharge. “Reminders” were sent by email once weekly for 6 weeks after discharge to potential responders of the Internet group, who had not completed the discharge questionnaire yet. BODY.SCORE CONSTRUCTION: The questionnaires were analyzed according to the French national recommendations of the Direction Générale de l'Offre de Soins [30]. Each item was rated from 0 to 10. Rates 1 to 5 corresponded to increasing ordinal rankings of satisfaction; rates 0, 6, 7, and 8 corresponded to answers of nonrelevancy of the item for the patient (depending on the item: never felt discomfort, no drugs were prescribed, no surgery, etc), and spontaneous answers “I don’t know” and “I don’t wish to answer the question” were rated 9 and 10. Rates 1, 2, 3, 4, and 5 were valued 0, 25, 50, 75, and 100, respectively, in the analysis. Rates 0, 6, 7, 8, 9, and 10 were handled into the analyses as a missing value. The scores of the dimensions “global care,” “information to patient,” “communication with health care providers,” “behavior of health care providers,” “hospital room convenience,” and “hospital catering” were calculated if at least three, three, three, three, two, and two items comprising the dimension were answered, respectively. The global score was calculated whenever every dimension score was calculated. BODY.STATISTICAL ANALYSIS: The participation rates observed in the Internet and telephone groups were compared using the Fisher exact test, as well as the proportions of nonrelevancy answers observed in these two groups. The delays of questionnaire completion observed in the Internet and telephone groups were compared using Wilcoxon-Mann-Whitney test. Internal consistency of questionnaires was measured by calculating Cronbach alpha [31], taking into account every score that could be calculated according to the abovementioned rules. An alpha coefficient value of greater than .7 was considered as satisfactory. Dimensions’ scores were calculated for each patient as the mean of the corresponding dimensions’ items, and global score was the mean of all answered items of the questionnaire. CIs were obtained by bootstrap. Standardized Cohen d-type effect size was measured between the scores of the two groups [32]. Comparisons between the Internet and telephone groups in terms of Cronbach alpha coefficients and in terms of satisfaction scores (including dimensions’ scores) were made using a permutation test [33], with the null hypothesis distribution (distribution of the difference between the two groups under the hypothesis of no difference) generated through 1,000,000 shuffled datasets. A P value of ≤.05 defined the significance of comparisons. Missing data were handled as follows: First, nonresponding patients were excluded from score analyses. Patients for whom less than 16 items were completed were also excluded from score analyses (ie, handled as nonresponders in the analyses). Second, the scores issued from the remaining partially completed questionnaires were calculated as mentioned above (see subsection on Score Construction). All analyses were made with the R statistical computing freeware version 3.3.0 [34]. BODY.ETHIC AND LEGAL APPROVALS: The SENTIPAT study was approved by the Comité de Protection des Personnes Ile de France IX (decision CPP-IDF IX 12-014, June 12, 2012); the Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé (Decision 12.365, June 20, 2012); and the Commission Nationale de l’Informatique et des Libertés (Decision DR-2012-582, December 12, 2012). BODY.RESULTS: Between February 25, 2013 and September 8, 2014, we managed to enroll in the SENTIPAT study 1680 eligible patients (840 randomized in the Internet group and telephone group each) and not opposed to participating in the trial. Among these, the baseline population of patients fulfilling the minimum LOS of 2 nights required for I-Satis investigation comprised 781 patients, with 392 and 389 patients in the Internet and telephone groups, respectively (Figure 1). Table 1 provides the details of baseline values of the patients who constituted the population investigated in this study. There were no missing data relating to baseline values. Considering all 781 patients, the median LOS was 5 days (interquartile range [IQR]: 2-9); there were as many men as women, participants were aged 19 to 97 years, and median age was 53 years (IQR: 37-64), and 711 patients (91.0%) had a complementary private health insurance in addition to the compulsory health insurance. There were 154 responders out of the 392 patients in the Internet group (response rate of 39.3%) and 344 responders out of the 389 patients in the telephone group (response rate of 88.4%; P<.001), and the corresponding median delays between hospital discharge and questionnaire completion were 6 days (IQR: 3-15.75) and 7 days (IQR: 7-9), respectively (P=.002). Missing data in responders concerned 10 patients of the Internet group: answer to question 13 (satisfaction about pain management) was missing in 2 responders, answer to question 20 (satisfaction about the answers of the surgeon about patient’s questions on surgery) was missing in 3 responders, and answer to both questions was missing in 5 responders. In addition, there were 13 (8.4%), 95 (61.7%), 43 (27.9%), and 3 (1.9%) responders in the Internet group with 0, 1 to 5, 6 to 10, and more than 10 answers, for which the answer code corresponded to nonrelevancy or refusal (further handled as a missing value in the analyses, see the section on Methods), respectively, whereas the corresponding responders observed in the telephone group were 3 (0.8%), 124 (36.0%), 200 (58.1%), and 15 (4.4%), respectively. Internet responders provided an answer code corresponding to nonrelevancy and refusal less frequently than telephone responders (P<.001). Figure 1Flow of participants through the study. Table 1Characteristics of study patients. Variable Total,N=781 Responders Internet, n=154 Nonresponders Internet, n=238 Responders telephone, n=344 Nonresponders telephone, n=45 LOSa, in days, median (IQRb) 5 (2-9) 5 (3-8) 5.5 (3-8) 5 (2-9) 7 (3-10) Sex, n (%) Male 385 (49.4) 71 (46.1) 115 (48.3) 176 (51.2) 23 (51) Age in years, median (IQR) 53 (37-64) 55 (38-64) 50 (36-64) 53 (38-65) 51 (33-67) Relationship status, n (%) Living alonec 342 (43.8) 59 (38.3) 112 (47.1) 146 (42.4) 25 (56) Living as a coupled 439 (56.2) 95 (61.7) 126 (52.9) 198 (57.6) 20 (44) Level of education, n (%) Lower secondary education 88 (11.3) 17 (11.0) 28 (11.8) 35 (10.2) 8 (18) Upper secondary education 249 (32.0) 46 (29.9) 80 (33.8) 110 (32.0) 13 (30) Postsecondary nontertiary education or short-cycle tertiary 113 (14.5) 23 (14.9) 32 (13.5) 53 (15.4) 5 (11) Bachelor’s degree or above 329 (42.2) 68 (44.2) 97 (40.9) 146 (42.4) 18 (41) Activity, n (%) Nonworking 321 (41.2) 64 (42.1) 101 (42.4) 139 (40.4) 17 (38) Employed or student 458 (58.8) 88 (57.9) 137 (57.6) 205 (59.6) 28 (62) Insurance, n (%) Precariouse 30 (03.8) 3 (02.0) 13 (05.5) 11 (03.2) 3 (07) Compulsory health insurance 40 (05.1) 7 (04.6) 15 (06.3) 14 (04.1) 4 (09) Compulsory health insurance plus complementary private health insurance 711 (91.0) 144 (93.5) 210 (88.2) 319 (92.7) 38 (84) Delay between hospital discharge and questionnaire completion, in days, median (IQR) N/Af 6 (3-15.75) N/A 7 (7-9) N/A aLOS: length of hospital stay. bIQR: interquartile range. cSingle, widowed, divorced, separated. dMarried, living together under a civil solidarity pact, simply living together without legal ties. eBenefit from state medical help or universal health insurance. fN/A: not applicable. Table 2 shows the values of Cronbach alpha in the Internet and telephone responders. All estimates, with the exception of those corresponding to room convenience, were >.7. The alpha estimates observed in the Internet group were always greater than those observed in the telephone group, the difference being statistically significant for the two dimensions, global care and room convenience (P=.003 and P=.03, respectively), and for the global satisfaction (P=.03). Table 3 summarizes the satisfaction scores observed in the Internet and telephone groups. The mean global satisfaction score was 68.89 (95% CI 66.36-71.36) in the Internet group and 72.01 (95% CI 70.36-73.58) in the telephone group. In both groups, the dimension that received the lowest score was hospital catering, with means of 45.77 (95% CI 42.18-49.39) and 45.70 (95% CI 43.32-48.06) in the Internet and telephone group, respectively. Conversely, in both groups, the theme that received the highest score was behavior of health care providers, with means of 87.49 (95% CI 85.05-89.73) and 92.14 (95% CI 90.81-93.39) in the Internet and telephone group, respectively. There were three dimension scores significantly smaller in the Internet group than in the telephone group: information to patients with a mean difference of −5.38 (P=.008), communication with health care providers with a mean difference of −7.16 (P=.003), and behavior of health care providers with a mean of −4.66 (P<.001). The global satisfaction score was significantly smaller in the Internet group with a mean difference of −7.16 (P=.003) and behavior of health care providers with a mean of −4.66 (P<.001). The global satisfaction score was significantly smaller in the Internet group, with a mean difference of −3.12 (P=.03). The absolute values of the effect sizes ranged from 0.003 to 0.352. The satisfaction scores observed in the group of Internet responders according to the delay of questionnaire completion are summarized in Multimedia Appendix 1, and whatever the type of score considered, the score difference between the two subgroups of Internet responders (questionnaire completed at day 7 after discharge or later versus questionnaire completed earlier) was not significant. Table 2Cronbach alpha coefficients. Dimension of the score (number of items involved) Internet responders: Cronbach alpha (95% CI); n Telephone responders: Cronbach alpha (95% CI); n Comparison between the two groups: P value Global care (6) .92 (0.87-0.96); 154 .79 (0.73-0.85); 344 .003 Information to patients (6) .89 (0.81-0.94); 131 .83 (0.76-0.88); 235 .27 Communication with health care providers (5) .74 (0.65-0.82); 152 .71 (0.64-0.77); 334 .56 Behavior of health care providers (5) .82 (0.71-0.92); 153 .73 (0.61-0.84); 344 .33 Hospital room convenience (4) .66 (0.53-0.75); 154 .48 (0.37-0.56); 344 .03 Hospital catering (2) .86 (0.79-0.91); 136 .76 (0.69-0.82); 285 .07 Global satisfaction score (28) .89 (0.86-0.91); 116 .84 (0.79-0.87); 189 .03 Table 3Satisfaction scores. Dimension of the score Internet responders: mean score (95% CI); n Telephone responders: mean score (95% CI); n Internet-telephone: mean score difference (95% CI), P value Effect size (95% CI) Global care 70.67 (68.01-73.34); 154 72.15 (70.56-73.75); 344 −1.48 (−4.59 to 1.64), .33 −0.094 (−0.290 to 0.105) Information to patients 59.62 (56.07-63.23); 131 65.01 (62.75-67.25); 235 −5.38 (−9.53 to −1.19), .009 −0.286 (−0.501 to −0.062) Communication with health care providers 67.42 (63.28-71.42); 152 74.58 (71.90-77.15); 334 −7.16 (−12.01 to −2.36), .003 −0.287 (−0.477 to −0.096) Behavior of health care providers 87.49 (85.05-89.73); 153 92.14 (90.81-93.39); 344 −4.66 (−7.40 to −2.02), <.001 −0.352 (−0.554 to −0.154) Hospital room convenience 61.03 (58.07-63.95); 154 60.97 (59.07-62.88); 344 0.05 (−3.46 to 3.50), .98 0.003 (−0.189 to 0.195) Hospital catering 45.77 (42.18-49.39); 136 45.70 (43.32-48.06); 285 0.07 (−4.24 to 4.44), .98 0.003 (−0.206 to 0.214] Global satisfaction score 68.89 (66.36-71.36); 116 72.01 (70.36-73.58); 189 −3.12 (−6.13 to −0.15), .03 −0.253 (−0.490 to −0.014] BODY.DISCUSSION: BODY.PRINCIPAL FINDINGS: The investigation of patient satisfaction after a hospital stay resulted in several differences when comparing the two modes of questionnaire administration: self-reported Internet completion or telephone interview. The comparison between these modes of administration may be discussed according to three topics: response rate, questionnaire reliability, and satisfaction scores. The response rate observed in the group of patients randomized in the Internet group (39.3%) was much lower than that observed in the group of patients randomized in the telephone group (88.4%). Such a difference might have resulted in unbalancing the initial comparability of responders in the two groups even if Table 1 indicates that baseline variables are similar in the responders of the two groups. Unsurprisingly, the observed difference between the two groups in terms of participation rate is in accordance with the previous results issued from the same cohort focusing on patient satisfaction with regard to the hospital discharge process [26]. The difference between the participation rates observed with the two administration modes of the survey might be, at least in part, owing to the fact that it is easier to ignore an email than a phone call scheduled at a date chosen by the patient. The participation rates observed in our study are also similar to those reported by Harewood et al [20] who investigated patient satisfaction with endoscopy and observed a response rate of 34% and 78% in the Internet group and telephone group, respectively. However, comparing the response rates observed in our study with other rates previously reported is probably of limited interest as the study design widely varies from one study to another, as response rates are likely to be highly sensitive to the detailed underlying procedures for selecting participants (eg, face-to-face enrollment vs random selection in an administrative database, issues related to the initial comparability of the participants allocated in the Internet and telephone arms), reaching/soliciting responders (including reminding procedures for soliciting Web participants to complete the survey or procedure for scheduling the phone calls), and collecting answers (eg, attractiveness of the website and ease in accessing/completing the questionnaire form). With the exception of the values for the hospital room convenience dimension, which raise concerns, the values of Cronbach alpha were satisfactory for all dimensions investigated and for the global satisfaction score, favoring the conduction of surveys with this questionnaire using either administration mode. Besides, interestingly, considering all six dimensions of the questionnaire, the values of Cronbach alpha were always higher in the Internet group than in the telephone group, with a statistical significance observed for two dimensions (global care and hospital room convenience) and for the global satisfaction score. Here, the adjunction of an interviewer in the telephone group (as compared with self-completion in the Internet group) might be considered as an undesired burden disturbing initial signal. The observed score differences between the Internet and telephone groups (see Table 3) are contrasted, depending on the dimension investigated. On the one hand, considering hospital room convenience and hospital catering dimensions, both telephone and Internet modes of administration resulted in very similar satisfaction scores, and the difference was only slight when considering global care dimension. On the other hand, in the three dimensions related to interactions with health care providers, that is, information to patients, communication with health care providers, and behavior of health care providers, scores were significantly lower in the Internet group than in the telephone group, although it is worth mentioning that corresponding effect sizes never exceeded 0.35, a value below the medium threshold proposed by Cohen [32]. Such an observation raises a general comment on the surveys conducted in this domain. Those surveys are deployed for investigating patient satisfaction with hospital services, for bringing into light the elements which require improvements and for assessing evolution with time. Additionally, French authorities require the analysis of 120 patients per medical center each year. In such a context, our study’s finding that a mean difference of 7 points based on a sample size of 498 responders is modest in terms of effect size, suggests that potential improvements on patient satisfaction are very difficult to evidence. Dynamic trends within a given center from one year to another should be interpreted with great caution and must take into account the underlying variability of the scores, and a similar caution should be required in the interpretation of differences between centers. A potential explanation for the higher scores observed in the telephone group is that a patient might be more reluctant to provide low scores to an interviewer (moreover potentially identified as a member of the hospital staff) than when completing a strict anonymous form via the Internet. Previous studies [35,36] have also mentioned such a social desirability bias [23] as a potential explanation for the higher patient satisfaction scores issued from a questionnaire administered by a telephone interviewer as compared with a self-completed form administered by mail [35] or via the Internet [36]. In addition, the distribution of the delay between hospital discharge and questionnaire completion was more variable in the Internet group. However, as shown in the Multimedia Appendix 1, the scores of the Internet responders did not significantly vary according to the delay of questionnaire completion, indicating that the wider variability in the delay of questionnaire completion observed in the Internet group had a very limited impact (if any) on the differences of scores that were observed between the telephone and Internet modes of administration. BODY.STRENGTHS AND LIMITATIONS: A strength of the study relies on the fact that it is the first randomized trial reported to date that compared inpatient satisfaction collected either via a telephone interview or via the self-completion of a similar questionnaire on a dedicated website and involved a reasonable sample of inpatients, both in terms of case-mix variability (patients originating from 5 very different hospital wards) and in terms of sample size (498 questionnaires were eventually analyzed). A home access to the Internet and a phone number were two required inclusion criteria for patient eligibility, ensuring the initial comparability of the individuals randomized in the two administration modes of the questionnaire. Moreover, to our knowledge, this study is the first to date that explores, in detail, critical issues relating to the I-Satis questionnaire, which is dedicated to be deployed in all inpatient structures in France; on the one hand, this constitutes an additional strength of the study, while on the other hand, the fact that this questionnaire is yet restricted to France constitutes a limitation of the study. BODY.CONCLUSIONS: In conclusion, our study shows that the lower response rate observed with the Internet mode of administration than that observed with the telephone mode of administration must be balanced with other positive features associated with the Internet. Using the latter mode of administration has a potential lower cost than telephone [14-17], and the quality of satisfaction estimates is likely improved because the potential veil of a telephone interviewer is discarded, allowing patients to express more freely. They are more likely to rate their satisfaction about hospital stay with lower scores. This study indicates that some of these score decreases are statistically significant but the corresponding effect sizes are small, indicating that the decreases relate to moderate differences. Communication habits are evolving, and the Web form is progressively adopted as a reference mode for administrating surveys as well as a reference mode for completing questionnaires. With the exception of the higher response rate observed with telephone interview in this study, all other study results support the deployment of Web-based questionnaires for exploring inpatient satisfaction.

TITLE: Oral liarozole in the treatment of patients with moderate/severe lamellar ichthyosis: results of a randomized, double-blind, multinational, placebo-controlled phase II/III trial ABSTRACT.SUMMARY: ABSTRACT.BACKGROUND: Oral liarozole, a retinoic acid metabolism-blocking agent, may be an alternative to systemic retinoid therapy in patients with lamellar ichthyosis. ABSTRACT.OBJECTIVE: To demonstrate the efficacy and safety of once-daily oral liarozole in the treatment of moderate/severe lamellar ichthyosis. ABSTRACT.METHODS: This was a double-blind, multinational, parallel phase II/III trial (NCT00282724). Patients aged ≥ 14 years with moderate/severe lamellar ichthyosis [Investigator's Global Assessment (IGA) score ≥ 3] were randomized 3 : 3 : 1 to receive oral liarozole (75 or 150 mg) or placebo once daily for 12 weeks. Assessments included: IGA; a five-point scale for erythema, scaling and pruritus severity; Short Form-36 health survey; Dermatology Life Quality Index (DLQI); and safety parameters. The primary efficacy variable was response rate at week 12 (responder: ≥ 2-point decrease in IGA from baseline). ABSTRACT.RESULTS: Sixty-four patients were enrolled. At week 12, 11/27 (41%; liarozole 75 mg), 14/28 (50%; liarozole 150 mg) and one out of nine (11%; placebo) patients were responders; the difference between groups (liarozole 150 mg vs. placebo) was not significant (P = 0·056). Mean IGA and scaling scores decreased from baseline in both liarozole groups at weeks 8 and 12 vs. placebo; erythema and pruritus scores were similar between treatment groups. Improvement in DLQI score was observed in both liarozole groups. Treatment with liarozole for 12 weeks was well tolerated. ABSTRACT.CONCLUSIONS: The primary efficacy variable did not reach statistical significance, possibly owing to the small sample size following premature termination. However, once-daily oral liarozole, 75 and 150 mg, improved scaling and DLQI and was well tolerated in patients with moderate/severe lamellar ichthyosis. BODY.WHAT'S ALREADY KNOWN ABOUT THIS TOPIC?: Oral liarozole, a retinoic acid metabolism-blocking agent, may be an alternative to systemic retinoid therapy for patients with lamellar ichthyosis. BODY.WHAT DOES THIS STUDY ADD?: While the primary endpoint was not met, compared with placebo, once-daily oral liarozole, 75 or 150 mg, decreased overall severity and scaling, but not erythema and pruritus, and improved Dermatology Life Quality Index in patients with lamellar ichthyosis. Oral liarozole was well tolerated. Ichthyoses comprise a large, heterogeneous group of inherited skin disorders resulting from an abnormality of the keratinization process.1,2 Lamellar ichthyosis, a member of the nonsyndromic autosomal recessive congenital ichthyosis group of ichthyoses, has an incidence of 1 : 100 000–300 000 live births.2–4 Patients with lamellar ichthyosis typically have severe generalized hyperkeratosis and dry, scaly skin across the entire body. The disorder may be associated with decreased quality of life (QoL).5,6 Treatment options for lamellar ichthyosis include mechanical scale removal, hydrating and lubricating creams or ointments, and topical keratolytic agents.2 Patients who do not respond adequately to topical agents may be treated with oral retinoids; however, dose-limiting side-effects, including mucocutaneous side-effects, increased serum triglycerides and liver enzymes, and potentially long-lasting teratogenic effects are major constraints of their use.2,7 Women are advised to delay pregnancy for a minimum of 2 years (Europe) or 3 years (U.S.A.) following therapy with acitretin.7,8 Liarozole, a retinoic acid (RA) metabolism-blocking agent (RAMBA) in clinical development, has been granted orphan drug designation for congenital ichthyosis by the European Commission and the U.S. Food and Drug Administration;9,10 such designation is not a judgement on drug effectiveness or safety. RAMBAs inhibit the CYP26-dependent 4-hydroxylation of all-trans-RA, which results in an increased concentration of endogenous all-trans-RA in tissues expressing CYP26, such as the skin.11 Azole RAMBAs, such as liarozole, and the more potent CYP26-specific compound talarozole, are expected to have less systemic toxicity than oral retinoids as they do not require high systemic exposure to RA to achieve therapeutic effects; furthermore, a long-term risk of teratogenic effects is not expected with RAMBAs, as they are quickly eliminated and all-trans-RA levels return to baseline levels within 24 h of treatment discontinuation.11 Previous studies observed clinical improvements in patients with ichthyosis receiving twice-daily oral liarozole, 75 and 150 mg, for 12 weeks.12,13 In the comparative study, liarozole was equally as effective as acitretin and showed a trend towards a more favourable tolerability profile.13 The objective of this study was to demonstrate the efficacy and safety of once-daily oral liarozole, 75 or 150 mg, vs. placebo in the treatment of patients with moderate/severe lamellar ichthyosis. BODY.METHODS: BODY.STUDY DESIGN: This was a randomized, double-blind, placebo-controlled, parallel phase II/III trial to evaluate the efficacy and safety of two once-daily doses of oral liarozole (75 and 150 mg) in the treatment of patients with moderate/severe lamellar ichthyosis (NCT00282724). The study was performed in accordance with Good Clinical Practice (ICH/CPMP/135/95) and the guiding principles of the Declaration of Helsinki,14 and had institutional review board approval from each participating centre. Patients were recruited from 16 treatment centres in nine countries between January 2006 and April 2007. Important study amendments included premature termination of the trial owing to slow recruitment and change of primary efficacy variable to response based on Investigator's Global Assessment (IGA) instead of overall scaling score. BODY.PATIENTS: Eligible patients were aged ≥ 14 years (Canada, Dominican Republic, France and Sweden) or ≥ 18 years (Belgium, Germany, Italy, the Netherlands and Norway) and had lamellar ichthyosis with an IGA score ≥ 3 (moderate to severe) at screening and baseline, a body weight ≥ 45 kg and otherwise good general health, and were free from any disease that could impair the evaluation of ichthyosis. Women of childbearing potential were required to use hormonal and barrier contraception during, and for 1 month after, the treatment period, and required a negative pregnancy test at screening and baseline. Exclusion criteria included: inflammatory skin disease unrelated to ichthyosis; use of topical (except emollient) or ultraviolet treatment for ichthyosis ≤ 2 weeks (≤ 4 weeks in Sweden) prior to baseline; use of systemic therapy for ichthyosis or vitamin A supplements ≤ 4 weeks prior to baseline; use of drugs metabolized by the CYP450 system during the treatment period; use of immunosuppressive drugs, including topical or systemic corticosteroids; retinoid hypersensitivity; significant hepatic, renal or immune disease, osteoporosis, or history of adrenal cortex dysfunction; heart disorders requiring treatment, myocardial infarction in the previous 24 weeks, or a history of heart failure/cardiac arrhythmia. BODY.INTERVENTIONS: Patients attended up to six study visits over 18–20 weeks (screening, baseline, weeks 4, 8 and 12, follow-up). At the screening visit, patients provided written informed consent, demographic characteristics and medical history, and were evaluated against the inclusion/exclusion criteria. Patients then entered a 2- to 4-week washout period to confirm ichthyosis severity before randomization. At baseline, patients were re-evaluated against inclusion/exclusion criteria. Eligible patients were randomized 3 : 3 : 1 to receive once-daily liarozole 75 mg (one liarozole 75-mg tablet and one placebo tablet), liarozole 150 mg (two liarozole 75-mg tablets), or placebo (two placebo tablets) for 12 weeks using dynamic minimization to ensure balanced treatment groups within centres. Randomization was weighted towards the liarozole treatment groups owing to ichthyosis severity in patients requiring systemic treatment. Liarozole and placebo tablets (provided by Barrier Therapeutics, Geel, Belgium) were identical in appearance and packaging. Following the treatment period, patients entered a 4-week follow-up period and attended a study visit at week 16. Patients could continue mechanical scale removal and use of emollients except ≤ 12 h preceding a study visit. BODY.ASSESSMENTS: At all study visits, patients provided medication usage, underwent a physical examination and vital signs measurements, and provided blood and urine samples for clinical laboratory analyses. Patients completed a daily diary for 7 days prior to each study visit (except screening), noting concomitant medications, emollient use and mechanical scale removal. Lamellar ichthyosis was assessed at all study visits using the IGA (5-point scale: 0, clear; 1, almost clear; 2, mild; 3, moderate; 4, severe); erythema, scaling and pruritus on the legs, trunk, palms and scalp were evaluated as marker areas on a 5-point scale (0, none; 1, mild; 2, moderate; 3, severe; 4, very severe), and an overall scaling score was calculated; individual scaling scores for each marker area were weighted (4 × score for legs + 3 × score for trunk + 1 × score for palms + 1 × score for scalp) and summed (minimum summed score, 0; maximum summed score, 36). Efficacy assessments for each individual patient were performed by the same investigator throughout the trial, or by a subinvestigator familiar with the study and subject. Quality of life was assessed at all study visits except week 8 using the acute (1-week) recall version of the Short Form (SF)-36 health survey,15 a 36-item questionnaire with eight domains (higher scores indicate better QoL) and the Dermatology Life Quality Index (DLQI),16,17 a 10-item questionnaire with six domains (higher scores indicate poorer QoL). Ophthalmological examinations (slit-lamp inspection for corneal opacity, Schirmer's tear test and fluorescein staining for corneal damage) and electrocardiographic (ECG) examinations were performed between screening and baseline, at week 4 (ECG; Germany only) and week 12. ECG examinations and clinical laboratory analyses were performed at follow-up if previous assessment revealed clinically relevant abnormalities. Concentrations of bone markers in serum [osteocalcin, intact procollagen type I N-terminal propeptide (PINP) and C-terminal cross-linking telopeptide of type I collagen (CTX)] and urine [N-terminal cross-linking telopeptide of type I collagen (NTX)] were assessed at baseline, weeks 4 and 12, and follow-up. Adverse events (AEs) were recorded and mucocutaneous symptoms possibly related to RA (cheilitis, epistaxis, hair loss) graded for severity on a 5-point Likert scale from 0 (absent) to 4 (very severe) at baseline, weeks 4, 8 and 12, and follow-up. A composite score for mucocutaneous symptoms was calculated as the sum of the severity grades. Plasma liarozole concentration was assessed prior to dosing at baseline and week 8; predose and 1–2 h postdose at weeks 4 and 12. Photographs of the two most severely affected body areas at screening were taken at all study visits. BODY.ANALYSIS POPULATIONS: The intent-to-treat (ITT) population included all patients who received one or more doses of study medication and had postbaseline efficacy data. The safety population included all patients who were randomized into the study. BODY.STUDY END VARIABLES: The primary efficacy variable was response rate at week 12. A patient was a responder if their IGA score decreased by ≥ 2 points from baseline. Secondary end variables were: IGA score at week 8; change from baseline in overall scaling score at weeks 8 and 12; change from baseline in severity scores for erythema and pruritus at weeks 8 and 12; SF-36 and DLQI scores; use of emollients and mechanical scale removal; safety and tolerability; and plasma liarozole concentration. BODY.SAMPLE SIZE AND STATISTICAL METHODS: A total of 98 patients were planned for enrolment to provide 90% power to detect a difference between a response rate of 0·20 (placebo group) and 0·75 (liarozole groups) and to accommodate a drop-out rate of 15%. Data from all centres were combined for analyses. For the primary efficacy analysis (ITT population), the 150-mg liarozole group was compared with the placebo group; if a significant difference was observed, the 75-mg liarozole group was tested against the placebo group. If IGA evaluation at week 12 was missing, last observation carried forward was used. The number of responders in each liarozole group was compared with those in the placebo group using Fisher's exact test. IGA and overall erythema, scaling and pruritus scores at weeks 8 and 12 were compared between liarozole and placebo groups using the Wilcoxon–Mann–Whitney rank sum test, provided the analysis of the primary efficacy variable showed a significant result for that dosage group. Changes from baseline in QoL scores were compared between each of the liarozole groups and the placebo group using analysis of covariance (ancova); covariates included sex, age and baseline value. The proportion of patients reporting one or more AEs was compared between the liarozole and the placebo groups using Fisher's exact test. Changes from baseline in vital signs were evaluated within each group using paired t-tests and vs. the placebo group using unpaired t-tests. Mucocutaneous symptoms were evaluated using the Wilcoxon signed rank test within each group and compared with placebo using the Wilcoxon–Mann–Whitney rank sum test. For bone markers, changes from baseline in treatment groups were compared using an ancova model; within-group changes from baseline were assessed with a paired t-test. All statistical analyses were performed using SAS software version 8.02 (SAS Institute, Cary, NC, U.S.A.) and significance set as P < 0·05. BODY.RESULTS: A total of 84 patients were screened; 64 patients were randomized and included in the safety and ITT populations (Fig.1). Baseline demographics were similar between treatment groups (Table 1). Patients were aged 17–70 years, predominantly white (81%; 52/64) and 52% (33/64) were male. No significant differences between treatment groups were observed for baseline bone markers. One patient (75-mg liarozole group) was taking prohibited medication at baseline (budesonide and salmeterol xinafoate aerosols for asthma). Table 1Baseline demographics and characteristics (intent-to-treat and safety population) CharacteristicPlacebo (n = 9)Liarozole 75 mg (n = 27)Liarozole 150 mg (n = 28)Age (years), mean (SD), range 36 (14), 19–59 40 (14), 18–65 36 (15), 17–70 Sex, n (%)  Male 5 (56) 14 (52) 14 (50) Race, n (%)  Moroccan – – 1 (4)  Hispanic – 4 (15) 5 (18)  White 8 (89) 22 (81) 22 (79)  Black 1 (11) 1 (4) – Height (cm), mean (SD), range 166 (15), 137–183 166 (17), 132–196 165 (17), 130–196 Weight (kg), mean (SD), range 73 (21), 55–122 73 (19), 35–116 70 (15), 52–109 Body mass index (kg m−2), mean (SD), range 26·4 (6·8), 18·2–36·6 26·5 (4·9), 15·6–36·9 25·9 (5·4), 18·0–40·0 IGA score, mean (SD), range 3·4 (0·5), 3–4 3·6 (0·5), 3–4 3·5 (0·5), 3–4 Scaling score, mean (SD), range 26·8 (6·7), 13–34 27·9 (5·7), 15–36 28·8 (6·2), 17–36 Erythema score, mean (SD), range 13·7 (9·2), 3–28 15·9 (9·6), 0–36 14·9 (8·9), 0–36 Pruritus score, mean (SD), range 13·3 (10·2), 0–32 14·7 (11·0), 0–34 14·2 (9·0), 0–33 Total symptoms score, mean (SD), range 53·8 (18·7), 26–85 58·5 (19·3), 27–104 57·9 (16·4), 20–95 Mechanical scale removal (proportion of days used during week before baseline),a mean (SD) 0·63 (0·42) 0·47 (0·42) 0·59 (0·37) Use of emollients during week before baseline (average score),a mean (SD) 1·68 (0·54) 1·43 (0·54) 1·32 (0·51) QoL scores, mean (SD)  SF-36 standardized physical component scale 50·8 (7·9) 49·6 (7·0) 50·9 (5·7)  SF-36 standardized mental component scale 41·4 (10·1) 47·5 (10·2) 46·0 (9·3)  DLQI 13·7 (6·7) 9·4 (6·7) 7·3 (4·1)b Mucocutaneous symptoms (composite score),c mean (SD) 0·0 (0·00) 0·36 (0·81)d 0·82 (1·31) Ophthalmological abnormalities, n (%) 3 (33) 14 (52) 15 (54) DLQI, Dermatology Life Quality Index; IGA, Investigator's Global Assessment; QoL, quality of life; SF-36, Short Form-36 health survey. aFor 7 days prior to the baseline visit (and subsequent study visits), patients completed a daily diary, noting the use of emollients (none, a little or much) and mechanical scale removal (yes or no); bn = 27; csum of severity scores for cheilitis, epistaxis and hair loss, each graded on a 5-point scale from 0 (absent) to 4 (very severe), maximum composite score = 12; dn = 25. Figure 1Patient flow diagram. BODY.PRIMARY EFFICACY VARIABLE: One of nine (11%) patients in the placebo group was a responder vs. 11/27 (41%) patients in the 75-mg liarozole group and 14/28 (50%) patients in the 150-mg group. However, the difference between the liarozole 150 mg and placebo groups was not significant (P = 0·056). Clinical improvement in a patient with lamellar ichthyosis following treatment with oral liarozole (150 mg) is shown in Figure2. Figure 2Patient with lamellar ichthyosis (a) at baseline and (b) after 12 weeks of treatment with once-daily liarozole (150 mg). Marked improvement of scaling from severe [Investigator's Global Assessment (IGA) score of 4] at baseline to almost clear (IGA score of 1) after treatment is observed. BODY.SECONDARY END VARIABLES: As no significant difference was demonstrated for the primary efficacy variable, formal statistical analyses were not planned to be performed on secondary efficacy variables, as per the study statistical analysis plan. Statistical analyses were, however, performed post hoc and are provided for information in Table S1 (see Supporting Information). Compared with the placebo group, mean IGA score decreased from baseline to weeks 8 and 12 in the 75- and 150-mg liarozole groups and returned to near baseline values at the follow-up visit (Fig.3). Mean scaling score, but not erythema or pruritus scores, also decreased from baseline to weeks 8 and 12 in the 75- and 150-mg liarozole groups compared with the placebo group (Fig.4). No obvious difference in the use of mechanical scale removal and emollients was observed between treatment groups. The use of emollients remained stable during the treatment period in the placebo and the 150-mg liarozole groups; a slight decrease was observed in the 75-mg liarozole group. Figure 3Mean Investigator's Global Assessment (IGA) score over time (intent-to-treat population, last observation carried forward). For results of post hoc Figure 4Mean symptom scores over time: (a) scaling, (b) erythema and (c) pruritus (intent-to-treat population; last observation carried forward). For results of post hoc statistical analysis, see Table S1. SF-36 summary measures remained mostly stable during the study in all treatment groups, except for transient improvement at week 4 in the physical component scale for patients receiving liarozole 75 mg, and in the mental component scale for patients receiving liarozole 150 mg. During the treatment period, mean DLQI score remained stable in the placebo group, whereas an improvement in mean DLQI score was observed in both liarozole groups, followed by worsening during the follow-up period (Figure S1; see Supporting Information). These trends were observed in all domains. Mean DLQI score improved significantly from baseline at week 12 in patients receiving liarozole 75 mg vs. patients receiving placebo [mean (SD) change, −4·3 (6·0); P = 0·014]. Significant differences were observed between the 75-mg liarozole and the placebo groups for all domains at week 12 (P < 0·05) except ‘personal relationships’ and ‘work and school’. No significant difference was observed between the 150-mg liarozole and the placebo groups in change from baseline of mean DLQI score at week 12 [mean (SD) change, −2·8 (3·4); P = 0·06]; however, the 150-mg liarozole group showed a significant improvement from baseline vs. the placebo group in the ‘daily activities’ (P = 0·008), ‘leisure’ (P = 0·026), and ‘symptoms and feelings’ (P = 0·023) domains. BODY.PHARMACOKINETICS: In the 75- and 150-mg liarozole groups, predose and near-peak plasma concentrations remained the same order of magnitude throughout treatment, and no accumulation occurred (Table S2; see Supporting Information). BODY.SAFETY: Overall, 178 AEs were reported for 45 patients (Table S3; see Supporting Information). Both liarozole groups presented a higher number of AEs than the placebo group; however, the difference between groups was not significant (liarozole 75 or 150 mg vs. placebo, P ≥ 0·062), and AE incidence did not appear to be related to liarozole dose. The most frequently reported AEs are shown in Table 2; with the exception of nasopharyngitis and nausea, all AEs occurred more frequently in the liarozole groups than in the placebo group. Treatment-emergent AEs were mostly mild to moderate in severity; four were severe (fungal infection, intermittent chest cramp, joint pain and dry skin). Two serious AEs occurred: one patient was hospitalized for deterioration of ichthyosis during the wash-out period and was not randomized to any study treatment; one patient in the 150-mg liarozole group became pregnant 10 days after the last administration of study medication and gave birth to a child with a dilated renal pelvis during the poststudy period. This condition was considered not related to liarozole treatment. Table 2Incidence of adverse events occurring in > 10% of patients and two or more patients within at least one treatment group (safety population) Preferred termaPlacebo group (n = 9)Liarozole 75 mg group (n = 27)Liarozole 150 mg group (n = 28)Liarozole, combined groups (n = 55)Nasopharyngitis 3 (22) 2 (7) 4 (11) 6 (9) Alopecia 1 (11) 1 (4) 5 (11) 6 (7) Fatigue – 3 (11) 3 (11) 6 (11) Arthralgia – 2 (7) 4 (11) 6 (9) Headache – 2 (7) 4 (11) 6 (9) Nausea 2 (22) 2 (7) 1 (4) 3 (5) Pruritus – 3 (11) 2 (7) 5 (9) Epistaxis – 4 (11) 1 (4) 5 (7) Cheilitis – 3 (11) 2 (7) 5 (9) Creatine phosphokinase increased – 1 (4) 3 (11) 4 (7) Hyperhidrosis – 3 (11) – 3 (5) Skin exfoliation – 3 (11) – 3 (5) aValues shown are the number of events (percentage of patients with event). No relevant changes from baseline were observed in the mean values of alanine aminotransferase, aspartate aminotransferase, γ-glutamyltransferase, creatine phosphokinase or plasma lipids in any treatment group. Overall, 58 patients (91%) had one or more abnormal laboratory parameters during the treatment or follow-up period, which were considered clinically relevant and related to study treatment in seven of the 64 patients (11%; Table 3). Table 3Most clinically relevant treatment-related adverse events (safety population) Placebo group (n = 9)Liarozole 75 mg group (n = 27)Liarozole 150 mg group (n = 28)Liarozole, combined groups (n = 55)Laboratory abnormalities, n  Increased plasma creatine phosphokinase 0 1 2 3  Increased alanine aminotransferase 0 0 1 1  Increased plasma lipids 0 0 2 2  Increased blood cell count 1 0 0 0 Physical examination abnormalities, n 0 1a 3b 4 Vital signs abnormalities, n 0 1c 1c 2 Ophthalmological abnormalities, n 0 0 1d 1 Electrocardiogram abnormalities, n 0 0 2e 2 Mucocutaneous symptoms, n 1f 6g 4h 10 aMild shoulder pain, mild arthralgia and mild pain in extremity; bmoderate rash (one patient), mild hypertension (one patient), moderate herpes simplex and moderate lymphadenopathy (one patient); chypertension (mild in 150-mg group, moderate in 75-mg group); dmoderate worsening of Schirmer's tear test and tearfilm break-up time; emoderate ventricular extrasystoles (one patient), mild abnormal electrocardiogram (not further specified) that was already reported at screening (one patient); fmild alopecia; gmild epistaxis (three patients), mild cheilitis (two patients), moderate cheilitis (one patient), moderate alopecia (one patient); hmoderate epistaxis (one patient), mild alopecia (one patient), mild alopecia and mild cheilitis (two patients). No relevant changes in ECG intervals were observed. Two ECG abnormalities were reported as treatment-related AEs (Table 3). Most physical examination, vital signs and ophthalmological abnormalities recorded during the treatment and follow-up phases, as appropriate, were reported at screening or baseline, and were not considered clinically relevant. Those reported as AEs possibly related to study drug are shown in Table 3. There were no significant differences between treatment groups regarding the change from baseline in severity scores for mucocutaneous symptoms. Eleven patients reported mucocutaneous symptoms that were recorded as AEs, all of which were considered to be related to the study treatment (Table 3). Slight increases in bone resorption biomarkers (serum CTX and urinary NTX) from baseline were observed in both liarozole groups; however, this effect was of low magnitude, not dose related and did not differ significantly from the placebo group except for CTX (75-mg liarozole group) 4 weeks after discontinuation of liarozole treatment. Liarozole induced a modest, significant, dose-related decrease in the bone formation marker serum PINP from baseline in both liarozole groups (75 mg: −11·72%, P = 0·0224; 150 mg: −16·00%, P = 0·0036); this decrease was transient and returned to baseline concentrations 4 weeks after termination of liarozole treatment. No significant changes in serum bone formation marker osteocalcin were observed, except a significant increase vs. baseline (P = 0·0005) and placebo (P = 0·04) in the 75-mg liarozole group 4 weeks after therapy discontinuation. This isolated increase was not dose related. BODY.DISCUSSION: The primary objective of this study was to demonstrate the efficacy of two different once-daily doses of oral liarozole vs. placebo in patients with lamellar ichthyosis. In the liarozole groups, 41% (75-mg liarozole group) and 50% (150-mg liarozole group) of patients were considered responders to treatment vs. 11% of patients in the placebo group. However, the difference between the 150-mg liarozole group and the placebo group at week 12 was not significant (P = 0·056). As the study was terminated prematurely as a result of slow recruitment, failure to reach significance may have been largely due to diminished statistical power of the reduced sample size. Furthermore, the definition of a responder (≥ 2-point decrease in IGA score) may not have been ideal owing to possible spontaneous fluctuation in ichthyosis severity and low sensitivity of a subjective 5-point scale. IGA and scaling scores decreased in both liarozole treatment groups vs. the placebo group. Changes in erythema and pruritus were similar between treatment groups, and no obvious difference in the use of emollients or mechanical scale removal was observed between treatment groups, possibly due to continued daily skincare routines of many patients with lamellar ichthyosis. While QoL remained mostly stable during the study when assessed using the SF-36 health survey, a disease nonspecific assessment,15 improvement in QoL was observed at week 12 in patients receiving both liarozole doses compared with placebo using a skin disease-specific assessment (DLQI). Once-daily liarozole, 75 or 150 mg, for 12 weeks was well tolerated. No undue accumulation of liarozole was observed in pharmacokinetic studies, and there were no relevant differences between the liarozole groups and the placebo group with regard to safety parameters. Most patients (91%) had at least one laboratory abnormality during the treatment and follow-up periods; these were considered clinically relevant in seven patients (11%) (five of whom were in the 150-mg liarozole group). Safety findings are consistent with those of previous 12-week studies of twice-daily oral liarozole at 150 and 75 mg.12,13 A modest transient decrease in PINP, a marker of type I collagen synthesis and bone formation, was observed during the trial; the long-term effect of liarozole on bone turnover is unknown. Reduced sample size limits the efficacy conclusions that can be drawn from the study; despite this, the sample size is considerable given the rarity of the disease and the stringent eligibility criteria for study participation. Future studies should consider more congruent and effective eligibility criteria, an assessment of treatment success more consistent with clinical practice, and the low prevalence of lamellar ichthyosis, when planning the size of the study population. When limited numbers of patients can be recruited, a different randomization scheme, for example with equal size placebo and active treatment groups, would increase the power to detect a treatment effect. In conclusion, statistical significance was not obtained for the primary efficacy variable (response rate), possibly due to decreased statistical power as a consequence of reduced sample size. Compared with placebo, oral liarozole, 75 or 150 mg, once daily for 12 weeks, reduced the overall severity of ichthyosis and scaling, but not erythema or pruritus, and improved DLQI in patients with moderate or severe lamellar ichthyosis. Oral liarozole was well tolerated.

TITLE: ProSeal™ laryngeal mask airway cuff pressure changes with and without use of nitrous oxide during laparoscopic surgery ABSTRACT.BACKGROUND:: The ProSeal™ laryngeal mask (PLMA) is increasingly being used as an airway device for laparoscopic surgery. Its silicone cuff allows diffusion of nitrous oxide, carbon dioxide and other gases with resultant rise in its intracuff pressure during anesthesia. The present study was designed to investigate the intracuff pressure changes during anesthesia with and without nitrous oxide in patients undergoing laparoscopic surgery lasting up to two hours. ABSTRACT.MATERIALS AND METHODS:: One hundred patients, American Society of Anesthesiologists physical status 2 and 3, undergoing general anesthesia with muscle paralysis, were randomized into two groups of 50 patients each to receive an anesthetic gas mixture containing either oxygen and nitrous oxide (group N) or oxygen and air (group A). Following insertion of an appropriate size PLMA, its cuff was inflated with air to an intracuff pressure of 45 mm Hg. The cuff pressure was measured every 10 minutes for the entire course of anesthesia. The position of the device was also assessed fiberoptically and postoperative airway complications were recorded. ABSTRACT.RESULTS:: The maximum intracuff pressure recorded in group N was 103 ± 4.7 mm Hg vs. 45.5 ± 1.5 mm Hg in group A. The percentage rise in cuff pressure every 10 minutes was also highly significant (P < 0.001) being maximum in first 10 min in group N. The incidence of postoperative airway complications was comparable between the two groups. ABSTRACT.CONCLUSION:: The results of this study demonstrate that the intracuff pressure of the PLMA increases progressively over time when the breathing gas mixture contains nitrous oxide. BODY.INTRODUCTION: Several cuffed supraglottic airway devices (SADs) have been introduced into clinical practice since the introduction of classic laryngeal mask airway (cLMA). An ideal cuff of a SAD should provide good airway seal during positive pressure ventilation and protect against pulmonary aspiration without causing trauma to the surrounding structures. A recent survey reported that the use of a supraglottic airway as a primary airway management device for general anesthesia is as high as 56.2%.[1] The ProSeal™ laryngeal mask airway (PLMA), is a directional perilaryngeal sealer.[2–4] Its special cuff presses firmly into the periglottic tissues allowing an oropharyngeal seal (≥ 30 cmH2O) without increasing directly measured mucosal pressure.[35] Inflating the cuff to an intracuff pressure of 60 cmH2O (45 mmHg) is recommended to prevent leak. Excessive intracuff pressure can result in malposition, suboptimal seal and pharyngolaryngeal morbidity, including sore throat, dysphagia and nerve injury.[2] Nitrous oxide, carbon dioxide and other gases can diffuse into the cuff of airway devices and may further increase the intracuff pressure. There are several reports of the use of PLMA in laparoscopic surgery[6–9] but there is paucity of data on the measurement of intracuff pressure changes during these procedures. The present study was designed to investigate the intracuff pressure changes during anesthesia with and without nitrous oxide in patients undergoing laparoscopic surgery. BODY.MATERIALS AND METHODS: This randomized prospective study was conducted after obtaining approval from the hospital ethics committee and written informed consent from the patients. One hundred adult patients, of either sex, in American Society of Anesthesiologists physical status 1-2, undergoing elective laparoscopic surgery with an anticipated duration of 30-120 minutes were recruited for the study. Exclusion criteria included obesity (Body Mass Index > 35), oropharyngeal pathology, limited mouth opening (inter-incisor gap < 20 mm) and patients at risk of aspiration (full stomach, previous upper gastrointestinal surgery, hiatus hernia). The patients were randomized by computer generated numbers to one of two groups of 50 patients each. They were to receive an anesthetic gas mixture of oxygen and air (group A) or oxygen and nitrous oxide (group N). Patient enrollment was performed by an investigating anesthesiologist blinded to patient allocation. Anesthetic technique was standardized. After intravenous (IV) access, patients were premedicated with ranitidine 50 mg, metoclopramide 10 mg, glycopyrrolate 0.2 mg and midazolam 1 mg IV. Monitoring included pulse oximetry, electrokardiograph, non-invasive blood pressure, capnography and neuromuscular monitoring with train of four ratios. Pre-use checkup and size selection of PLMA was done as recommended by the manufacturer.[2] The transducer (Pressure Monitoring Kit, Edwards Life Sciences, US) to measure the cuff pressure was set up as follows. The transducer was fixed to the operating table by means of clamp (provided by manufacturer) mounted on a steel rod with its height adjusted 10 cm above the operating table top, corresponding to the level of the glottis with the patient lying supine. The transducer cable was attached to the monitor. Other end of transducer was attached to the PLMA cuff with the help of pressure monitoring line, 100 cm long and a three way stop-cock. Zeroing of the transducer was done by opening transducer to the atmosphere and was repeated every 30 minutes or whenever changes were made in patient's position. The entire set-up was filled with air only. The cuff was deflated and the cuff pressure was set at -25 mmHg with the help of a transducer connected to the monitor. Anesthesia was induced with fentanyl citrate 1.5 mcg/kg and propofol 2 mg/kg IV. Neuromuscular blockade was achieved with vecuronium bromide 0.1 mg/kg IV. One anesthesiologist (out of two anesthesiologists in the study) well versed with PLMA use, inserted and fixed the device with the following two anesthesiologists well versed with PLMA use, inserted and fixed the device. A hand-held cuff manometer (Mallinckrodt Medical, Athlone, Ireland) was connected to the pilot balloon of the PLMA via a three-way stopcock and the cuff was inflated with air to an intracuff pressure of 45 mm Hg (60 cmH2O). Anesthesia was maintained with 1-2% sevoflurane in oxygen/air or oxygen/nitrous oxide (FiO2 = 0.3). Aliquots of vecuronium bromide 0.02 mg/kg were given for maintenance of neuromuscular blockade, as and when required. The PLMA was connected to the gas delivery circuit of the anesthesia machine (Prima SP, Penlon, Abingdon, UK). Proper placement of the PLMA was confirmed by; capnography, bilateral chest wall movements and absence of audible leak from the drain tube with the peak airway pressure <20 cmH2O. A maximum of three attempts were allowed. If insertion failed after 3 attempts, the airway was secured by a tracheal tube. Posterior folding of mask was ruled out by passing a gastric tube through the drain tube and its correct position confirmed by aspiration of gastric contents or by auscultating the epigastrium while injecting air. The patients were mechanically ventilated with a tidal volume of 8 ml/kg (volume control mode) with I:E ratio of 1:2. Respiratory rate was adjusted to maintain EtCO2 between 35-45 mmHg. The oropharyngeal seal pressure (OSP) was measured by giving a fresh gas flow of 4 l/min, closing the expiratory valve of the circuit and noting the pressure at which there was an audible leak. The OSP was not allowed to exceed 40 cmH2O to avoid barotrauma. The position of the PLMA was evaluated by a flexible fiberoptic scope (Olympus LF-2, Japan) introduced into the airway tube, for viewing the laryngeal structures. The fiberoptic view was graded on the following scoring system; 4 = vocal cords only; 3 = vocal cords plus posterior epiglottis (PE); 2 = vocal cords plus anterior epiglottis (AE); 1 = vocal cords not seen.[10] The peak airway pressure and cuff pressure were monitored. The cuff pressure was recorded every 10 minutes for the entire course of anesthesia, using a transducer which was zeroed every 30 minutes and with every change in patient's position. The measurements were made with the head and neck in neutral position. In both groups, the intra-abdominal pressure was held constant at 15 mmHg by an automatic, high flow insufflation unit (Olympus, UHI 3, Tokyo, Japan). If surgery lasted more than 2 hours, the anesthesiologist was allowed to adjust the intracuff pressure of the PLMA to 45 mmHg but the data collection was terminated at that point. After the completion of procedure, the neuromuscular block was reversed with neostigmine 0.05 mg/kg and glycopyrrolate 0.02 mg/kg and the PLMA was removed with the cuff inflated. Post-operatively, the patients were assessed by an independent observer blinded to the technique, in the post-anesthesia care unit for sore throat (constant pain, independent of swallowing), dysphagia (difficulty in, or pain provoked by swallowing), hoarseness of voice, dysphonia (difficulty in or pain on speaking) and numbness of the tongue. Based on a pilot study, the sample size was calculated to detect a difference of 55 % increase in the intracuff pressure in group N when compared to group A at 30 minutes duration of surgery to achieve 90% power at 5% level of significance. The primary variable was the cuff pressure. Statistical tests were performed using SPSS software (17.0 version; SPSS Inc., Chicago, IL, USA). Statistical techniques included quantitative and qualitative analysis. Continuous variables were presented as mean ± SD and categorical variables were expressed as frequencies. Unpaired t-tests were used for comparison of continuous variables between the two groups. Differences between groups were assessed with Chi-square or Fisher's exact test for categorical variables as appropriate. P value of < 0.05 was taken as significant. BODY.RESULTS: Complete data were obtained from all patients. Data collection was terminated in one patient of group N at 120 min and the cuff pressure was reset to 45 mmHg as the surgery lasted for 220 minutes. The patient characteristics, methods of insertion, and number of insertion attempts, anesthesia time, the mean OSP and fiberoptic position [Table 1] were comparable between the two groups. Table 1Patients characteristics and anesthesia details In group N, [Table 2] there was a consistent and significant increase in intracuff pressure over time (P < 0.001) while in group A, it remained stable throughout the procedure (maximum intracuff pressure of 45 mmHg). The maximum intracuff pressure recorded in group N was 103 mmHg which was 129% higher than the baseline (p < 0.001). The percentage rise in cuff pressure every 10 minutes was also highly significant (p < 0.001). It was highest (19%) in the first 10 min, decreasing over time to 5% at 120 minutes. The rise in peak airway pressure after carboperitoneum was comparable between the two groups. There was no evidence of displacement of device in either group. Table 2Intracuff pressure (mmHg) changes of PLMA over time in groups N and A In the post anesthesia period, five (10%) patients in Group N and one (2%) patient in group A complained of sore throat (p = 0.2). The incidence of sore throat increased with increased number of insertion attempts (p = 0.04) Hoarseness of voice was reported in one (2%) patient in group N. There was no incidence of dysphagia, dysphonia and numbness of tongue in either group. BODY.DISCUSSION: We observed a significant and progressive increase in intracuff pressure of the PLMA over time when nitrous oxide was used as a part of balanced anesthesia technique for laparoscopic surgery. However, the cuff pressure did not change when air was used instead of nitrous oxide (p < 0.001). The incidence of sore throat was comparable between the two groups. Airway devices have cuffs which are permeable to a variety of gases depending on their partial pressure, and solubility. The composition and thickness of the cuff material (latex, silicone or polyvinyl chloride) play a significant role in the intracuff pressure changes during anesthesia. Nitrous oxide and other gases diffuse into air filled cuffs of tracheal tubes and supraglottic devices, increasing their volume and pressure.[11–13] The addition of plasticizers to the polyvinyl chloride cuff material of the tracheal tubes and disposable LMAs softens it and renders it less permeable to nitrous oxide.[1415] The reusable cLMA and PLMA cuffs are made up of silicone. The elastance for the ProSeal™ has been reported to be lower than that of the cLMA, probably due to its larger cuff size.[5] In a case report of a 5h general anesthesia, with gas mixture containing 57-63% nitrous oxide, it was observed that the intracuff pressure of PLMA increased from base line of 45 mmHg to approximately 76 mmHg after one hour, 87 mmHg after two hours, and remained stable thereafter.[12] A similar increase in intracuff pressure from 45 mmHg to 91 mmHg within two hours of surgery has been reported using the laryngeal tube with a silicone cuff.[13] In our study too, the intracuff pressure remained around 45 mmHg in group A while in group N, the percentage rise in cuff pressure every 10 minutes from the baseline was significantly higher (P < 0.001), reaching 103 mmHg, i.e. an increase of 129% from baseline at the end of the 120 min study period. The maximum increase, at first 10 min was due to the increased pressure gradient at initial low intracuff volume. With the passage of time, it declined as the pressure gradient decreased with further diffusion of nitrous oxide into the PLMA cuff. The rise in the intracuff pressure of the tracheal tubes and supraglottic devices is known to increase the ischemic damage to the surrounding pharyngolaryngeal mucosa.[16–20] Unlike the tracheal tube cuff which expands within the rigid confines of the tracheal rings, the PLMA cuff inflates in the compliant potential space of the pharynx allowing the cuff walls to match the contours of pharyngeal and laryngeal surfaces.[21] A progressive reduction in the pharyngeal mucosal perfusion has been reported when mucosal pressure increases from 25 to 60 mmHg while using a cuffed oropharyngeal airway.[18] The cuffs of cLMA and PLMA exert pressure on the pharyngeal mucosa causing a concomitant decrease of pharyngeal perfusion and increase in the incidence of post-operative complications including sore throat, dysphonia, and nerve damage.[18–20] Recurrent laryngeal nerve neuropraxia resulting from high cuff pressure due to nitrous oxide influx during anesthesia has been reported following the use of PLMA in a patient with CREST syndrome (Calcinosis, Raynaud's syndrome, Esophageal Dysmotility, Sclerodactyly, Telangiectasia).[22] However, another study with the PLMA suggested that directly measured mucosal perfusion pressure rarely exceeded 25 mmHg[19] and therefore, did not increase pharyngeal mucosal injury, while others recommend reducing the cuff volume until it just seals the leak.[2324] A cadaveric study comparing the mucosal pressure induced by cuffs of seven airway devices reported that the cuff of PLMA inflated with maximum recommended cuff volumes, exerted lower pressures (predominantly below 15 mm Hg) on the pharyngeal and hypopharyngeal mucosa than the cuff of cLMA. It was also reported that PLMA along with easy tube, induced significantly higher pharyngeal pressures in the posterior location when compared to other devices. This may be because of the additional cuff on the posterior part of the PLMA.[25] Higher cuff pressure and higher incidence of postoperative sore throat has also been reported after use of PLMA in children while breathing 50% nitrous oxide and oxygen mixture in comparison to patients whose breathing gases were composed of oxygen and air.[26] Tekin et al. recommended inflating the PLMA cuff with nitrous oxide and oxygen mixture to avoid further increase in cuff pressure when nitrous oxide was a part of general anesthesia technique.[27] Carbon dioxide used during the laparoscopic procedures gets absorbed into the blood to increase the PaCO2 as well as the end-tidal carbon dioxide, and may diffuse into the cuff to increase intracuff pressure. However, it does not contribute to rise in intracuff pressure as the cuff pressure remained unchanged in group A. The rise in intracuff pressure in group N can be attributed to the diffusion of nitrous oxide which is more diffusible than carbon dioxide.[28] Moreover, the inspired/expired concentration of carbon dioxide was low (4-6.5% vs. 66% for nitrous oxide). The reported incidence of sore throat with the PLMA is 14-23%.[829] In our study, the incidence of sore throat was low and comparable between the two groups. Since we had limited our study to two hours duration, no conclusion can be drawn regarding the incidence of sore throat being related to the duration of anesthesia. As the device is being increasingly used for procedures longer than two hours, vigilance is required during its use and excessive gas should be regularly removed from the cuff. Our study has a few limitations. We did not record the pharyngeal mucosal pressure or analyze the intracuff gas mixture due to the non-availability of the appropriate equipment (microchip sensors or gas analyzer). Since the number of attempts at insertion also has a significant relationship with the incidence of postoperative sore throat, the study cannot exclusively relate sore throat with rise in intra-cuff pressures. BODY.CONCLUSION: In conclusion, the results of this study demonstrate that the intracuff pressure of PLMA increases progressively over time when the breathing gas mixture contains nitrous oxide. Use of cuff pressure monitor is recommended for initial cuff inflation as well as for intraoperative monitoring during prolonged surgery when using a nitrous oxide based anesthesia technique. Further studies confined to single attempt of PLMA insertion would be necessary to evaluate the relationship of sore throat with prolonged duration of nitrous oxide based anesthesia.

TITLE: Effects of risk-based multifactorial fall prevention on health-related quality of life among the community-dwelling aged: a randomized controlled trial ABSTRACT.BACKGROUND: This study aimed to assess the effects of a risk-based, multifactorial fall prevention programme on health-related quality of life among the community-dwelling aged who had fallen at least once during the previous 12 months. ABSTRACT.METHODS: The study is a part of a single-centre, risk-based, multifactorial randomised controlled trial. The intervention lasted for 12 months and consisted of a geriatric assessment, guidance and treatment, individual instruction in fall prevention, group exercise, lectures on themes related to falling, psychosocial group activities and home exercise. Of the total study population (n = 591, 97% of eligible subjects), 513(251 in the intervention group and 262 in the control group) participated in this study. The effect of the intervention on quality of life was measured using the 15D health-related quality of life instrument consisting of 15 dimensions. The data were analysed using the chi-square test or Fisher's exact test, the Mann-Whitney U-test and logistic regression. ABSTRACT.RESULTS: In men, the results showed significant differences in the changes between the intervention and control groups in depression (p = 0.017) and distress (p = 0.029) and marginally significant differences in usual activities (p = 0.058) and sexual activity (p = 0.051). In women, significant differences in the changes between the groups were found in usual activities (p = 0.005) and discomfort/symptoms (p = 0.047). For the subjects aged 65 to 74 years, significant differences in the changes between the groups were seen in distress (p = 0.037) among men and in usual activities (p = 0.011) among women. All improvements were in favour of the intervention group. ABSTRACT.CONCLUSION: Fall prevention produced positive effects on some dimensions of health-related quality of life in the community-dwelling aged. Men benefited more than women. BODY.BACKGROUND: The assessment of health-related quality of life (HRQOL) has become common in clinical trials, although in fall prevention trials it is still less commonly used. In fall prevention studies HRQOL has usually been classified as a secondary outcome and only parts (for example physical or functional ability) of the quality of life (QOL) instruments have been used [1-3]. Prevention of falls in later life is a key public health priority Presenting and measuring outcomes of quality of life is relevant because physical frailty and fall-related injuries are two of the biggest threats to QOL or HRQOL, or to functioning of older people in general. Due to consequences of falls such as fractures and fear of falling the physical, psychological and social functional abilities decrease which can have a considerable impact on perceived QOL [4]. Randomized controlled trials (RCTs) involving fall prevention interventions including the assessment of quality of life among the community-dwelling aged [5-14] have shown some positive effects on quality of life only in two RCTs [5,6]. These RCTs have comprised exercise and Comprehensive Geriatric Assessment (CGA) programmes and have shown significant improvements in physical function, physical health, mental health, social function, vitality and energy/fatigue of HRQOL in the intervention groups. Interventions comprising exercise and information programmes, performed among nursing home residents and hospitalised participants, have shown improvements in role-physical, role-emotional, pain and general health of HRQOL [7,8]. A risk-based, multifactorial randomised controlled trial among the previously fallen community-dwelling aged was conducted in Pori, in western Finland. The purpose of the trial was to assess the effects of a multifactorial fall prevention programme on the incidence of falls and injurious falls, selected risk factors of falling, physical, psychological and social functional abilities, HRQOL, use of health services and mortality [15]. A fall was defined as an unexpected event where a person falls to the ground from an upper level or same level [16]. In this study HRQOL was conceptually defined as part of the overall QOL by WHO (the World Health Organisation): "An individual's perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns. A broad ranging concept affected in a complex way by the person's physical health, psychological state, personal beliefs, social relationships and their relationship to salient features of their environment" [17-19]. This study is part of the randomised controlled trial and the paper reports the effects of fall prevention programme on the dimensions of HRQOL measured with the 15D instrument in a risk group of the community-dwelling aged. BODY.METHODS: BODY.STUDY POPULATION: The participants were 65 years or older, who had fallen at least once during the previous 12 months, had moderate or good physical abilities (able to walk 10 meters independently with or without walking aids) and cognitive abilities (Mini-Mental State Examination MMSE ≥17) and were living at home or in sheltered housing (sheltered home or homelike living). The sample size, based on a power calculation, was estimated based on the results of previous fall prevention studies showing that every third fall or fall injury could be prevented [20-22]. The sample was estimated to consist of at least 458 persons, representing about 10% of the persons aged over 65 years or older who had fallen during the previous year in the town of Pori in western Finland, where this trial was conducted (total population of over 65-year-old persons was 13547 at the baseline of the intervention). The persons willing to participate and fulfilling the inclusion criteria (n = 591) were randomised into an intervention group (IG) (n = 293) and a control group (CG) (n = 298) separately in two age groups (65–74 years and ≥75 years) after the baseline assessment using consecutively numbered sealed envelopes (Figure 1). The design and the predictors of adherence to this randomised controlled trial have been described in more detail elsewhere [15,23]. Figure 1Progression of the study. BODY.HRQOL MEASUREMENT: HRQOL measurements in both groups were completed immediately before and after the 12-month intervention. The 15D health-related quality of life instrument was used as a secondary outcome measure in the trial. QOL deserves more attention in fall prevention RCTs and that is why this paper focuses only the effects of HRQOL. The 15D is a generic, comprehensive, multi-dimensional (physical, mental and social well-being), standardised and self-administered instrument and it is described in a greater detail in Sintonen 1994, 2001. It can be used to elicit both a profile and single index scores and it includes the following 15 dimensions: mobility, seeing (vision), hearing, breathing, sleeping, eating, speech (communication), elimination, usual activities, mental function, discomfort/symptoms (pain), depression, distress, vitality and sexual activity. The 15D items have five response categories [24,25]. BODY.INTERVENTION FOR FALL PREVENTION: The one-year intervention consisted of seven parts and was based on an individual risk factor analysis. The intensity of the activities was planned separately for each participant and increased progressively according to his/her health status. 1. A thorough individual geriatric assessment, guidance and treatment (recommendations concerning risk medications and visual, hearing and nutritional problems) were done by an experienced geriatrician. 2. Participants received individual guidance on fall prevention (e.g. importance of peroral calcium, hip protectors, home safety) by a trained public health nurse. 3. Physical group exercise was performed twice a month supervised by a physiotherapist. For the exercise groups the subjects were divided into three levels according to their physical functional abilities at baseline assessments (balance, muscle strength and respiratory function). Each exercise session began with warm up exercises (5–10 min), followed by exercises designed to improve lower leg muscle strength, balance and coordination (30 min) and ended up by cool-down exercises (5–10 min). Exercises could be performed in a sitting or standing position according to the subject's health and functional status. 4. Lectures on falling-related themes once a month were given by health professionals. The subjects in the intervention group were offered lectures on preventive aspects of falling, such as walking aids, nutrition in old age, home hazards and physical exercise and overall fall prevention. 5. Psychosocial group activities once a month were held by nursing students. Subjects were divided into two groups according to their psychological health, amount of depressive symptoms, feelings of loneliness and level of social activity. Those having few contacts with other people and feeling themselves lonely and whose sum score was over 10 in the Geriatric Depression Scale (GDS) (20) were advised to join a smaller "support" group. All the others were advised to join a bigger psychosocial group. Activities included discussions on different themes and actual events, group singing, quizzes, reading poems and a summer party. 6. The subjects were advised to perform physical exercises similar to those performed in groups at home three times a week. The subjects were given a brochure based on the exercise class content and encouraged to record the amount of their physical activity in the physical exercise diaries daily. 7. Home hazards assessment with a control visit approximately at six months was performed by trained nursing students. The assessment of the home environment consisted of questions about lighting, stairs, thresholds, corridors, floors, carpets, furniture and availability of handrails. Written suggestions for modifications were given to each subject and an additional home visit was performed to reinforce the modifications. The control group attended one counselling session at the baseline and had no other contacts with study personnel during the 12-month intervention [15]. BODY.ETHICS: The permission to conduct the study was issued by the chief physician of Pori Health Center and ethics approval was obtained from the Ethics Committee of Satakunta Hospital District. The participants gave written informed consent, and the study was conducted in accordance with the guidelines of the Declaration of Helsinki. BODY.DATA ANALYSES: The data were analysed on an intention-to-treat basis, to provide a realistic indication of the generalisability and effectiveness of the intervention [26]. Differences in the categorical sociodemographic variables between the IG and CG groups were analysed using the chi-square test or Fisher's exact test. The Mann-Whitney U-test was used to test the differences in the continuous variables between the groups. In the analysis of HRQOL outcome variables, the baseline differences between the groups were assessed using logistic regression. During the follow-up, the changes in HRQOL items were examined by logistic regression analyses with generalised estimation equations (GEE) to account for correlations between repeated measurements [27]. Cumulative logistic models were used for ordinal dependent items consisting of three categories and binary logistic models for dependent items having two categories. Differences in the changes between IG and CG were tested with interaction terms between the group and measurement. The results of logistic models were quantified by calculating cumulative odd ratios (COR) and odds ratios (OR) with their 95% confidence intervals (95% CI). Corresponding analyses were also performed in two age groups (65–74 years and ≥75 years). All analyses were carried out separately for men and women. P-values of less than 0.05 were considered statistically significant, and P-values between 0.05 and 0.080 marginally significant. SAS System for Windows, version 9.1 (SAS Institute Inc., Cary, NC) was used for statistical analyses. The statistical methods used were chosen because they are appropriate methods to analyse the differences between the groups. Chi-square test or Fisher's exact test are basic statistical methods for analysing the differences between the categorical variables. Due to skewed distributions continuous, variables were analysed using non-parametric Mann-Whitney U-test. HRQOL outcome variables had two or three categories and the baseline differences between the groups were assessed using binary and cumulative logistic regression. For longitudinal data of HRQOL variables logistic regression analyses with generalized estimation equations to account for correlations between repeated measurements were used. BODY.RESULTS: BODY.DATA COLLECTION: Participants with baseline and follow-up data of at least one item of the 15D were included in the analyses (n = 513). Of the 513 subjects, 251 (36 men and 215 women) belonged to IG and 262 (46 men and 216 women) belonged to CG. A total of 78 subjects dropped out (42 in IG and 36 in CG) during the 12 months follow-up. The 15D was used to elicit profiles of participants. For statistical analyses the items were combined from 5 categories into 3 categories because of the small numbers of observations in the categories 4 and 5. The categories 3 to 5 were combined except for some items. The items of depression, distress and vitality were placed into 2 categories (the categories 2 to 5 were dichotomised) among men and the items of eating and speech among women. Among men, the items of eating and speech could not be used at all. It was not reasonable to recode the categories from 5 to 2 in all profiles without loosing some information. In the subgroup analyses among younger subjects, the item on depression was not included and the item on eating was divided into 2 categories among women. BODY.DROPOUT ANALYSES: Among men, there was a difference between those who completed the study (n = 82) and those who dropped out (for reasons other than death) (n = 11) in MMSE (Median, lower quartile-upper quartile) (28.0, 26.0–29.0 and 26.0, 24.0–28.0, respectively) (p = 0.042). Cognitive functional ability was better among those completing the study compared to those who dropped out. Of those completing the study, 84% lived with a spouse or some other person and 16% lived alone, while the corresponding proportions among the dropouts were 45% and 55% (p = 0.008). Among women, there were differences between those who completed the study (n = 431) and the dropouts (n = 58) in median age (72.0, 68.0–76.0 and 77.0, 73.0–84.0) (p < 0.001), MMSE (28.0, 26.0–29.0 and 27.0, 24.0–28.0) (p = 0.006), the Geriatric Depression Scale (GDS) scores (4.0, 1.0–8.0 and 5.0, 2.0–11.0) (p = 0.031) and the ability to manage activities of daily living (ADL) (32.0, 31.0–32.0 and 30.0, 25.0–32.0) (p < 0.001). Those who completed the study were younger and had better cognitive, mental and physical abilities compared to the dropouts. BODY.BASELINE CHARACTERISTICS AND PARTICIPATION RATES: The intervention and control groups (n = 513) did not differ from each other in the baseline characteristics (Additional file 1). The majority of the participants (84%) were female, and 96% lived at home. The mean participation rate (the number of attended sessions divided by the number of sessions offered during the intervention period) of IG subjects were 65% among men and 63% among women in group exercises, 27% and 38% in lectures and 23% and 29% in psychosocial group activities, respectively. Men performed home exercises on an average of 2.5 (SD 2.2) and women 2.6 (2.0) times per week. BODY.DIFFERENCES IN CHANGES IN HRQOL: Differences in the changes between IG and CG were found in depression (p = 0.017) and distress (p = 0.029) and marginal differences in usual activities (p = 0.058) and sexual activity (p = 0.051) among men (additional file 2). In women, the analyses showed differences in the changes between the groups in usual activities (p = 0.005) and discomfort/symptoms (p = 0.047) (additional file 3.). These differences in changes were in favour of IG. The analyses were also done by age groups 65–74 or ≥75 years. There were differences in changes among men in distress (p = 0.037) and among women in usual activities (p = 0.011) in the age group of 65–74 years. These differences in the changes were also in favour of IG. BODY.DISCUSSION: This study is part of the randomised controlled trial, which is the largest risk-based, multifactorial fall prevention programme conducted in Finland. The aim of this paper was to assess the effects of fall prevention intervention on the dimensions of HRQOL measured with the 15D instrument in a risk group of the community-dwelling aged. The trial has several strengths. Randomisation was successful, and the intervention aimed to apply the best current evidence of fall prevention. The preventive methods were multifactorial, individually tailored and based on an individual risk factor assessment by a geriatrician, nurse and physiotherapist. The duration of the intervention was long and the effects of some outcomes will be followed up for five years. The outcome assessments were wide and the methods are suitable for primary care with some modification. The large sample size also enabled analysis of the effects in subgroups of participants. However, when implementing a trial in a small town, contamination of the control group by family members, friends or newspapers must be taken into account [15]. Attrition is a potential problem in studies where outcome measures are obtained over time. The sample size was estimated using power calculations. The dropout analyses showed that over a half of the men who did not complete the study lived alone and had a lower MMSE than study participants. The women who dropped out were older and had worse cognitive, psychological and functional abilities than the participants. Hence the subjects most likely to benefit from the intervention were not reached. The health and HRQOL of the participants in this study were good at baseline (most answers of the participants were in categories signifying the best HRQOL). Thus, it is difficult to produce a change during the intervention. We combined the 15D items into 3 and even 2 categories because the frequencies in the categories four and five were too small for statistical analyses. It was noteworthy that men had more favourable changes in HRQOL than women, even though the male sample was considerable smaller than that of women. Men probably benefited from the physical exercise and group activities of the intervention. This may also have increased their social contacts. An increase in social contacts and changes in the social roles of men may at least partly explain the effects of the programme on depressive symptoms [28,29] and distress among men. HRQOL instruments, different from that used in this study, namely SF-36 (The 36-Item Short-Form Health Survey), RAND-36 (36-Item Health Survey) and Euroqol (EQ-5D, European Quality of Life Instrument) were used in the previous studies, which were not multifactorial [5,14]. Thus, it is not easy to compare their results to ours. Structured quality of life questionnaires may also be geographically or culturally sensitive and inadequate in capturing all perspectives of the subjects' experiences [11]. In our study QOL is the secondary outcome measure of the trial and deserves more attention. Bacause of that the manuscript focuses on the effects of QOL. The 15D instrument was used to elicit profiles of participants and it is recommended by the Washington Panel and is valid, reliable, sensitive and responsive to change and generalisable at least in western-type societies. The 15D is particularly valid for deriving quality-adjusted life years (QALY's), which is applicable to cost effective analyses planned to be performed later. The characteristics and the validity of the instrument verified before the beginning of the study to make sure that results are reliable. Conceptually, the 15D subscribes to the definition of health by WHO, and it is available in several languages for population and clinical economic evaluation studies. It has been developed in Finland and validated among Finnish people but it has not been designed especially for the aged [24,25]. The 15D and most other HRQOL instruments used among older people have commonly measured health status, functional ability, impact of illness on people's lives or behaviour and influences on HRQOL rather than HRQOL itself [30-32]. Many valid instruments have been designed for measuring HRQOL in working-aged populations or among those suffering from a certain disease. They may not measure all dimensions of HRQOL, and it is important to assess the content of the instrument before application to older people [33-36]. However, it is likely that the participants' experiences may be best addressed by using both quantitative measures and qualitative methods, such as theme or in-depth interviews. In order to obtain results based on qualitative methods, random samples should be interviewed in clinical trials, even though additional economic resources are needed for that. The analyses were performed for men and women separately because we were interested in knowing whether the effects of the intervention differ between men and women. The results were different and that is why they are also reported separately for men and women. The findings indicated improvement on the following dimensions of HRQOL: depression, distress, usual activities and sexual activity in men and usual activities and discomfort/symptoms in women in the intervention group compared with the control group. The subgroup analyses were planned before the beginning of the study. Two age groups (65–74 and ≥75 years) showed positive changes in distress among men aged 65–74 years and in usual activities among women aged 65–74 years in the IG. The results showed improvements in the intervention groups compared with the control groups, and they are supported by several previous intervention studies [5-8]. These interventions, single-based, exercise-oriented or Comprehensive Geriatric Assessment-oriented, had positive effects on the HRQOL of the community-dwelling aged in such domains as physical functioning, physical health, vitality, energy/fatigue, mental health and social functioning [5,6]. Some other fall prevention trials have not shown significant improvements in HRQOL [9,12-14]. The results of this study cannot be generalised to the unselected aged population. The intervention was targeted to persons with at last one previous fall, and the results represent the effects of a prevention program on a risk group. The social dimension of HRQOL is currently highlighted, particularly among the aged but it is usually lacking in assessments or has been measured too narrowly [37,38]. The 15D instrument contains four items about the psychological dimension and only one item about the social dimension. This lack has been compensated for in our trial by using additional questions about the psychosocial domains of HRQOL. The results on these domains will be analysed later. Fall prevention may affect quality of life by different mechanisms. The group activities included in the programme may directly improve quality of life. Decrease in falls and fall injuries can also be a significant factor in improvement of quality of life. Furthermore maintenance of functional abilities and social activities by decreasing fall injuries may have a positive effect on quality of life. If the participants consider the prevention too demanding, requiring e.g. significant changes in health and other habits, the effects on the QOL may be negative. The effects of the intervention on falls and fall injuries have not yet been assessed. Thus, we do not know, if a reduced amount of injuries have been a factor affecting the positive results on HRQOL. We may say that our results on HRQOL show that the participants have not considered 39]. Thus increasing social contacts may have more positive effects on men than on women. BODY.CONCLUSION: The measurement of quality of life is important because fall prevention intervention may have multi-level effects on HRQOL. The fall prevention intervention implemented here produced positive effects on some dimensions of HRQOL in a risk group of the community-dwelling aged. Men benefited more than women. BODY.COMPETING INTERESTS: The author(s) declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: S-LK planned and organised the multifactorial prevention trial. SV and MS conceptualised the design of this study together with S-LK. Statistical analyses and interpretation of data were made by SV, MS and TV. Writing and critical revision of the manuscript were accomplished by SV, MS and S-LK, and TV, NS, RI and PA participated in the writing and revising. Funding was obtained by S-LK together with RI and PA. Supervision and decision making to submit for publication were the tasks of SV, MS and NS. All authors read and approved the final manuscript. BODY.SUPPLEMENTARY MATERIAL: BODY.ADDITIONAL FILE 1: Baseline characteristics in the intervention and control groups by gender. Click here for file BODY.ADDITIONAL FILE 2: Health related quality of life measured with 15D-instrument at baseline and after 12-month intervention in intervention and control groups among men. Click here for file BODY.ADDITIONAL FILE 3: Health related quality of life measured with 15D-instrument at baseline and after 12-month intervention in intervention and control groups among women. Click here for file

TITLE: No effects of functional exercise therapy on walking biomechanics in patients with knee osteoarthritis: exploratory outcome analyses from a randomised trial ABSTRACT.AIM: To assess the effects of a functional and individualised exercise programme on gait biomechanics during walking in people with knee OA. ABSTRACT.METHODS: Sixty participants were randomised to 12 weeks of facility-based functional and individualised neuromuscular exercise therapy (ET), 3 sessions per week supervised by trained physical therapists, or a no attention control group (CG). Three-dimensional gait analyses were used, from which a comprehensive list of conventional gait variables were extracted (totally 52 kinematic, kinetic and spatiotemporal variables). According to the protocol, the analyses were based on the ‘Per-Protocol’ population (defined as participants following the protocol with complete and valid gait analyses). Analysis of covariance adjusting for the level at baseline was used to determine differences between groups (95% CIs) in the changes from baseline at follow-up. ABSTRACT.RESULTS: The per-protocol population included 46 participants (24 ET/22 CG). There were no group differences in the analysed gait variables, except for a significant group difference in the second peak knee flexor moment and second peak vertical ground reaction force. ABSTRACT.CONCLUSION: While plausible we have limited confidence in the findings due to multiple statistical tests and lack of biomechanical logics. Therefore we conclude that a 12-week supervised individualised neuromuscular exercise programme has no effects on gait biomechanics. Future studies should focus on exercise programmes specifically designed to alter gait patterns, or include other measures of mobility, such as walking on stairs or inclined surfaces. ABSTRACT.TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT01545258. BODY.WHAT ARE THE NEW FINDINGS?: A 12-week functional exercise programme does not alter the walking biomechanics in patients with knee osteoarthritis. BODY.HOW MIGHT IT IMPACT ON CLINICAL PRACTICE IN THE NEAR FUTURE?: The biomechanical rationale for delivering this kind of exercise programme is weak, and should not be used to inform clinical decisions with the patients. BODY.INTRODUCTION: Osteoarthritis (OA) of the knee is a chronic disease associated with significant mobility limitations including impaired walking ability. Because walking is the most natural and convenient way of ambulation, limitations in walking ability is a major source of restrictions in an individual’s independency and participation in everyday life. While patient-reported physical functioning is included in the core set of outcome measures,1 detailed evaluation of walking performance following treatments in knee OA is seldom reported. Gait analysis provides objective and quantitative data on the walking biomechanics, reflecting the biomechanical function of the single joints, and of the gait pattern as a whole. Knee OA cannot be cured wherefore its management is an enormous challenge for clinicians and society. The ultimate treatment for knee OA is surgical joint replacement but conservative treatments are recommended before surgery.2 One recommended conservative non-pharmacological treatment for OA is exercise.3 A fundamental aim of exercise therapy is to improve mobility in relation to activities of daily living, such as walking. Comprehensive analyses of how exercise affects walking biomechanics in knee OA could inform strategies to exercise optimisation. The effects of exercise on walking biomechanics have been assessed in a number of studies.4–8 The focus has been on strengthening exercises, and the biomechanical outcomes have focused on knee adduction moment (KAM), but have not shown any effects of exercise on these outcomes. While the KAM during walking has been a specific focus in relation to knee OA, more comprehensive analyses of the effects of exercise on the walking biomechanics of the entire lower extremity has not been investigated before. One study compared strengthening exercises with individualised functional exercises in people with arthritis and lower extremity impairments and found beneficial effects of functional and individualised exercises compared with strengthening exercises on walking speed and mechanical work at the ankle, knee and hip joints.9 In contrast, a recent study compared similar functional exercises with quadriceps strengthening, and found no group differences in KAM, KAM angular impulses, knee flexor moments or walking speed, but did not report on other lower extremity gait analysis outcomes.6 Thus, it remains ambiguous if functional and individualised exercise affects lower extremity gait biomechanics. Therefore, the objective of this exploratory outcome analysis of a randomised study is to assess the effects of a functional and individualised therapeutic exercise programme on lower extremity gait biomechanics in people with knee OA. BODY.METHODS: This is the report of exploratory outcome analyses of a randomised controlled study (ClinicalTrials.gov: NCT01545258). The primary outcome is reported elsewhere.10 Participants were recruited through March–December 2012 from the OA outpatient clinic of Copenhagen University Hospital at Frederiksberg, Copenhagen, Denmark. Eligible participants were adults aged 40 years or over with a clinical diagnosis of knee OA confirmed by radiography, and a body mass index between 20 kg/m2 and 35 kg/m2. The exclusion criteria included (but were not restricted to) participation in exercise therapy within the previous 3 months, inflammatory and autoimmune diseases, and lower extremity joint replacement. Participants were randomly assigned (1:1) to one of two groups stratified by gender; an exercise therapy group (ET) receiving exercise therapy for 12 weeks, or a control group (CG) receiving no attention for 12 weeks. At inclusion, the participants’ most symptomatic knee was deemed target of all subsequent assessments and measurements. All patients gave written informed consent and the study was approved by the local research ethics committee and performed in compliance with the Helsinki Declaration. BODY.INTERVENTION: The patients assigned to the ET were offered facility-based exercise therapy supervised by a trained physiotherapist three times weekly for 12 weeks. The exercise was group-based and the participants consecutively joined the group as they were included. The exercise programme lasted approximately 1 hour and consisted of a warm-up phase (bicycle ergometer at moderate intensity) followed by a circuit training programme focusing on strength and coordination exercises of the trunk, hips and knees. The exercises were performed with free weights, elastic rubber bands or body weight as resistance. Progression was made on an individual basis, according to a prespecified progression protocol. The participants assigned to the CG received no attention during the 12 weeks. BODY.GAIT ANALYSIS: Kinematic data were acquired using a six-camera three-dimensional motion analysis system (MX-F20, Vicon, Oxford, UK) operating at 100 Hz synchronised with two force platforms (AMTI OR 6-5-1000, AMTI, USA) embedded in the laboratory floor (1500 Hz). Three-dimensional orientations of 7 body segments of interest (pelvis, thighs, shanks, feet) were obtained by tracking trajectories of markers placed according to a common commercially available kinematic model (Plug-In-Gait, Vicon, Oxford, UK). Markers were placed directly on the skin and patients wore their own comfortable shoes during all trials. Participants walked a 10 m walkway freely until a stable and comfortable walking speed was obtained. A photocell system registered the walking speed with a digital display providing the subjects with immediate visual feedback. The starting point was adjusted for each subject to ensure a clean foot strike on either of the two force platforms. Once walking speed and starting points were determined, a series consisting of 10 acceptable trials (within ±0.1 km/h of target speed) were recorded. The analyses focused on one gait cycle (one platform heel strike to the next) and gait variables were calculated using the Plug-In-Gait model (Vicon, Oxford, UK). We extracted a comprehensive list of conventional variables related to hip, knee and ankle kinematics (joint angles), kinetics (joint moments and work) and spatiotemporal variables (table 1). All trials were analysed individually and subsequently each variable was averaged across the 10 accepted trials for each participant. Table 1Baseline characteristics Variable Control group Exercise group Randomised (n=29) Per protocol (n=22) Randomised (n=31) Per protocol (n=24) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Age, years 61.3 (7.1) 61.4 (7.2) 65.9 (8.5) 64.9 (9.1) Female sex, no. (%) 21 (72%) 16 (73%) 27 (87%) 22 (92%) Height, m 1.72 (0.09) 1.71 (0.09) 1.69 (0.08) 1.69 (0.08) Weight, kg 83.3 (15.0) 83.6 (15.7) 81.9 (14.1) 83.1 (14.0) Body mass index, kg/m2 28.1 (4.5) 28.4 (4.6) 28.7 (4.2) 29.1 (4.1) Pain, 0–100 mm 63.3 (12.4) 62.9 (11.0) 56.5 (14.8) 56.8 (16.1) Gait analysis (n=28)* (n=30)* Hip kinematics (stance and swing) Max. flexion angle, degrees 40.6 (9.1) 38.7 (9.3) 39.7 (5.8) 40.2 (6.2) Max. extension angle, degrees −8.3 (9.1) −9.8 (9.0) −8.8 (7.9) −7.5 (7.8) Max. adduction angle, degrees −11.2 (5.2) −10.6 (4.4) −11.6 (3.8) −12.0 (3.7) Max. abduction angle, degrees 4.0 (4.2) 4.3 (3.9) 3.9 (5.0) 3.1 (4.8) Max. external rotation angle, degrees 16.6 (10.2) 15.6 (10.3) 13.8 (14.7) 12.6 (14.8) Max. internal rotation angle, degrees −14.9 (14.4) −15.0 (13.5) −13.5 (14.9) −12.1 (16.2) Hip kinetics (stance only; net internal moments) Peak extensor moment, Nm/kg 1.09 (0.32) 1.03 (0.29) 1.03 (0.27) 1.00 (0.25) Peak flexor moment, Nm/kg −1.00 (0.25) −0.99 (0.26) −1.08 (0.26) −1.05 (0.27) First peak abductor moment, Nm/kg 0.90 (0.15) 0.89 (0.14) 0.90 (0.20) 0.86 (0.15) Second peak adductor moment, Nm/kg 0.65 (0.14) 0.67 (0.14) 0.64 (0.14) 0.62 (0.10) First peak lateral rotation moment, Nm/kg −0.13 (0.04) −0.14 (0.04) −0.13 (0.05) 0.86 (0.15) Second peak medial rotation moment, Nm/kg 0.17 (0.05) 0.17 (0.05) 0.16 (0.05) 0.62 (0.10) First peak resultant moment, Nm/kg 1.20 (0.26) 1.15 (0.24) 1.14 (0.24) 1.09 (0.19) Second peak resultant moment, Nm/kg 1.15 (0.23) 1.15 (0.23) 1.22 (0.27) 1.18 (0.26) Positive work, Joule 0.23 (0.07) 0.22 (0.08) 0.23 (0.08) 0.21 (0.07) Negative work, Joule −0.20 (0.12) −0.19 (0.12) −0.22 (0.11) −0.20 (0.12) Knee kinematics (stance and swing) Angle at heel strike, degrees 13.5 (6.8) 12.2 (5.7) 13.9 (5.7) 13.8 (5.9) First peak flexion angle, degrees 23.7 (6.2) 23.1 (6.2) 25.7 (6.9) 25.8 (7.6) Mid-stance peak extension angle, degrees 7.0 (6.9) 6.2 (6.3) 9.6 (6.9) 10.1 (7.0) Peak swing phase flexion angle, degrees 59.6 (8.5) 59.5 (8.9) 61.4 (8.5) 61.4 (9.4) Knee kinetics (stance only; net internal moments) First peak flexor moment, Nm/kg −0.40 (0.13) −0.39 (0.10) −0.40 (0.14) −0.40 (0.13) First peak extensor moment, Nm/kg 0.82 (0.26) 0.80 (0.25) 0.88 (0.36) 0.65 (0.22) Second peak flexor moment, Nm/kg −0.11 (0.18) −0.14 (0.16) −0.05 (0.19) −0.03 (0.16) First peak abductor moment, Nm/kg 0.69 (0.24) 0.67 (0.25) 0.66 (0.22) 0.65 (0.22) Second peak abductor moment, Nm/kg 0.49 (0.21) 0.50 (0.23) 0.45 (0.18) 0.44 (0.18) Abductor angular impulse, Nm*s/kg 24.8 (10.7) 24.7 (11.4) 23.0 (9.0) 23.2 (9.4) First peak medial rotation moment, Nm/kg 0.10 (0.06) 0.10 (0.06) 0.09 (0.05) 0.09 (0.05) Second peak lateral rotation moment, Nm/kg −0.08 (0.05) −0.09 (0.05) −0.08 (0.04) −0.07 (0.04) First peak resultant moment, Nm/kg 1.07 (0.27) 1.04 (0.26) 1.12 (0.26) 1.11 (0.29) Second peak resultant moment, Nm/kg 0.58 (0.16) 0.58 (0.17) 0.57 (0.12) 0.56 (0.12) Positive work, Joule 0.24 (0.12) 0.23 (0.10) 0.27 (0.15) 0.26 (0.17) Negative work, Joule −0.28 (0.09) −0.26 (0.08) −0.29 (0.09) −0.29 (0.10) Ankle kinematics (stance and swing) Max. plantarflexion (early stance), degrees 2.1 (4.9) 1.6 (5.1) 2.2 (3.9) 2.2 (4.0) Max. dorsiflexion (late stance), degrees 18.3 (4.7) 17.7 (3.9) 20.4 (5.9) 20.9 (6.4) Swing dorsiflexion angle, degrees 7.6 (8.1) 7.1 (8.3) 9.8 (7.6) 9.4 (8.3) Ankle kinetics (stance only; net internal moments) Peak dorsiflexor moment (early stance), Nm/kg −0.24 (0.10) −0.25 (0.11) −0.20 (0.12) −0.19 (0.13) Peak plantarflexor moment (late stance), Nm/kg 1.42 (0.18) 1.41 (0.18) 1.47 (0.22) 1.45 (0.21) Peak resultant moment (late stance), Nm/kg 1.43 (0.18) 1.41 (0.18) 1.48 (0.22) 1.46 (0.21) Positive work, Joule 0.34 (0.06) 0.34 (0.06) 0.34 (0.09) 0.33 (0.08) Negative work, Joule −0.19 (0.07) −0.18 (0.06) −0.21 (0.06) −0.21 (0.07) Spatiotemporal variables Walking speed, m/s 1.37 (0.17) 1.36 (0.14) 1.37 (0.25) 1.35 (0.27) Step length, m 0.72 (0.07) 0.71 (0.05) 0.72 (0.1) 0.71 (0.11) Cadence, steps/min 116.8 (10.2) 116.4 (10.4) 116.9 (9.4) 116.5 (10.3) Double support, % 0.26 (0.05) 0.25 (0.05) 0.26 (0.09) 0.26 (0.10) Single support, % 0.39 (0.05) 0.39 (0.05) 0.39 (0.05) 0.39 (0.05) Foot progression angle, degrees 4.0 (6.1) 4.1 (4.8) 5.3 (5.0) 5.8 (5.4) Ground reaction forces and support moment (stance only) First peak vertical ground reaction force, N 113.7 (9.5) 112.4 (9.3) 116.9 (12.1) 116.1 (13.0) Second peak vertical ground reaction force, N 106.5 (7.7) 106.9 (7.6) 108.1 (9.1) 106.7 (8.2) First peak a-p ground reaction force, N 20.8 (4.0) 20.5 (3.4) 21.1 (6.1) 20.6 (6.6) Second peak a-p ground reaction force, N −20.9 (3.5) −21.0 (3.3) −20.9 (5.9) −20.5 (6.1) First peak support moment, Nm/kg 1.34 (0.40) 1.30 (0.42) 1.34 (0.39) 1.34 (0.43) Second peak support moment, Nm/kg 0.69 (0.25) 0.69 (0.28) 0.70 (0.31) 0.68 (0.32) *One invalid gait analysis at baseline (see figure flow diagram) Figure 1Flow diagram of the study participants. BODY.SAMPLE SIZE: The sample size was based on the primary outcome,10 and accounting for possible dropouts the sample was 60 participants. BODY.RANDOMISATION: For allocation of the patients, a computer-generated list of random numbers is used. Randomisation sequence was stratified by gender with a 1:1 allocation using random block sizes of 2, 4 and 6. The allocation sequence was concealed from the researchers enrolling and assessing participants in sequentially numbered, opaque and sealed envelopes. To prevent subversion of the allocation sequence, the name and date of birth of the participant were written on the envelope. Allocation occurred only after a participant completed all baseline assessments. Whereas the participating patients were aware of their group allocation, outcome assessors and data analysts were kept blinded to the allocation. BODY.STATISTICAL ANALYSES: As this study aims at exploring the mechanistic effects of exercise on gait biomechanics, the analyses were based on the ‘per protocol' population, defined as those participants that had followed the study protocol (attendance to at least 24 exercise sessions in the ET; no exercise in the CG) and with complete data sets at baseline and follow-up. The analyses focused on group mean differences in changes from baseline in gait variables, calculated as the baseline value subtracted from the follow-up value. General linear models were used with a factor for group and adjusting for the baseline value. All analyses were done using SAS software (v 9.2), and statistical significance was accepted at p <0.05. Because this study did not work under any prespecified hypotheses, no adjustments for multiple statistical tests were done. BODY.RESULTS: Of the 60 included participants, 31 were randomised to ET and 29 to CG. Baseline characteristics and gait variables are presented in table 1. The analyses involved participants who adhered to the protocol, and had complete data recordings. In the ET group, six participants were lost to follow-up and one had invalid baseline gait analysis. The remaining 24 participants all adhered to the protocol (ie, attendance to at least 24 sessions) and defined the per-protocol population in the ET group. In the CG one participant had invalid gait analysis at baseline and one at follow-up, four were lost to follow-up, and there was one protocol violation; a man in the control group disclosed participation in exercise outside the study. The remaining 22 participants defined the per-protocol population in the CG. Thus, 46 participants constituted the per protocol population. Except from a statistically significant group difference in the second peak knee flexor moment and second peak vertical ground reaction force, there were no group differences in the analysed gait variables as presented in table 2. Table 2Change from baseline in gait variables Variable Change from baseline Control group (n=22) Exercise group (n=24) Group difference Mean (95% CI) Mean (95% CI) Mean (95% CI) p Value Hip kinematics (stance and swing) Max. flexion angle, degrees −0.5 (−3.4 to 2.4) −1.7 (−4.4 to 1.1) 1.2 (−2.8 to 5.1) 0.56 Max. extension angle, degrees −1.6 (−5.0 to 1.8) −0.6 (−3.9 to 2.6) −0.9 (−5.6 to 3.8) 0.69 Max. adduction angle, degrees −0.1 (−2.1 to 1.9) 0.1 (−1.7 to 2.0) −0.2 (−3.0 to 2.5) 0.86 Max. abduction angle, degrees −0.2 (−1.7 to 1.3) −0.4 (−1.9 to 1.0) 0.2 (−1.9 to 2.3) 0.84 Max. external rotation angle, degrees −2.7 (−8.2 to 2.7) −0.1 (−5.4 to 5.1) −2.6 (−10.2 to 5.0) 0.50 Max. internal rotation angle, degrees −3.1 (−9.0 to 2.8) −0.8 (−6.4 to 4.9) −2.4 (−10.5 to 5.8) 0.56 Hip kinetics (stance only; net internal moments) Peak extensor moment, Nm/kg −0.03 (−0.16 to 0.09) −0.00 (−0.12 to 0.12) −0.03 (−0.21 to 0.14) 0.72 Peak flexor moment, Nm/kg −0.05 (−0.16 to 0.07) −0.04 (−0.15 to 0.06) −0.00 (−0.16 to 0.16) 0.99 First peak abductor moment, Nm/kg −0.00 (−0.09 to 0.09) 0.01 (−0.07 to 0.10) −0.02 (−0.14 to 0.11) 0.81 Second peak adductor moment, Nm/kg −0.05 (−0.12 to 0.02) 0.04 (−0.03 to 0.10) −0.09 (−0.18 to 0.01) 0.07 First peak lateral rotation moment, Nm/kg −0.01 (−0.03 to 0.01) −0.01 (−0.02 to 0.01) −0.00 (−0.03 to 0.02) 0.88 Second peak medial rotation moment, Nm/kg −0.01 (−0.03 to 0.01) 0.02 (−0.00 to 0.04) −0.03 (−0.05 to 0.00) 0.09 First peak resultant moment, Nm/kg −0.01 (−0.12 to 0.10) −0.02 (−0.13 to 0.08) 0.01 (−0.14 to 0.17) 0.86 Second peak resultant moment, Nm/kg 0.02 (−0.11 to 0.14) 0.05 (−0.07 to 0.16) −0.03 (−0.20 to 0.15) 0.77 Positive work (stance), Joule −0.01 (−0.05 to 0.02) 0.00 (−0.03 to 0.04) −0.02 (−0.07 to 0.03) 0.50 Negative work (stance), Joule −0.04 (−0.10 to 0.01) −0.02 (−0.07 to 0.03) −0.03 (−0.10 to 0.05) 0.45 Knee kinematics (stance and swing) Angle at heel strike, degrees 1.3 (−1.3 to 3.9) −0.2 (−2.8 to 2.3) 1.5 (2.1 to 5.2) 0.41 First flexion angle (early stance), degrees 1.7 (−0.8 to 4.2) −1.2 (−3.6 to 1.3) 2.9 (−0.7 to 6.4) 0.11 Mid-stance extension angle, degrees 1.1 (−1.6 to 3.7) −1.3 (−3.8 to 1.3) 2.3 (−1.4 to 6.1) 0.22 Swing phase flexion angle, degrees −0.6 (−3.8 to 2.7) −1.1 (−4.2 to 2.1) 0.5 (−4.0 to 5.1) 0.81 Knee kinetics (stance only; net internal moments) First peak extensor moment (early stance), Nm/kg 0.04 (−0.10 to 0.18) −0.01 (−0.15 to 0.12) 0.05 (−0.14 to 0.25) 0.59 Second peak flexor moment (late stance), Nm/kg 0.05 (−0.02 to 0.11) −0.5 (−0.11 to 0.01) 0.10 (0.01 to 0.19) 0.04 First peak abductor moment, Nm/kg 0.05 (−0.03 to 0.12) 0.06 (−0.01 to 0.13) −0.01 (−0.11 to 0.09) 0.84 Second peak abductor moment, Nm/kg 0.07 (0.01 to 0.014) 0.09 (0.02 to 0.16) −0.02 (−0.12 to 0.08) 0.72 Abductor angular impulse, Nm×s/kg 2.91 (−0.60 to 6.41) 3.09 (−0.27 to 6.45) −0.18 (−5.04 to 4.58) 0.94 First peak medial rotation moment, Nm/kg 0.004 (−0.02 to 0.03) 0.025 (0.004 to 0.05) −0.02 (−0.05 to 0.01) 0.17 Second peak lateral rotation moment, Nm/kg −0.02 (−0.04; −0.00) −0.013 (−0.03 to 0.01) −0.01 (−0.04 to 0.02) 0.48 First peak resultant moment, Nm/kg 0.05 (−0.05 to 0.15) 0.04 (-0.06 to 0.14) 0.01 (−0.14 to 0.15) 0.91 Second peak resultant moment, Nm/kg 0.04 (−0.02 to 0.10) 0.06 (0.01 to 0.11) −0.02 (−0.10 to 0.06) 0.59 Positive work (stance), Joule 0.01 (−0.05 to 0.07) 0.02 (-0.04 to 0.08) −0.01 (−0.09 to 0.08) 0.84 Negative work (stance), Joule 0.003 (−0.04 to 0.05) −0.01 (-0.05 to 0.03) 0.01 (−0.05 to 0.08) 0.69 Ankle kinematics (stance and swing) Max. plantarflexion (early stance), degrees −0.5 (−2.3 to 1.3) −1.6 (−3.3 to 0.2) 1.1 (−1.4 to 3.6) 0.38 Max. dorsiflexion (late stance), degrees −0.1 (−1.9 to 1.7) −1.3 (−3.0 to 0.5) 1.1 (−1.5 to 3.7) 0.38 Swing phase dorsiflexion angle, degrees 2.3 (−0.5 to 5.0) −0.4 (−3.0 to 2.2) 2.7 (−1.2 to 6.5) 0.17 Ankle kinetics (stance only; net internal moments) Peak dorsiflexor moment (early stance), Nm/kg 0.02 (−0.03 to 0.07) −0.00 (−0.05 to 0.05) 0.02 (−0.05 to 0.10) 0.48 Peak plantarflexor moment (late stance), Nm/kg −0.07 (−0.18 to 0.04) −0.00 (−0.11 to 0.10) −0.07 (−0.22 to 0.08) 0.37 Peak resultant moment (late stance), Nm/kg −0.07 (−0.19 to 0.04) −0.00 (-0.11 to 0.10) −0.07 (−0.23 to 0.083) 0.36 Positive work (stance), Joule −0.01 (−0.04 to 0.03) 0.00 (-0.03 to 0.04) −0.01 (−0.06 to 0.04) 0.67 Negative work (stance), Joule 0.00 (−0.03 to 0.04) −0.01 (-0.04 to 0.02) 0.02 (−0.03 to 0.06) 0.46 Spatiotemporal variables Walking speed, m/s −0.05 (−0.14 to 0.04) −0.03 (−0.12 to 0.06) −0.02 (−0.15 to 0.11) 0.79 Step length, m −0.02 (−0.07 to 0.02) −0.01 (−0.06 to 0.03) −0.01 (−0.07 to 0.05) 0.74 Cadence, steps/min −2.9 (−7.1 to 1.4) −1.8 (−5.8 to 2.3) −1.1 (−6.9 to 4.8) 0.71 Double support, % 0.01 (−0.02 to 0.05) −0.00 (−0.03 to 0.03) 0.02 (−0.03 to 0.06) 0.45 Single support, % 0.01 (−0.01 to 0.03) 0.01 (−0.00 to 0.03) −0.00 (−0.03 to 0.02) 0.74 Foot progression angle, degrees 1.0 (−1.4 to 3.4) −1.4 (−3.7 to 0.9) 2.4 (−0.9 to 5.7) 0.15 Ground reaction forces and support moment (stance only) First peak vertical ground reaction force, N −1.5 (−6.6 to 3.7) 1.0 (−4.0 to 5.9) −2.4 (−9.7 to 4.7) 0.50 Second peak vertical ground reaction force, N −2.7 (−5.9 to 0.6) 2.3 (−0.9 to 5.4) −4.9 (−9.5:−0.4) 0.03 First peak a-p ground reaction force, N −0.9 (−3.4 to 1.5) −0.0 (−2.4 to 2.3) −0.9 (−4.3 to 2.4) 0.59 Second peak a-p ground reaction force, N 1.2 (−1.0 to 3.5) 0.8 (−1.4 to 2.9) 0.4 (−2.6 to 3.5) 0.77 First peak support moment, Nm/kg −0.03 (−0.25 to 0.20) 0.04 (−0.18 to 0.25) −0.06 (−0.4 to 0.2) 0.68 Second peak support moment, Nm/kg 0.02 (−0.13 to 0.17) 0.09 (−0.05 to 0.23) −0.07 (−0.28 to 0.13) 0.49 BODY.DISCUSSION: This study presents a comprehensive analysis of the effects of functional exercises on gait biomechanics in knee OA. Overall, there were no group differences in the gait changes, except for two variables (second peak knee flexor moment and second peak vertical ground reaction force). Our confidence in these two statistically significant observations is limited, as the likelihood of these occurring by chance is not negligible due to multiple statistical tests. Also, the group difference in the change of the second peak vertical ground reaction force is very small and probably without any clinical significance. Further, while these findings may be plausible we would expect changes in other variables, such as the ankle plantarflexor moment, in the late stance phase to happen concurrently to be biomechanically meaningful. While several previous studies have assessed the effects of exercise on gait, the studies have all focused on selected variables and on quadriceps strengthening.4–8 No studies have found effects of exercise and thus corroborate the present results. One study suggested beneficial effects of the functional exercises compared with strengthening exercises on ankle, knee and hip power,9 whereas a recent comparison of functional and strengthening exercises did not show any differences in knee joint moments.6 While we assessed both positive and negative work in ankle, knee and hip joints, we did not assess power in the same way as by McGibbon et al.9 However, our comprehensive analyses report a range of widely used gait variables, and the results do not suggest any group differences to support changes in joint powers as reported by McGibbon et al.9 Bennell et al 6 compared gait changes between functional and strengthening exercises, applying a rigorous clinical trial design and intention-to-treat analyses, yet without group differences in selected gait variables. In our study we compared functional exercises with a no attention control group, included a comprehensive evaluation of gait variables and constrained our analyses to the participants that adhered to the protocol. Thus, the chances of detecting any effects of functional exercises on gait biomechanics were maximised, yet no differences were found. Although the functional exercise programme that we employed includes specific exercises aiming at improving walking performance, the majority of the exercises are not specifically aimed at walking biomechanics. This may explain the absence of effects in the gait biomechanics in this and previous studies.4–8 Another explanation could be that the participants in our study did not have significant gait aberrations at baseline. Thus, it is therefore not surprising that the functional exercise programme had no effects on the gait as this was not anomalous to begin with. It is possible that the exercise programme may have affected the biomechanics of other locomotor tasks, such as stair ambulation or walking on inclined or declined surfaces. Conventional gait analysis may not necessarily reflect the mobility limitations experienced by the patients with knee OA . While walking is a basic activity of daily living, the inclusion of other measures of everyday mobility could have shown beneficial effects of exercise and would have enhanced our study. Further, our small sample size is a limitation to the study although there are no indications in our results or among previous studies, that a larger sample size would return different results. In conclusion, a functional and individualised therapeutic exercise programme had no effects on lower extremity gait biomechanics in people with knee OA. If gait biomechanics are to be changed, future studies should focus on exercise programmes specifically designed to alter gait patterns, or include other measures of mobility, such as stair negotiation or on inclined surfaces.

TITLE: The effect of lidocaine on reducing the tracheal mucosal damage following tracheal intubation ABSTRACT.BACKGROUND:: The aim of this study was to investigate the efficacy of lidocaine solution in the cuff of the endotracheal tube in reducing mucosal damage following tracheal intubation. ABSTRACT.MATERIALS AND METHODS:: This was a randomized controlled trial study undertaken in the intensive care unit patients. Participants, who met all eligibility criteria, were randomly assigned to one of two groups of patients, according to whether lidocaine or air was used to fill the tracheal tube cuff. The tracheal mucosa at the site of cuff inflation was inspected by fiberoptic bronchoscopy and scored at the 24 h and 48 h after intubation. ABSTRACT.RESULTS:: In all, 51 patients (26 patients in the lidocaine group and 25 patients in the control group) completed the study. After 24 h, erythema and/or edema of tracheal mucosa were seen in 2 patients (7.7%) of lidocaine group and 6 patients (24%) of air group (P = 0.109). Binary logistic regression analysis showed that lidocaine has a significant protective effect against mucosal damage (odds ratio = 0.72, confidence interval = 0.60-0.87). ABSTRACT.CONCLUSION:: The inflation of the tracheal tube cuff with lidocaine was superior to air in decreasing the incidence of mucosal damage in the 24 h and 48 h post intubation. BODY.INTRODUCTION: Tracheal intubation is one of the most frequently performed procedures in the intensive care unit (ICU) and has an essential role in airway management in critically ill patients.[123] Hemodynamic instability, hypoxemia and acidosis in ICU patients indicate the need for maintaining a secure airway for these patients.[4] Poor physical conditions, emergent intubation, and medication in ICU bring the high risk of intubation complications as high as 54%.[5] On the other hand, high incidence of tracheal mucosal lesions is attributed to high cuff pressure compression.[6] The cuff is designed to provide an airway seal and reduces aspiration of orotracheal secretions.[7] Hyperinflation of the endotracheal cuff to pressures more than 25 cm H2O or greater is contributed to mucosal ischemia and subsequent destruction of the tracheal wall.[8] Mucosal ischemia is associated with inflammation, hemorrhage, and necrosis.[9] These circumferential lesions heal by fibrosis, leading to a progressive tracheal stenosis, which occurs in 1-4% of the patients after treatment at ICUs.[10] Several interventions have been used to reduce the cuff pressure and decrease the incidence of associated adverse consequences. Providing the stable cuff pressure by filling the cuffs with anesthetic gas mixture[11] and tight control of cuff pressure was effective in minimizing the incidence and severity of tracheal mucosal damage.[12] In another study, inflating cuffs with either air or saline was examined in two groups. In saline group, cuff pressure remained stable during anesthesia and tracheal lesions were significantly lower at the time of extubation.[13] The role of lidocaine in reducing the frequency of postoperative cough and sore throat was demonstrated in several studies.[141516] Lidocaine is also thought to be useful in reducing injury to the tracheal mucosa with vasodilation mechanism.[17] Until now, there has not been any study regarding the effect lidocaine on mucosal damage considering bronchoscopy evaluation. Therefore, the aim of this study was to investigate the efficacy of lidocaine solution in the cuff of the endotracheal tube in reducing mucosal damage following tracheal intubation and compare it with the air. BODY.MATERIALS AND METHODS: This was a double-blinded randomized controlled trial study that was undertaken in the ICU of University Hospital, Isfahan, Iran. The target population was hospitalized patients in the ICU, requiring tracheal intubation and mechanically ventilation for acute respiratory failure. Eligibility criteria included age more than 18 years and less than 70 years, no history of hematologic, cardiovascular and pulmonary diseases, undergoing surgery rather than thoracic surgery, stable hemodynamic condition, no prior intubation in the last year, not pregnant, and not suspected as difficult intubation and the absence of any contraindication for performing bronchoscopy. Patients who were extubated earlier than 48 h were excluded. Other exclusion criteria were: Cuff pressure more than 25 mmHg, unstable hemodynamic condition during the study, persistent hypoxemia, coagulation abnormalities or bleeding during the study and death. For a statistically significant level of 5%, a statistical power of 80%, (P1 = 0.4, P2 = 0.1) a sample size of 25 patients per group was required. Between March 2012 and October 2012, participants, who met all eligibility criteria, were randomly assigned to one of two groups, according to whether lidocaine or air was used to fill the tracheal tube cuff. The randomization was performed by a ratio of 1:1, using Excel software random number generation. All patients were intubated with a standard 8.0 mm polyvinyl chloride oral cuffed Endo tracheal Tube [ETT] for men and 7.5 mm for women. The same anesthetist performed all tracheal intubations. In the control group, after orotracheal intubation, the tracheal tube cuffs were inflated with air, up to a pressure of 25 mmHg. Patients in the intervention group underwent intubation using a tracheal tube, which filled with 2% lidocaine (Caspian tamin, Pharmaceutical company, Rasht, Iran) associated to a volume that was sufficient to cause a cuff pressure of 25 mmHg the tracheal tube cuff pressures were measured using a control-inflator device after intubation, and then after 24 h and 48 h. Data relating to oxygen saturation, systolic, and diastolic blood pressure, were obtained immediately after orotracheal intubation, and then 6, 12, 18, 24, 30, 36, 42, and 48 h after the intubation [Table 1]. Table 1Demographic and clinical characteristics of patients in two groups during the study All patients underwent fiberoptic bronchoscopy at 24 and 48 h after the study. Bronchoscopy examination was performed by an ICU specialist who was blind to the study group. The fiberscope was fed in the tracheal tube as the endoscope went below the end of the endotracheal tube, the cuff was deflated. The endotracheal tube and the endoscope were gently withdrawn concomitantly; the tracheal mucosa at the site of cuff inflation was inspected and scored according to the following scoring: 0 = normal, 1 = erythema and/or edema, 2 = erosion and/or hemorrhage on one side of tracheal wall, 3 = erosion and/or hemorrhage on both anterior and posterior tracheal walls.[18] Demographic and clinical data of participants (age, gender, etiology of disease, medical history) were collected by a questionnaire. BODY.STATISTICAL ANALYSIS: All descriptive statistics are presented as means and standard deviations (SD) for quantitative variables and as relative frequencies and percentages for categorical variables. Student t-test and Chi-square test were conducted to compare the baseline characteristics of two groups. Repeated measure analysis of variance was used to examine any difference in the study outcomes (systolic and diastolic blood pressure, oxygen saturation and cuff pressure) between the intervention and the control groups over the evaluation period. The severity of tracheal mucosal damage was compared between two groups using Chi square analysis of variance. Binary logistic regression analysis was applied to assess the predictors of mucosal damage. Considering the presences of mucosal lesions after 48 h as the dependent variable, the independent (predictor) variables were entered in the model, including the age of participants, gender and etiology of hospitalization in ICU. The level of significance was set at P < 0.05 and all tests were two-tailed. The analysis of data was performed by the predictive analytic software (SPSS version 18) for Windows. BODY.ETHICS: The design of the study was approved in Ethics committee of Vice Chancellor for Research, Isfahan University of Medical Sciences (project no. 391341). Legally authorized representatives were provided written informed consent and the confidentiality of all information was managed carefully by researchers. BODY.RESULTS: Seventy-five patients acquired the inclusion criteria and were enrolled in the study. Seven patients were excluded due to extubation earlier than 48 h. Other reasons of exclusion include: Hemoglobin concentration less than 10 (n = 10), cardiac arrest (n = 4) and cuff pressure more than 25 mmHg (n = 3). In all, 51 patients (26 patients in the lidocaine group and 25 patients in the control [air] group) actually completed the study and were evaluated for the outcome [Figure 1]. Figure 1CONSORT trial flow diagram The mean age of patients in the intervention group and control group was 49.0 years (SD = 13.5) and 45.1 (SD = 13.8), respectively (P = 0.31). Of whom, 14 patients were female (27.5%) and 37 patients were men (72.5%) [Table 1]. Baseline clinical and demographic characteristics were balanced across treatment arms, indicating the success of randomization. There were no differences in demographic data between the patients who attended and those who dropped out (P > 0.05). The most common causes of ICU hospitalization were: Intracranial hemorrhage (12%) and multiple trauma (10%). There was no statistically significant difference between two groups regarding the etiology of ICU hospitalization (P = 0.15). The mean arterial blood pressure was not statistically significant different between two groups (P = 0.72). The cuff pressure remained stable in two groups (25 mmHg) and there were no gas leaks at the tracheal tube during the intubation. Consequently, oxygen saturation was not statistically significant different between lidocaine and air groups during the 48 h (P = 0.134). After 24 h, erythema and/or edema of tracheal mucosa were seen in 2 patients (7.7%) in lidocaine group and 6 patients (24%) of air group. Severe lesions were not seen in both groups. There was no statistically significant difference between two groups regarding the bronchoscopy findings after 24 h (P = 0.109). Bronchoscopy findings after 48 h indicated erythema and edema of tracheal mucosa in the same patients (2 patients (7.7%) in lidocaine group and 6 patients (24%) of air group). In one of the patients of air group, erosion and hemorrhage on the anterior wall of trachea was present (P = 0.147). No exposed cartilage in the cuff contact area was seen in any patient [Table 2]. Table 2Frequency of tracheal mucosal damage after 24 h and 48 h Binary logistic regression analysis showed that lidocaine has a significant protective effect against mucosal damage (odds ratio [OR] = 0.72, confidence interval [CI] = 0.60-0.87). Age of participants (OR = 1.02, CI = 0.95-1.08), gender (OR = 0.34, CI = 0.02-7.36) and etiology of hospitalization (OR = 1.06, CI = 0.92-1.22) were not significant predictors of tracheal mucosal damage. BODY.DISCUSSION: The aim of this study was to study the efficacy of intra-cuff lidocaine solution in reducing postoperative mucosal damage of trachea. The tracheal mucosal damage was more frequent in the control group in comparison to the lidocaine group (24% vs. 7.7%). Although this difference was not statistically significant, logistic regression analysis showed that being in the lidocaine group has a significant protective effect against mucosal damage. However, in our study, most patients did not experience tracheal damage, which may be due to tight control of cuff pressure during the study. Sajedi and Maaroffi confirmed that tight control of cuff pressure can minimize the incidence and severity of tracheal mucosal damage at the site of cuff inflation.[12] The tracheal mucosa is extremely fragile. Previous studies showed that even a mild pulling of a cotton swab along it causes loss of epithelium.[19] Tracheal intubation and respiratory movements may cause mucosal damage and abrasion of tracheal epithelium following intubation especially in patients with poor general status.[20] Esteller-Moré et al. examined the incidence of laryngotracheal injuries following intubation and/or tracheotomy in ICU. Endoscopic exploration of the upper airway 6-12 months after extubation revealed laryngotracheal injuries in 11% of the patients.[21] In the ICU care, the cuff pressure could not be decreased due to the risk of aspiration. Hence, the only way to reduce the tracheal mucosal damage is shortening the period in which patient is intubated. In our study, we examined another idea to reduce the mucosal damage by vasodilatory effect of lidocaine. Our study showed that lidocaine is effective in reducing the incidence of intubation-induced mucosal damage. Lidocaine is the most widely used drug for local anesthesia.[22] This drug can diffuse across the cuff in a time- and concentration-dependent manner and influence the local tracheal receptors to induce local anesthesia and ultimately reduce the tube discomfort.[23] In addition to having analgesic effects, it is also may cause vasodilation by blocking sodium channels at clinically relevant doses.[24] This vasodilatory effect of lidocaine is also mediated by the release of nitric oxide from vascular endothelium.[25] The protective effect of lidocaine against mucosal damage in our study may be due to the vasodilation mechanism of this drug. The vasodilator effects of lidocaine are believed to be due mainly to the sodium channel blocking in vasoconstrictor sympathetic nerves inhibiting the production and propagation of action potentials.[26] Furthermore, peripheral microcirculation is through the release of nitric oxide, which nitric oxide release also contributes to the vasoactivity of this drug.[25] Intra cuff lidocaine has other beneficial effects. Several randomized controlled trial studies showed that filling the cuff with lidocaine could prevent postoperative sore throat resulting from intubation.[15272829] Furthermore, the effectiveness of lidocaine in reducing cough and incidence of cardiac arrhythmia was reported.[303132] Lidocaine can diminish hemodynamic response after tracheal intubation by several mechanisms: Inhibiting sodium influx in the neuronal cell membrane and inhibiting signal conduction,[33] decreasing the sensitivity to the heart muscle to electrical pulses,[34] direct cardiac depression and peripheral vasodilatation properties.[35] The toxicity of local anesthetic must be considered. In the event of a cuff rupture, a relatively high dose of lidocaine can be delivered into the trachea and bronchium leading to toxicity. However, lidocaine induced cuff rupture has never been reported either in vivo or in vitro.[36] The major strength of this study was the use of a randomized controlled design, which enhanced its generalizability. Our study was limited because of the small sample size that prevents more elaborate subgroup analysis. Furthermore, this study was not powered enough to detect a significant difference between two groups regarding the bronchoscopy findings. Performing bronchoscopy after 48 h and 1 week later may reveal more frequent tracheal mucosal damage. Further large clinical trial studies are also needed to evaluate the effectiveness of lidocaine in reducing the mucosal damage after intubation. In conclusion, the present study demonstrated that the inflation of the tracheal tube cuff with lidocaine was superior to air in decreasing the incidence of mucosal damage in the 24 h and 48 h post intubation. Further large clinical trial studies are warranted to evaluate the effectiveness of lidocaine in reducing the mucosal damage after intubation.

TITLE: Effects of a New Metabolic Conditioning Supplement on Perioperative Metabolic Stress and Clinical Outcomes: A Randomized, Placebo-Controlled Trial ABSTRACT.BACKGROUND:: Insulin resistance is a measure of metabolic stress in the perioperative period. Before now, no clinical trial has determined the summative effects of glutamine, L-carnitine, and antioxidants as metabolic conditioning supplements in the perioperative period. ABSTRACT.OBJECTIVES:: The purpose of this study was to determine the effects of a new conditioning supplement on perioperative metabolic stress and clinical outcomes in non-diabetic patients. ABSTRACT.PATIENTS AND METHODS:: In this randomized controlled trial, 89 non-diabetic patients scheduled for coronary artery bypass grafting, with ejection fractions above 30%, were selected. Using the balanced block randomization method, the patients were allocated to one of four study arms: 1) SP (supplement/placebo): supplement seven days before and placebo 30 days after the surgery; 2) PS: placebo before and supplement after the surgery; 3) SS: supplement before and after the surgery; and 4) PP: placebo before and after the surgery. The supplement was composed of glutamine, L-carnitine, vitamin C, vitamin E, and selenium, which was manufactured for the first time by this research team. Five blood samples were drawn: seven days preoperatively, at the entrance to the operating room, while leaving the operating room, seven days postoperatively, and 30 days postoperatively. Levels of glucose, insulin, and HbA1c were measured in blood samples. Insulin resistance and sensitivity were calculated using a formula. Surgical complications were assessed 30 days postoperatively. Data analysis was done using one-way ANOVA, the Chi-square test, and a general linear model repeated-measures analysis with Bonferroni adjustment. ABSTRACT.RESULTS:: Blood glucose levels were increased postoperatively in the four groups (< 0.001), but a significantly higher increase occurred in the PP group compared to the SP (0.027), PS (0.026), and SS (0.004) groups. The superficial wound infection rate was significantly different between the four groups (0.021): 26.08% in PP, 9.09% in SP, 4.54% in PS, and 0% in SS. ABSTRACT.CONCLUSIONS:: Our new metabolic conditioning supplement, whether given pre- or postoperatively, led to better perioperative glycemic control and decreased postsurgical wound infections in non-diabetic patients. BODY.1. BACKGROUND: Insulin resistance is a measure of metabolic stress in patients undergoing surgery (1). The magnitude of insulin resistance is associated with the severity and degree of the surgery. Major operations cause severe insulin resistance, while minor surgeries are correlated with mild resistance (1, 2). Insulin resistance increases 7- to 8-fold in adult patients undergoing elective major surgery (1), and it usually extends to 3 weeks after the surgery (3, 4). The surgical technique is also important in this regard. Compared to laparoscopic techniques, open surgery techniques cause higher degrees of insulin resistance (1). In response to any trauma, such as surgery, several neuroendocrine changes occur, including increased plasma concentrations of cortisol, glucagon, and catecholamines, as well as increased levels of cytokines and immunologic responses (1, 2). These changes lead to a decreased insulin-to-glucagon ratio, and thus insulin resistance occurs (5, 6). Other factors also affect postoperative insulin resistance, such as postoperative oxidative stress, impaired function of skeletal muscle mitochondria, complete bed rest, and pain (1, 3, 7). As a stress marker, postoperative insulin resistance is closely associated with clinical outcomes (8). In a study by Sato et al., insulin sensitivity that is decreased by 50% in patients undergoing abdominal surgery was related to 5 - 6 times as many major complications and 10 times as many severe infections. In other words, with a 1 mg/kg/minute decrease in insulin sensitivity, the incidence rates of major complications (OR: 2.23), severe infections (OR: 4.98), and minor infections (OR: 1.99) were increased (2). Perioperative insulin resistance is accompanied by hyperglycemia (5). Acute hyperglycemia leads to increased inflammation, vulnerability to infection, and multiorgan system dysfunction via decreased nitric oxide production in the endothelium, decreased vasodilation, decreased complement function, increased cytokine levels, increased expression of adhesion molecules in leukocytes and endothelium, and impaired neutrophil chemotaxis and phagocytosis (6, 9). Based on Van den Berghe et al.’s study, even a moderate (6 - 8 mM) increase in perioperative glucose concentration leads to increased mortality and morbidity (10). Different approaches have been proposed to control perioperative insulin resistance, such as preoperative carbohydrate loading and early postoperative enteral feeding (3, 11). Studies have also revealed that metabolic conditioning using arginine, glutamine, and antioxidants helps to control insulin resistance in the perioperative period (3). Glutamine can control insulin resistance by metabolic regulation pre- and postoperatively via decreased muscle cell injury, increased antioxidant capacity, and increased peripheral glucose utilization (3, 12). The results of a meta-analysis of 14 clinical trials showed that postoperative intravenous glutamine leads to decreased length of hospital stays and fewer complications in surgical patients (3, 13). It is also reported that using antioxidants, such as vitamin C and E, as preconditioning agents reduces free-radical-induced complications from surgery (14, 15). A newly recognized preoperative metabolic regulatory factor, which is also considered an adjunctive therapy for type 2 diabetes mellitus, is L-carnitine (3, 16-18). L-carnitine can control insulin resistance via increased fatty acid oxidation (19). It also facilitates glucose utilization via increased pyruvate dehydrogenase activity (19), and it can control postoperative oxidative stress (20). Before now, no clinical trial has evaluated the summative effects of carnitine, glutamine, and antioxidants. In a trial by Awad et al. (21), the effects of an oral nutritional supplement containing carbohydrate, glutamine, and antioxidants were assessed in 20 patients undergoing laparoscopic cholecystectomy. Patients received either 600 mL of the supplement or a placebo the evening before surgery and an additional 300 mL 3 - 4 hours before anesthesia. A 300-mL aliquot of ONS contained 50 g of carbohydrate, 15 g of glutamine, and antioxidants. Intraoperative liver glycogen reserves, plasma glutamine, and antioxidant concentrations increased after supplement ingestion. Muscle pyruvate dehydrogenase kinase (PDK4), forkhead transcription factor 1 (FOXO1), and metallothionein 1A (a marker of cellular oxidative stress) expressions were lower in the supplement group. It was proposed that preoperative ingestion of this oral nutritional supplement can reduce insulin resistance through lowering muscle PDK4 mRNA and protein expression (21). BODY.2. OBJECTIVES: The role of insulin resistance is established in postoperative glycemic control and postsurgical morbidity and mortality, while metabolic conditioning agents such as glutamine, L-carnitine, and antioxidants play roles in the postoperative phase. In consideration of this, the present study aimed to determine the effects of a new metabolic conditioning supplement composed of glutamine, L-carnitine, vitamin C, vitamin E, and selenium, taken pre- and postoperatively, on perioperative metabolic stress and clinical surgical outcomes in non-diabetic patients. BODY.3. PATIENTS AND METHODS: BODY.3.1. STUDY PARTICIPANTS: The participants in the present study were selected from male and female patients scheduled for on-pump coronary artery bypass grafting (CABG) surgery at three government hospitals: Namazi, Faghihi, and Al-Zahra. These are referral hospitals for Shiraz University of Medical Sciences. The inclusion criteria were age of 30 - 70 years, ejection fraction above 30%, serum creatinine level less than 1.5 mg/dL, undergoing on-pump CABG in one of the above-named hospitals, not taking antioxidant supplements in the previous month, and being included in the surgery schedule at least eight days before the operation. Patients scheduled for off-pump CABG, valve repair, or emergency operations, as well as those with diabetes mellitus, infectious disease, metabolic disease, humoral disease, immunological disease, or stroke, were excluded from the study. The sample size was determined based on a trial on the effect of L-carnitine on HOMA-IR, an insulin resistance index (19). Considering α = 0.05, 1-β = 0.90, difference = 0.8, and SD = 0.8, the sample size was calculated to be 22 patients in each arm (88 patients in the four groups). After eligible patients were identified, they were given oral and written explanations of the study, including its goal, benefits, and procedure, and were asked to read and sign an informed consent document. The study protocol was reviewed and approved by the Human Ethics Committee of the Research Council of the Deputy of Research Affairs of Shiraz University of Medical Sciences. From June 2013 to March 2014, the patients who met all of the inclusion criteria and none of the exclusion criteria were enrolled consecutively, except for those who refused to enter the trial. BODY.3.2. SUPPLEMENT: The supplement used in the present study was composed of glutamine (15 g), L-carnitine (3 g), vitamin C (750 mg), vitamin E (250 mg), and selenium (150 μg). This supplement was manufactured for the first time by the research team, under the supervision of a pharmaceutics specialist. The powders were mixed with a cubic mixer, and a homogenous powder was packed into sachet forms for daily use. The patients’ instructions were to suspend the contents of one sachet in a glass of cold water and drink it after a meal. The placebo sachets contained 5 g of starch powder. The same flavoring agent was used in the supplement and the placebo, and both types of sachet (drug and placebo) were tested blindly in volunteers to confirm that they were not distinguishable. There was no distinguishable difference in the packaging of the supplement and placebo. BODY.3.3. INTERVENTION DESIGN: A randomized, placebo-controlled trial was carried out after the eligible patients were recruited between June 2013 and March 2014. We used balanced block randomization for random allocation of the patients into four groups. With a block size of four per group (A, B, C, D), there were 24 possible combinations. Each number in the random-number sequence in turn selected the next block, determining the next four participant allocations. Group SP received the supplement (one sachet daily) starting seven days before the surgery and the placebo (one sachet daily) for 30 days after the surgery. Group PS received the placebo for seven days before the surgery and the supplement for 30 days post-surgery. Group SS received the supplement for seven days before and 30 days after the surgery. Group PP received the placebo for seven days before and 30 days after the surgery. A total of five blood samples were drawn from each patient. The first 7-mL venous sample was taken seven days before the operation, after eight hours of fasting. Then, depending on the patient’s allocation, seven sachets of supplement or placebo were provided, with instructions to take one sachet daily. The supplement or placebo was taken from the day of the first blood sample until the day before the operation. On the day of the operation, the second blood sample was drawn upon arrival in the operating room. The third blood sample was drawn at the end of the operation, as the patient was leaving the operating room. The supplement or placebo was restarted after extubation on the second day postoperatively. At discharge, additional supplement/placebo sachets were given to the patients, and the fourth blood sample was drawn seven days after the operation. The fifth sample was collected at 30 days. Complications from the surgery were assessed 30 days postoperatively, including mortality rate, myocardial infarction, requirement for intra-aortic balloon pump (IABP), cerebrovascular accident, dialysis, serious or minor infections, and blood-product transfusions. The demographic data were collected with interviews. Anthropometric assessments included measurement of weight and height. Body weight was measured to the nearest 0.1 kg using the Seca 713 scale, with the subjects minimally clothed. Height was determined using non-stretchable measuring tape, without shoes, and body mass index was then calculated by dividing weight (kg) by squared height (m2). All of the equipment was calibrated each morning. Based on the Centers for Disease Control and Prevention (CDC) guidelines, a superficial wound infection was defined in this study as an infection limited to the skin and subcutaneous tissue with a clinical presentation of erythema, drainage, low-grade fever, and sternal instability, while a deep sternal wound infection (DSWI) reached the sternum but did not involve it. The diagnosis of DSWI required one of the following criteria: (1) detection of an organism in a culture of mediastinal tissue or fluid; (2) mediastinitis seen during the operation; or (3) either chest pain, sternal instability, or fever of > 38°C, and either purulent drainage from the mediastinum, isolation of an organism present in a blood culture, or culture of the mediastinal area (22, 23). Based on the CDC criteria for the diagnosis of nosocomial pneumonia, clinical factors (such as fever and leukocytosis), radiological criteria (including persistent new findings on chest radiograph), and microbiologic evidence were all taken into account (24). The clinical definition of urinary tract infection (UTI) was based on the presence of a minimum of one of the following characteristics: specific and nonspecific micturition-related symptoms and signs, a positive test (nitrite test, leukocyte esterase test, dipslide, or culture), antibiotic treatment for UTI, or UTI reported in the medical record (25). Specific symptoms and signs were pain before, during, or after micturition; increased frequency of micturition; abdominal pain; hematuria; foul smell; and common signs of sickness (fever > 37.9°C or 1.5°C above baseline temperature, chills, nausea, and vomiting) (25). The diagnoses of the above-mentioned outcomes were confirmed by the surgeon. We also recorded the duration of the operation; the graft number; the cardiopulmonary bypass (CPB) time; use of exogenous insulin; administration of epinephrine, dexamethasone, and hydrocortisone during the operation and in the ICU; total blood loss; duration of intubation; and length of ICU and hospital stays. BODY.3.4. BIOCHEMICAL ASSESSMENT: Blood was collected for measurement of glucose, insulin, and HbA1c. After transferring adequate amounts of the blood to EDTA-containing tubes for HbA1c measurement (for all samples except the third), blood samples were collected for serum separation. All blood samples were first centrifuged immediately after blood collection at 40 rpm for 10 - 30 minutes, and serum aliquots were stored at -70°C. To avoid day-to-day laboratory variables, all of the blood samples were analyzed in a single batch at the end of the clinical phase of the study. Serum glucose levels were measured spectrophotometrically on the BT 1500 autoanalyzer. Insulin was measured using ELISA (DRG kit), and HbA1c was measured by HPLC (Agilent, affinity technique). Before the start of measurement-taking, the instruments were calibrated using a reference standard with a known value to cover the range of interest. The measurement of that standard was performed with the instrument, and the result was compared with the known value. Insulin resistance and sensitivity were calculated as follows (26): QUICKIindex(quantitativeinsulinsensitivitycheckindex)=1Log(insulin (μU/mL))+log(glucose (mg/dL))HOMA-IR(homeostasismodelassessment-insulinresistance)=(fastinginsulin [μU/mL] ×fastingglucose [mmol/L])22.5FIRI(fastinginsulinresistanceindex)=insulin(μU/mL) ×glucose(mmol/L)25Bennettsindex=1log[insulin(μU/mL)] ×log[glucose(mmol/L)] BODY.3.5. DIETARY INTAKE ASSESSMENT: The patients’ dietary intakes were evaluated upon enrollment and at the end of the study using 24-hour recall questionnaires. The food-processor software Nutritionist-4, modified by incorporating the Iranian food table, was used to calculate macro- and micronutrient consumptions. BODY.3.6. STATISTICAL ANALYSIS: Data processing and analysis were done using SPSS version 17 for windows (SPSS Inc., Chicago, IL, USA). Normal distribution of the data was checked using the Kolmogorov-Smirnov test. In normally distributed data, one-way ANOVA was used to compare the variables between the four groups. Chi-square and Fisher Exact tests were used for categorical variables. General linear model repeated-measures analysis with Bonferroni adjustment was used to compare changes in time within and between the four groups. Skewed data were compared using the Kruskal-Wallis test between the four groups. The significance level was set at P < 0.05. This trial is registered with Clinicaltrial.gov (number NCT02184507), where the trial protocol can be accessed. BODY.4. RESULTS: In this clinical trial, as shown in Figure 1, 105 patients were randomized, and then 16 were excluded, so that a total of 89 patients remained in the trial for analysis. The reasons for the exclusions are demonstrated in the flow diagram. There were no significant differences between the four groups at baseline (Table 1). Also, there were no significant differences between the four groups in the number of grafts, duration of surgery, administration of blood-glucose-increasing or -decreasing hormones during the operation and in the ICU, or blood loss (Table 2). Figure 1.BODY.FLOW DIAGRAM OF THE TRIAL: Table 1.BODY. COMPARISON OF DEMOGRAPHIC VARIABLES AND BASELINE CHARACTERISTICS IN THE FOUR GROUPS:a GroupSPPSSSPPP Value Number 22 22 22 23 Age, y 56.90 ± 7.5 58.72 ± 8.5 58.59 ± 6.4 55.21 ± 8.3 0.391 Gender 0.397 Male 12 17 16 16 Female 10 5 6 6 Body mass index, kg/m 2 26.19 ± 4.56 24.37 ± 2.55 25.69 ± 3.47 25.82 ± 3.77 0.382 Ejection fraction, % 51.47 ± 9.90 52.00 ± 7.40 49.95 ± 9.38 49.91 ± 10.05 0.830 Comorbidities Hypertension 11 (50.0) 10 (45.4) 10 (45.4) 16 (69.5) 0.340 Hyperlipidemia 9 (40.9) 9 (40.9) 8 (36.3) 11 (47.8) 0.891 Smoking history Previous smoker 1 (4.5) 5 (22.7) 4 (18.1) 3 (13.0) 0.305 Current smoker 12 (54.5) 8 (36.36) 11 (50.0) 11 (47.8) 0.744 Creatinine, mg/dL 1.02 ± 0.16 1.04 ± 0.21 1.00 ± 0.26 1.04 ± 0.20 0.902 aData are expressed as mean ± SD or No. (%). Table 2.BODY. COMPARISON OF SURGICAL PARAMETERS IN THE FOUR GROUPS:a VariablesSPPSSSPPP Value Number of grafts 3 (2 - 3) 3 (3 - 3) 3 (3 - 4) 3 (3 - 3) 0.209 Duration of surgery, min 174.77 ± 39.47 180.77 ± 56.8 178.57 ± 46.5 166.08 ± 51.7 0.390 CPB time, min 72.72 ± 19.29 76.13 ± 26.90 74.76 ± 21.13 68.34 ± 25.57 0.703 During operation Epinephrine 0.611 Epinephrine 0.01 - 0.05, μg/kg/min 10 (45.45) 7 (31.81) 5 (22.72) 11 (47.82) Epinephrine 0.06 - 0.1, μg/kg/min 2 (9.09) 3 (13.63) 2 (9.09) 2 (8.69) Dexamethasone, 8 - 16 mg 4 (18.18) 6 (27.27) 4 (18.18) 6 (26.08) 0.838 Hydrocortisone, 200 mg 0 0 0 1 (4.34) 1.00 ICU Epinephrine 0.505 Epinephrine 0.01 - 0.04, μg/kg/min 12 (54.54) 9 (40.9.) 11 (50.0) 14 (60.86) Epinephrine 0.05 - 0.08, μg/kg/min 4 (18.18) 9 (40.90) 8 (36.36) 7 (30.43) Epinephrine 0.08 - 0.11, μg/kg/min 6 (27.27) 4 (18.18) 3 (13.63) 2 (8.69) Dexamethasone 2 - 8, mg 2 (9.09) 2 (9.09) 0 3 (13.04) 0.504 Hydrocortisone 10 - 200, mg 3 (13.63) 3 (13.63) 3 (13.63) 3 (13.04) 1.00 Insulin 3 - 15, units 4 (18.18) 5 (22.72) 2 (9.09) 4 (17.39) 0.720 Blood loss, mL b 815 (465 - 1185) 770 (500 - 1190) 770 (495 - 1172) 770 (540 - 1650) 0.980 Abbreviation: CPB, cardiopulmonary bypass. aData are expressed as No. (%), median (interquartile range), or mean ± SD. bTotal drainage during the first 48 hours postoperatively in patients with chest tubes. The changes in the measured parameters during the trial are shown in Table 3. The time-treatment interaction was significant for none of the parameters. Blood glucose levels increased postoperatively in the four groups, and the between-group differences were significant (0.003). In the post hoc analysis, there was a significant difference between the PP group and the SP (0.027), PS (0.026), and SS (0.004) groups. Table 3.BODY. COMPARISONS OF MEASURED PARAMETERS BETWEEN AND WITHIN GROUPS:a Group−7 Days (1)Pre OP (2)Post OP (3)+7 Days (4)+30 Days (5)Within GroupsTime-Treatment InteractionBetween Groups Blood glucose < 0.001; 3: 1,2,4,5 (< 0.001) b; 4: 1,2,3,5 (< 0.001) 0.236 0.003; SP: PP (0.027) b; PS: PP (0.026); SS: PP (0.004) SP 93.18 ± 9.9 93.95 ± 15.8 164.50 ± 44.4 106.31 ± 21.6 95.72 ± 12.8 PS 92.19 ± 11.3 92.04 ± 13.0 167.76 ± 42.0 108.47 ± 27.1 92.00 ± 11.3 SS 99.00 ± 12.6 91.66 ± 22.8 153.80 ± 37.2 99.95 ± 12.9 94.71 ± 15.6 PP 101.68 ± 13.1 110.45 ± 34.8 189.31 ± 43.3 116.50 ± 21.9 102.13 ± 16.1 Insulin (μU/mL) < 0.001; 3: 1,2,5 (< 0.001) b; 3: 4 (0.001); 4: 1,3 (0.001); 4: 5 (0.018) 0.686 0.855 SP 7.90 ± 3.9 11.77 ± 5.8 16.51 ± 8.4 10.84 ± 6.0 8.94 ± 4.5 PS 7.81 ± 3.9 8.49 ± 5.3 16.11 ± 11.6 12.66 ± 6.0 9.03 ± 3.6 SS 11.00 ± 11.1 9.32 ± 6.3 16.60 ± 12.6 11.50 ± 8.1 9.96 ± 5.9 PP 8.5 ± 3.4 11.05 ± 6.6 17.04 ± 11.6 12.15 ± 4.9 11.1 ± 5.0 HbA1c, % 0.007; 1: 2 (0.033) b 0.375 0.090 SP 6.10 ± 0.3 6.14 ± 0.4 - 5.96 ± 0.4 5.87 ± 0.3 PS 5.93 ± 0.4 5.79 ± 0.3 - 5.90 ± 0.4 5.67 ± 0.4 SS 6.00 ± 0.4 6.06 ± 0.5 - 6.00 ± 0.7 5.64 ± 0.5 PP 6.12 ± 0.4 6.11 ± 0.5 - 5.94 ± 0.7 6.05 ± 0.5 QUICKI index < 0.001; 3: 1,2,4,5 (< 0.001) b; 4: 1,3 (< 0.001) 0.607 0.184 SP 0.35 ± 0.03 0.34 ± 0.04 0.29 ± 0.02 0.33 ± 0.03 0.35 ± 0.05 PS 0.36 ± 0.04 0.36 ± 0.05 0.30 ± 0.02 0.33 ± 0.05 0.34 ± 0.02 SS 0.35 ± 0.04 0.36 ± 0.05 0.30 ± 0.03 0.34 ± 0.04 0.35 ± 0.04 PP 0.34 ± 0.02 0.33 ± 0.03 0.29 ± 0.03 0.32 ± 0.03 0.33 ± 0.03 Bennett’s index < 0.001; 3: 1,2,4,5 (< 0.001) b 0.303 0.300 SP 2.04 ± 1.7 1.70 ± 1.0 0.95 ± 0.2 1.44 ± 0.5 1.60 ± 0.4 PS 2.25 ± 2.0 2.84 ± 2.9 1.04 ± 0.3 1.38 ± 0.6 1.59 ± 0.4 SS 1.83 ± 0.9 2.62 ± 3.1 1.15 ± 0.4 1.68 ± 0.8 1.78 ± 0.9 PP 1.61 ± 0.6 1.59 ± 0.7 1.18 ± 1.4 1.50 ± 1.3 1.45 ± 0.6 HOMA-IR < 0.001; 3: 1,2,4,5 (< 0.001) b; 4: 1,3,5 (< 0.001) 0.861 0.798 SP 1.80 ± 0.8 2.87 ± 1.8 6.98 ± 4.8 2.87 ± 1.7 2.13 ± 1.1 PS 1.73 ± 0.9 1.94 ± 1.4 6.42 ± 4.7 3.34 ± 1.6 2.01 ± 0.8 SS 2.29 ± 1.9 2.12 ± 1.7 6.26 ± 5.5 2.84 ± 2.3 2.29 ± 1.4 PP 2.08 ± 0.8 2.63 ± 1.7 6.62 ± 3.3 3.49 ± 1.7 2.77 ± 1.2 FIRI < 0.001; 3: 1,2,4,5 (< 0.001) b; 4: 1,3,5 (< 0.001) 0.845 0.601 SP 1.62 ± 0.8 2.59 ± 1.6 6.28 ± 4.3 2.58 ± 1.6 1.92 ± 1.0 PS 1.56 ± 0.8 1.75 ± 1.2 5.77 ± 4.2 3.00 ± 1.4 1.81 ± 0.7 SS 2.06 ± 1.7 1.91 ± 1.5 5.64 ± 5.0 2.56 ± 2.1 2.06 ± 1.3 PP 1.93 ± 0.7 2.85 ± 2.7 5.99 ± 2.9 3.18 ± 1.5 2.58 ± 1.1 aData are expressed as mean ± SD. bPair-wise comparisons. The surgical outcomes are demonstrated in Table 4. Two deaths occurred: one in the SS group on the patient’s first night in the ICU, and the second one in the PP group two weeks after discharge from the hospital. With regard to infectious complications, DSWI was seen in only one patient from the PP group. The superficial wound infection rate was significantly different in the four groups: 26.08% in PP, 9.09% in SP, 4.54% in PS, and 0% in SS. Table 4.BODY. CLINICAL OUTCOMES AND INCIDENCE OF ADVERSE EVENTS IN THE STUDY PATIENTS:a SP PS SS PP P Value Number 22 22 22 23 NA Death 0 0 1 (4.54) 1 (4.3) 0.865 MI 0 0 0 1 (4.3) 1.00 IABP 0 0 0 0 NA Dialysis 0 0 0 0 NA CVA 1 (4.54) 0 0 1 (4.34) 1.00 Severe infection Pneumonia (requiring ventilation) 0 0 0 0 NA DSWI 0 0 0 1 (4.34) 1.00 Minor infection Pneumonia (not requiring ventilation) 2 (9.09) 2 (9.09) 1 (4.54) 3 (13.04) 0.956 Superficial wound infection 2 (9.09) 1 (4.54) 0 6 (26.08) 0.025 UTI 2 (9.09) 0 0 1 (4.34) 0.455 Blood transfusion RBC, mL/patient Number (percent) 20 (90.90) 19 (86.36) 18 (81.81) 21 (91.30) 0.886 Unit/Patient 3.55 ± 2.36 2.72 ± 1.79 2.74 ± 1.72 3.84 ± 2.15 0.176 FFP, mL/patient Number (percent) 3 (13.63) 4 (18.18) 2 (9.09) 6 (26.08) 0.502 Unit/Patient 3.15 ± 0.78 3.85 ± 1.30 3.00 ± 1.41 4.14 ± 2.11 0.786 Platelets (mL/patient) number (percent) 1 (4.54) 1 (4.54) 2 (9.09) 1 (4.34) 0.935 unit/patient 2.0 4.0 4.00 ± 2.8 4.0 0.734 Intubation time, h 10 (8 - 14) 9.9 (8.2 - 12.0) 9.7 (7.1 - 11.1) 10.0 (6.5 - 13.5) 0.886 ICU stay, h 43.0 (40.5 - 48.0) 43.0 (39.7 - 51.0) 42 (41 - 43) 43 (39 - 44) 0.598 Hospital stay, d 3 (3 - 4) 4 (3 - 4) 3 (3 - 4) 4 (3 - 4) 0.311 Abbreviations:CVA, cerebrovascular accident; DSWI, deep sternal wound infection; FFP: fresh frozen plasma; IABP, intra-aortic balloon pump; MI, myocardial infarction; RBC, red blood cell; UTI, urinary tract infection. aData are expressed as No. (%), median (interquartile range), or Mean ± SD. With regard to the participants’ dietary intake, there were no significant differences in the intake of total calories, carbohydrates, protein, fat, fiber, vitamin C, vitamin E, or selenium among the four groups at the beginning and the end of the trial. In addition, there were no supplement-related complications. BODY.5. DISCUSSION: The results of the present study revealed for the first time that administration of a new metabolic conditioning supplement, whether pre- or postoperatively, led to better perioperative glycemic control and decreased post-CABG wound infection in non-diabetic patients. Concerning glycemic control in the perioperative period, few studies have investigated the effects of metabolic conditioning using insulin-resistance-reducing agents such as arginine, glutamine, and antioxidants (3, 21, 27). With the introduction of L-carnitine as a novel conditioning agent (3), no clinical trial before now has evaluated the summative effects of glutamine, L-carnitine, and antioxidants as a metabolic conditioning supplement on surgery-induced metabolic stress. Our trial is also unique in its relatively long period of supplement administration pre- and post-surgery. Postoperative blood glucose increased significantly in all four groups with the same pattern, but compared to the SP, PS, and SS groups, a significantly higher increase occurred in the PP group. Serum insulin levels and insulin resistance markers (HOMA-IR, FIRI) also increased significantly postoperatively, and again decreased to near the preoperative level after 30 days, but there were no significant differences between the four groups. Insulin sensitivity indices (QUICKI, Bennett’s) likewise decreased immediately postoperatively, then increased by 30 days postoperatively, without any significant differences between the four groups. In addition, there were no significant differences between the four groups with regard to HbA1c levels. As expected, our metabolic conditioning supplement led to better glycemic control in the perioperative period. In a study by Alvez et al., intravenous administration of 50 g of L-Alanyl glutamine three hours prior to surgery, in patients with critical limb ischemia undergoing operative revascularization, resulted in reduced muscle cell damage, enhanced antioxidant capacity, and improved glucose utilization by the peripheral tissues (28). Glutamine supplementation has been proven to increase insulin-dependent glucose utilization in the peripheral tissues, leading to better glycemic control without changing the insulin concentration, via increasing insulin sensitivity in the periphery (12, 29). Insulin resistance is associated with impaired mitochondrial function, which leads to decreased fatty acid oxidation. Inadequate and inefficient oxidative phosphorylation probably leads to oxidative stress and triglyceride accumulation in the skeletal muscles, which decreases insulin sensitivity (19, 30). Studies have revealed that L-carnitine concentration is reduced in diabetic patients (19). Low fatty acid oxidation is secondary to reduced carnitine palmitoyltransferase (CPT) activity (30), and it is proposed that exogenous carnitine supplementation may eliminate the deficit (19). We used HOMA-IR, QUICKI, Bennett’s and FIRI to measure insulin resistance and sensitivity. Ljunggren et al. showed that HOMA-IR and QUICKI are unable to reflect the magnitude of surgery-induced insulin resistance. Compared to the clamp method, which is the gold standard method of assessing insulin sensitivity, HOMA-IR and QUICKI detected only 10% of postoperative insulin resistance. These static methods reflect the balance between insulin and glucose levels in the absence of any metabolic stress, while the dynamic method (the clamp technique) measures insulin resistance in hyperinsulinemic situations (31). In fact, these approaches reflect different aspects of insulin resistance. The clamp method measures insulin resistance developing in peripheral tissues, such as muscle and adipose tissue, while HOMA-IR and QUICKI act as markers of hepatic insulin resistance (31, 32). Ljunggren concluded that surgery-induced insulin resistance must be attributed to metabolic alterations in the peripheral tissues rather than to hepatic metabolism (31). Considering the established role of glutamine and L-carnitine in alleviating insulin resistance in the peripheral tissues, we can claim that our metabolic conditioning supplement played a role in controlling peripheral-tissue insulin resistance, and its effects were reflected in the glycemic control and infection rates. However, the static methods we used to measure insulin resistance and sensitivity (HOMA-IR, FIRI, QUICKI, and Bennett’s) were unable to detect our supplement’s effect on peripheral insulin resistance. We can also conclude that our metabolic conditioning agent did not have any effect on hepatic insulin resistance. Since HbA1c represents the mean glycemia level over the preceding 3-4 months (33), it seems logical that changes in its value are not statistically significant. Surgical-site infection is the third most prevalent (17%) type of all nosocomial infections in hospitalized patients, and is a major cause of morbidity and mortality in the postoperative period (34, 35). Perioperative hyperglycemia is recognized as a modifiable risk factor for surgical-site infection (36). In a cohort of 149 diabetic patients undergoing colorectal resection, the mean 48-hour postoperative capillary glucose of > 200 mg/dL was independently associated with a greater than 3-fold increase in surgical-site infections, and this relationship was independent of the dose and regimen of the postoperative insulin administration (37). This means that glycemic control, rather than the dosage and regimen of the administered insulin, plays a role in infection control. Several studies have also shown that by controlling postoperative blood glucose to levels below 11.0 mmol/L, the rate of sternal wound infections is reduced up to three times in cardiac surgery patients (38, 39). The largest randomized controlled trial (the Leuven trial), which included more than 1,500 critically ill patients, showed that with strict blood glucose control (<110 mg/dL), compared to a level of < 200 mg/dL, bloodstream infections were decreased and the mortality rate was reduced (40). The effects of hyperglycemia on a wound take place when the wound is in the inflammatory phase of healing (37). Hyperglycemia upregulates the release of pro-inflammatory mediators and suppresses the immune system (36). In our study, DSWI occurred only in one patient from the PP group, and the minor wound infection rate was significantly higher in the PP group (26.08%) compared to the SP (9.09%), PS (4.54%), and SS (0%) groups. This result is in agreement with that of blood glucose levels in our study. Since the infection rate was the lowest in the SS group, we can conclude that our novel supplement must be taken both before and after the operation to exert its best effect on postoperative infection control. In this trial, the metabolic conditioning supplement was taken seven days prior to the operation, which is in contrast to other studies in which the conditioning agents were ingested some hours before the surgery. Therefore, the effects seen in our study are not the acute effects of the ingredients. The strengths of our study were its new metabolic conditioning supplement and the relatively long period of its administration. A limitation of our study was the use of formulas for estimating insulin resistance and sensitivity, instead of the hyperinsulinemic normoglycemic clamp technique. It is recommended that future studies evaluate the effects of our new supplement in diabetic patients undergoing major surgery. The effect of carbohydrate-loading while taking this metabolic conditioning supplement is also worthy of further study. Our new metabolic conditioning supplement, whether taken pre- or postoperatively, led to better perioperative glycemic control and to decreased post-CABG wound infections in non-diabetic patients. The supplement’s best effect on the prevention of wound infection was seen when it was ingested both before and after the surgery.

TITLE: Randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of etanercept in patients with moderately active rheumatoid arthritis despite DMARD therapy This study evaluated the efficacy and safety of adding etanercept to disease-modifying antirheumatic drugs (DMARDs) in patients with moderately active rheumatoid arthritis (RA). This randomized, double-blind, placebo-controlled study (ClinicalTrials.gov #NCT01313208) enrolled RA patients with Disease Activity Score using 28 joints with C-reactive protein (DAS28-CRP) >3.2 and ≤5.1 (moderate disease) despite stable DMARD therapy. Patients were randomized to etanercept 50 mg or placebo weekly for 12 weeks; all patients then received etanercept 50 mg weekly through week 24. Primary endpoint was low disease activity (LDA) at week 12; secondary endpoints included DAS28-CRP remission at week 12; Clinical Disease Activity Index (CDAI) and Simplified Disease Activity Index (SDAI) LDA; American College of Rheumatology (ACR) responses; change in Health Assessment Questionnaire Disability Index (HAQ-DI), and safety. For 210 patients with moderate disease at screening, (104 placebo; 106 etanercept), only 58% still had moderate disease at baseline. At week 12, 33% on etanercept and 21% on placebo achieved LDA (P = 0.055); remission was achieved in 19% and 12%, respectively (P = 0.14). At week 12, ACR20, ACR50, and ACR70 responses were observed in 29%, 13%, and 1% respectively, in patients on placebo, and 41%, 21%, and 6% of patients on etanercept. Mean (SD) change from baseline in HAQ-DI score was −0.20 (0.43) for placebo patients and −0.39 (0.54) for etanercept patients at week 12. No new safety signals were observed. LDA was achieved by more patients on etanercept than placebo in patients with moderate disease at screening, but the difference was not statistically significant at week 12. BODY.INTRODUCTION: The American College of Rheumatology (ACR) recommends a treatment goal of either low disease activity (LDA) or remission in all patients with early rheumatoid arthritis (RA) and established RA by using disease-modifying antirheumatic drugs (DMARDs) or biologic agents (Singh et al. 2012). Etanercept, a modified p75 receptor of tumor necrosis factor (TNF) that inhibits the action of TNF, has been shown to be efficacious for the treatment of moderate to severe RA in patients with early (Bathon et al. 2000) and established (Moreland et al. 2006) disease. Subgroup analyses have indicated that patients with moderately active disease may be more likely to achieve better disease status (LDA or remission) with etanercept treatment than patients with more severe disease, despite smaller absolute improvements in disease severity (Keystone et al. 2009). The original trials did not stratify by disease activity and the subgroup of patients with moderate disease activity was relatively small. A prospective trial would better characterize the efficacy profile of etanercept in patients with moderately active disease, a medically important subset of RA patients. The objective of this study was to evaluate whether adding etanercept 50 mg per week to standard-of-care DMARD therapy in patients with moderately active RA is superior in inducing very good control of disease compared with continued DMARD therapy. BODY.METHODS: BODY.STUDY DESIGN: This was a phase 4, prospective, randomized, double-blind, placebo-controlled study. After completing all assessments during the screening window of up to 31 days, patients were randomized (1:1) to receive etanercept 50 mg weekly or placebo administered subcutaneously for 12 weeks. After week 12, all patients received etanercept 50 mg weekly for an additional 12 weeks. Patients were followed for an additional 4 weeks following the last dose of investigational product to monitor safety. Randomization was accomplished using an Interactive Voice Response System. Assignment to treatment arm was based on a computer-generated randomization schedule that was prepared by the sponsor before the start of the study, and used randomly permuted blocks. Randomization was stratified by use of methotrexate at baseline. Patients, site personnel, and investigators were blinded to treatment assignment. BODY.PATIENTS: Eligible patients were ≥ 18 and ≤ 80 years of age at screening and had a diagnosis of RA per the 1987 ACR classification criteria (Arnett et al. 1988) for ≥ 6 months before screening. Patients were required to have moderately active disease as defined by a Disease Activity Score based on 28 joints with C-reactive protein as the indicator of inflammation (DAS28-CRP) > 3.2 and ≤ 5.1 (Fransen and van Riel 2005) and ≥ 3 swollen joints and ≥ 3 tender joints. CRP levels were measured using a central laboratory. Samples for CRP testing were collected at screening (for investigators to identify patients with moderately active disease based on DAS28-CRP calculations) and at baseline. Several days elapsed between collection of samples and availability of CRP results, so baseline DAS28-CRP was calculated retrospectively and randomization at baseline was based solely on swollen/tender joints at the screening visit. Patients had to be taking methotrexate for ≥ 12 weeks with a stable dose of 15–25 mg weekly for ≥ 8 weeks prior to baseline (lower doses were allowed at the investigator’s discretion); patients with contraindications to methotrexate were allowed to enroll if they were using sulfasalazine, leflunomide, minocycline, and/or hydroxychloroquine. Exclusion criteria included: prosthetic joint infection within 5 years or native joint infection within 1 year of screening, Class IV RA according to ACR revised criteria (Hochberg et al. 1992), diagnosis of Felty’s syndrome, use of > 1 commercially available or experimental biologic DMARD (use of 1 biologic DMARD was allowed if the patient had received no more than 8 weeks of treatment and did not use the DMARD within 2 months of the first dose in this study), serious infection requiring hospitalization or intravenous antibiotics within 8 weeks before screening, active infection requiring anti-infectives within 28 days prior to first dose, significant concurrent medical conditions, or laboratory abnormalities at screening. BODY.STUDY ENDPOINTS: Endpoints included the proportion of patients with DAS28-CRP LDA (DAS28-CRP < 3.2; primary endpoint) and remission (DAS28-CRP < 2.6; key secondary endpoint) (Aletaha and Smolen 2005) at week 12. Additional secondary endpoints included rates of Clinical Disease Activity Index (CDAI) LDA (score ≤ 10) and remission (score ≤ 2.8) (Aletaha and Smolen 2005); rates of Simplified Disease Activity Index (SDAI) LDA (score ≤ 11) and remission (score ≤ 3.3) (Aletaha and Smolen 2005); rates of 20%, 50%, and 70% improvement in ACR criteria (ACR20, ACR50, and ACR70) (Felson et al. 1995); changes in Health Assessment Questionnaire Disability Index (HAQ-DI) (Wolfe et al. 1988); and safety. Safety endpoints included the nature, frequency, severity and relationship to treatment of all adverse events (AEs). Efficacy and patient-reported outcomes were assessed at baseline and weeks 2, 4, 8, 12, 16, 20, and 24. BODY.STATISTICAL CONSIDERATIONS: The hypothesis tested was that adding etanercept in RA patients with moderately active disease despite DMARD therapy would yield a greater proportion of patients with LDA and remission than continued DMARD therapy only, as measured by DAS28-CRP at week 12. A sample size of 100 patients per treatment arm was estimated to provide 83% power to detect a difference in the proportion of patients achieving DAS28-CRP LDA with alpha of 0.05. The primary efficacy endpoint was compared between the two treatment arms using the Mantel-Haenszel test stratified by methotrexate use (yes or no) at baseline. DAS28-CRP was also summarized as a continuous variable by treatment group. Last observation carried forward was used to impute missing data for the primary analysis for patients without an assessment at week 12. Primary and secondary endpoints were tested sequentially, i.e. secondary endpoints were tested only if the primary endpoint was statistically significant. All efficacy endpoints were evaluated using the primary analysis set, which comprised all randomized patients. Additional efficacy analyses were performed on the subset of patients who had moderate disease at baseline. Safety endpoints were analyzed using the safety analysis set, which included all randomized patients who received at least one dose of any investigational product, and were analyzed based on treatment received. AEs were summarized and coded using the Medical Dictionary for Regulatory Activities (MedDRA) version 15.1. Statistical analyses were performed using SAS version 9.2 (SAS Institute Inc., Cary, NC, USA). BODY.ETHICAL STANDARDS: This study was conducted in accordance with the Helsinki declaration. The study protocol and consent were approved by the institutional review board at each study site. All patients provided signed informed consent prior to initiation of any study-related procedures. BODY.RESULTS: BODY.PATIENTS: A total of 210 patients were enrolled in the study from 38 centers in the US and Canada; 104 patients were randomized to the placebo-etanercept group and 106 were randomized to the etanercept-etanercept group. The study was conducted from March 31, 2011 (first patient enrolled) through May 22, 2013 (last patient visit). Most patients were women (77%), most were white (86%), the mean (standard deviation [SD]) age was 56.0 (12.4) years, and the mean (SD) duration of RA was 7.8 (9.8) years (Table 1). Patients were required to have moderate disease activity at screening to qualify for enrollment in the study; however, at baseline only 122 patients (58%) had moderate disease when administration of study drugs was initiated. The screening period ranged up to 35 days for most patients, with a median of 15 days. Of the 88 patients who no longer had moderate disease activity at baseline, 3 had improved to LDA and 85 had worsened. During the double-blind portion of the trial (weeks 1–12), 6.7% of patients discontinued treatment, and during weeks 13–24, 6.2% of patients discontinued treatment (Figure 1). All 210 patients were included in the primary analysis set and the safety analysis set.Table 1 Demographic and clinical characteristics at screening (primary analysis set) Placebo-etanercept (N = 104) Etanercept-etanercept (N = 106) All patients (N = 210) Age, mean years (SD) 55.5 (12.8) 56.5 (12.1) 56.0 (12.4) Sex, n women (%) 86 (82.7) 75 (70.8) 161 (76.7) Race, n (%)  White 90 (86.5) 91 (85.8) 181 (86.2)  Black/African American 8 (7.7) 9 (8.5) 17 (8.1)  Asian 3 (2.9) 2 (1.9) 5 (2.4)  Other 3 (2.9) 4 (3.8) 7 (3.3) BMI, mean kg/m2 (SD) 29.3 (6.6) 30.6 (7.7) 30.0 (7.2) Duration of RA, mean years (SD) 7.4 (8.1) 8.3 (11.2) 7.8 (9.8) DAS28-CRP, mean (SD) 4.9 (0.8) 4.9 (0.7) 4.9 (0.8) SD: standard deviation; BMI: body mass index; RA: rheumatoid arthritis; DAS28-CRP: Disease Activity Score based on 28 joints with C-reactive protein. Figure 1 Patient disposition. *One patient was counted as having both completed etanercept and discontinuing etanercept. The patient received all 12 doses of etanercept during weeks 1 through 12 of the study, but ended treatment on the day of the last dose because of an adverse event that required protocol-prohibited treatments. BODY.EFFICACY OUTCOMES: The study failed to meet the primary endpoint. At week 12, the percentage of patients who achieved LDA was not significantly different between the placebo-etanercept group (21%) and the etanercept-etanercept group (33%; P = 0.055). Rates of DAS28-CRP LDA were statistically significantly different at week 8 (placebo-etanercept group 16%, etanercept-etanercept group 34%; nominal P = 0.003) (Figure 2); however, improvement in DAS28-CRP in the placebo-etanercept group and lack of continued improvement in the etanercept-etanercept group was observed between weeks 8 and 12 (Figure 3). In a post hoc analysis of patients who had moderate disease at baseline, 18% of patients in the placebo-etanercept group and 42% of patients in the etanercept-etanercept group had achieved DAS28-CRP LDA at week 12 (P = 0.005) (Figure 2).Figure 2 Rates of DAS28-CRP LDA and remission. The percentages of patients with DAS28-CRP < 3.2 (LDA; top panels) and < 2.6 (remission; bottom panels) are shown. Data are shown for the primary analysis set (left panels) and the subset of patients with moderate RA at baseline (right panels). Patients in the placebo-etanercept group (black bars in left panels; gray bars in right panels) received placebo (hashed bars) in the first 12 weeks and etanercept through week 24 and patients in the etanercept-etanercept group received etanercept (gray bars in left panels; white bars in right panels) throughout the study. *P < 0.05; † P < 0.01; ‡ P < 0.001 for comparison between groups. DAS28-CRP: Disease Activity Score based on 28 joints with C-reactive protein; LDA: low disease activity; RA: rheumatoid arthritis. Figure 3 DAS28-CRP values. Mean DAS28-CRP values are shown for the placebo-etanercept (circles) and etanercept-etanercept (squares) groups. Dotted lines indicate the period when the placebo-etanercept group received placebo. Error bars represent standard deviations. *P < 0.05; † P < 0.01 for comparison between groups. DAS28-CRP: Disease Activity Score based on 28 joints with C-reactive protein; SD, standard deviation. Because the primary endpoint did not reach statistical significance, formal testing of secondary endpoints was not performed. Key secondary endpoints of CDAI and SDAI rates of LDA and remission and ACR responses are shown in Table 2. Similar to results of the primary endpoint, differences between treatment groups were greatest at week 8 for all key secondary endpoints.Table 2 Key secondary endpoints: rates of CDAI and SDAI LDA and remission, ACR responses, improvements in HAQ-DI (primary analysis set; LOCF imputation) Placebo-etanercept (N = 104) Etanercept-etanercept (N = 106) CDAI LDA (score ≤10), n (%)  Week 2 9 (9.1) 13 (12.3)  Week 8 16 (15.4) 31 (29.2)  Week 12 22 (21.2) 27 (25.5)  Week 24 43 (41.3) 49 (46.2) CDAI remission (score ≤2.8), n (%)  Week 2 1 (1.0) 2 (1.9)  Week 8 0 (0.0) 3 (2.8)  Week 12 1 (1.0) 4 (3.8)  Week 24 4 (3.8) 9 (8.5) SDAI LDA (score ≤11), n (%)  Week 2 8 (8.2) 13 (12.4)  Week 8 15 (14.4) 32 (30.2)  Week 12 22 (21.2) 25 (23.6)  Week 24 41 (39.4) 48 (45.3) SDAI remission (score ≤3.3), n (%)  Week 2 1 (1.0) 1 (1.0)  Week 8 0 (0.0) 6 (5.7)  Week 12 2 (1.9) 6 (5.7)  Week 24 7 (6.7) 11 (10.4) ACR20 response, n (%)  Week 2 15 (14.9) 30 (28.6)  Week 8 24 (23.1) 53 (50.0)  Week 12 30 (28.8) 43 (40.6)  Week 24 48 (46.2) 53 (50.0) ACR50 response, n (%)  Week 2 3 (3.0) 6 (5.7)  Week 8 5 (4.8) 21 (19.8)  Week 12 13 (12.5) 22 (20.8)  Week 24 30 (28.8) 35 (33.0) ACR70 response, n (%)  Week 2 0 (0.0) 2 (1.9)  Week 8 0 (0.0) 6 (5.7)  Week 12 1 (1.0) 6 (5.7)  Week 24 13 (12.5) 17 (16.0) Change from baseline in HAQ-DI, mean score change (SD)  Week 2 −0.09 (0.39) −0.27 (0.43)  Week 8 −0.21 (0.42) −0.37 (0.47)  Week 12 −0.20 (0.43) −0.39 (0.54)  Week 24 −0.45 (0.52) −0.48 (0.58) CDAI: Clinical Disease Activity Index; SDAI: Simplified Disease Activity Index; LDA: low disease activity; ACR: American College of Rheumatology; HAQ-DI: Health Assessment Questionnaire Disability Index; LOCF: last observation carried forward; SD: standard deviation. BODY.PATIENT-REPORTED OUTCOMES: Patients in the etanercept-etanercept group showed improvements from baseline in HAQ-DI score throughout the study (Table 2). Patients in the placebo-etanercept group showed minimal improvements from baseline through week 12, but at week 24 had similar improvements from baseline as patients in the etanercept-etanercept group. BODY.SAFETY RESULTS: During the 12-week double-blind portion of the study, 61% of patients in the placebo-etanercept group and 67% of the etanercept-etanercept group reported an AE (Table 3). The most commonly reported AEs through week 12 included injection site erythema (1.0% placebo-etanercept; 11.3% etanercept-etanercept), headache (10.6%; 7.5%), injection site pruritus (1.9%; 6.6%), and injection site rash (1.0%; 6.6%). Overall, 78% of all patients reported an AE through 24 weeks of treatment. The most commonly reported AEs through week 24 included injection site erythema (10.6% placebo-etanercept; 12.3% etanercept-etanercept), RA worsening/flare (11.5%; 11.3%), headache (15.4%; 8.5%), and upper respiratory tract infection (12.5%; 7.5%). Serious AEs were reported in 7 patients (3 patients in the placebo-etanercept group and 4 patients in the etanercept-etanercept group) through 24 weeks of treatment. No opportunistic infections, malignancies, or deaths were reported during the study. No new or unexpected safety signals were observed.Table 3 Summary of safety (safety analysis set) Placebo-etanercept (N = 104) Etanercept-etanercept (N = 106) All patients (N = 210) Patients reporting an AE, n (%)  Weeks 1–12 (double-blind portion) 63 (60.6) 71 (67.0) 134 (63.8)  Weeks 1–24 80 (76.9) 83 (78.3) 163 (77.6) Patients reporting an SAE, n (%)  Weeks 1–12 (double-blind portion) 2 (1.9) 3 (2.8) 5 (2.4)  Weeks 1–24 3 (2.9) 4 (3.8) 7 (3.3) Patients reporting an SIE, n (%)  Weeks 1–12 (double-blind portion) 0 (0) 0 (0) 0 (0)  Weeks 1–24 0 (0) 1 (0.9) 1 (0.5) Patients reporting an infection, n (%)  Weeks 1–12 (double-blind portion) 23 (22.1) 30 (28.3) 53 (25.2)  Weeks 1–24 46 (44.2) 39 (36.8) 85 (40.5) AE: adverse event; SAE: serious adverse event; SIE: serious infectious event. BODY.DISCUSSION: Patients with moderate disease activity despite treatment with DMARDs represent a medically important subset of patients with RA, as their first-line therapy has failed to achieve or sustain LDA or remission. As these patients have been shown to achieve better clinical responses to etanercept therapy than patients with severe disease (Keystone et al. 2009), this study therefore was designed to further investigate the efficacy and safety of adding etanercept to methotrexate in this patient population. Although the primary endpoint of the study was not reached for the entire study population, a post hoc analysis of the subset of patients who fulfilled the entry criterion of moderate disease activity at baseline showed that the addition of etanercept to methotrexate resulted in a greater proportion of patients achieving DAS28-CRP LDA at week 12. Several possible reasons for the failure of the study to meet its primary endpoint have been identified. There was a protocol failure with respect to how patients with moderate disease were screened and enrolled. The protocol required the site to calculate the DAS28-CRP and confirm moderate disease only at screening but should have required this assessment at both screening and at baseline prior to enrollment. A solution to this issue could have been the use of erythrocyte sedimentation rate (ESR), which can be performed locally. Measuring the level of inflammation with ESR instead of CRP would have avoided the time delay in measuring the level of inflammation for calculating disease severity. Another potential solution would have been to require that moderate disease activity was stable over a specific period, such as 3 months, before enrollment in the study. Moderate disease activity may represent a transient state, with some patients rapidly worsening to severe disease or improving to LDA. Additionally, patients were allowed to receive nonbiologic DMARDs at study entry; DMARDs initiated within 3 months of enrollment into this study may not have reached their full effect when patients were evaluated during the screening window. Use of corticosteroids did not appear to influence the study results (data not shown). Finally, the measurement of disease activity was based on a composite global measurement and may not have been sensitive enough to provide a clear distinction between moderate and high disease activity. The primary limitation of the study was the insufficient number of patients with moderate disease at baseline based on the power analysis requirement to determine the minimum number of patients required to test the hypothesis. Surprisingly, rates of DAS28-CRP LDA were statistically significant between etanercept and placebo at week 8, but failed to reach significance at week 12. Between weeks 8 and 12 of this study, unexpected improvements in DAS28-CRP in the placebo-etanercept group and lack of continued improvements in the etanercept-etanercept group were observed. All lots of drugs dispensed in the study were scrutinized, and 1999), 66% of patients on combination therapy achieved an ACR20 response at week 12 compared with only 41% of patients in our study. Notably, ACR20 response rates at week 12 were similar in patients receiving placebo plus methotrexate in the pivotal trial (33%) and our study (29%). The results of this study revealed a trend toward better clinical outcomes in patients on etanercept plus methotrexate therapy compared with methotrexate alone. Within the spectrum of moderately active disease, changes in status (ie, moderate to severe disease) can be frequent and disease activity scores at any given time may not accurately reflect these changes. Protocols that do not confirm the hypothesis such as this may offer results that are clinically significant but difficult to reconcile. However, they also provide information for improving designs of future studies.

TITLE: Effect of a sport-for-health intervention (SmokeFree Sports) on smoking-related intentions and cognitions among 9-10 year old primary school children: a controlled trial ABSTRACT.BACKGROUND: Preventing children from smoking is a public health priority. This study evaluated the effects of a sport-for-health smoking prevention programme (SmokeFree Sports) on smoking-related intentions and cognitions among primary school children from deprived communities. ABSTRACT.METHODS: A non-randomised-controlled trial targeted 9-10 year old children from Merseyside, North-West England. 32 primary schools received a programme of sport-for-health activities over 7 months; 11 comparison schools followed usual routines. Data were collected pre-intervention (T0), and at 8 months (T1) and one year post-intervention (T2). Smoking-related intentions and cognitions were assessed using an online questionnaire. Intervention effects were analysed using multi-level modelling (school, student), adjusted for baseline values and potential confounders. Mixed-sex focus groups (n = 18) were conducted at T1. ABSTRACT.RESULTS: 961 children completed all assessments and were included in the final analyses. There were no significant differences between the two study groups for non-smoking intentions (T1: β = 0.02, 95 % CI = -0.08–0.12; T2: β = 0.08, 95 % CI = -0.02–0.17) or for cigarette refusal self-efficacy (T1: β = 0.28, 95 % CI = -0.11–0.67; T2: β = 0.23, 95 % CI = -0.07–0.52). At T1 there was a positive intervention effect for cigarette refusal self-efficacy in girls (β = 0.72, 95 % CI = 0.21–1.23). Intervention participants were more likely to ‘definitely’ believe that: ‘it is not safe to smoke for a year or two as long as you quit after that’ (RR = 1.19, 95 % CI = 1.07–1.33), ‘it is difficult to quit smoking once started’ (RR = 1.56, 95 % CI = 1.38–1.76), ‘smoke from other peoples’ cigarettes is harmful’ (RR = 1.19, 95 % CI = 1.20–2.08), ‘smoking affects sports performance’ (RR = 1.73, 95 % CI = 1.59–1.88) and ‘smoking makes ‘no difference’ to weight’ (RR = 2.13, 95 % CI = 1.86–2.44). At T2, significant between-group differences remained just for ‘smoking affects sports performance’ (RR = 1.57, 95 % CI = 1.43–1.72). Focus groups showed that SFS made children determined to remain smoke free and that the interactive activities aided children’s understanding of smoking harms. ABSTRACT.CONCLUSION: SFS demonstrated short-term positive effects on smoking attitudes among children, and cigarette refusal self-efficacy among girls. Although no effects were observed for non-smoking intentions, children said that SFS made them more determined not to smoke. Most children had strong intentions not to smoke; therefore, smoking prevention programmes should perhaps target early adolescents, who are closer to the age of smoking onset. BODY.BACKGROUND: Smoking is an addiction often initiated in childhood, with approximately 207,000 children taking up smoking each year in the UK [1]. The earlier a child starts to smoke, the higher their chances of becoming a regular smoker and the more difficult it becomes to quit [2]. Early onset of smoking and persisting in the habit in adulthood increases the risk of developing lung cancer and other smoking-related diseases [3, 4]. Preventing smoking uptake in children by de-normalising tobacco use is therefore a key public health priority for the UK Government [5], which is aiming for a tobacco-free generation by 2025 [6]. Although only 0.3 % of 8-10 year old UK primary school children have ever smoked [7], some children develop intentions to start smoking [8]. Behavioural intentions to smoke are worth paying special attention to as they are theorised to be the first step in smoking initiation [9]. Intentions to smoke may be explained by individual smoking-related cognitions such as attitudes towards smoking and cigarette refusal self-efficacy [10–12]. Since children living in socially deprived areas have a high intention to smoke [13] and harbour misconceptions about the harms of smoking [14], implementing a smoking prevention intervention in primary school may prevent children from starting to smoke. Schools are considered an appropriate setting for smoking prevention because they can provide an efficient means of reaching large numbers of children [15] and provide an opportunity to ‘set’ healthy and enduring patterns of behaviour [16, 17]. Consequently, numerous school-based smoking prevention programmes have been developed and implemented over the last decade to discourage smoking uptake and deter regular use [18, 19]. Previous interventions have predominantly targeted adolescents, whilst programmes that have been developed for primary school aged children have been implemented outside the UK [18, 19]. Waiting until secondary school to intervene with smoking prevention programmes can be too late, since by then adolescents may have developed deep rooted smoking expectancies and norms [20–22] and, for some the behaviour is already underway (8 % of 15 year olds smoke) [23]. Sport-for-health programmes use sport as a mechanism to promote health and prevent disease [24–27]. Interventions typically use participatory approaches like game-based learning and activities with sport coaches, who represent important role models for youth, to transmit health promotion messages and positively shape attitudes [28, 29]. The use of sport as an educational platform for tobacco control has previously been trialled in the US and Canada with initiatives such as Tobacco Free Sports [30], Tobacco Free Athletes [31] and Play, Live, Be Tobacco Free [32]. Sport-for-health interventions have several potential benefits over traditional classroom-based smoking prevention education approaches. First, participation in physical activity improves health not only directly but also through its protective effect against smoking initiation in youth [33, 34]. Second, given that all primary school children are required to participate in physical education, this lesson could provide a forum to integrate smoking education messages within the primary school curriculum. Third, the use of sport as a smoking prevention strategy encourages active engagement with the intervention as well as interactions with other pupils and teaching staff, and is consistent with National Institute for Health and Care Excellence (NICE) recommendations [35] to deliver interactive and participatory smoking prevention interventions. Finally, integrating physical activity into the learning process may enable children to efficiently retain and retrieve learned information [28, 36–38]. To the authors’ knowledge, no published study has evaluated the use of sport-for-health programmes for smoking prevention in the UK. SmokeFree Sports (SFS) was a sport-for-health smoking prevention intervention for youth in Liverpool, which is one of the most deprived communities in England [39] where addressing inequalities in tobacco use is a public health priority. Established in October 2010, SFS was commissioned as part of the ‘SmokeFree Liverpool’ public health campaign. The intervention was designed in accordance with the NICE guidance [35] and the Medical Research Council (MRC) framework for developing and evaluating complex interventions [40]. Phase one of SFS (February-June 2011) was a community feasibility trial in five youth clubs, which received 12 weeks of coaching activities (dance, dodge-ball and boxing) delivered by trained sports coaches [29, 41]. A formative evaluation demonstrated that the intervention helped to prevent youth from initiating smoking and had positive benefits on their attitudes and knowledge about smoking [29, 41]. However, coaches reported challenges associated with its delivery in youth clubs and recommended that the intervention be trialled in schools. Phase two (February-April 2012) therefore examined the feasibility of a six week intervention in three primary schools [42]. Trained coaches delivered twelve sessions of sports (football and dance). Similar positive benefits for children were observed, whilst teachers and coaches perceived SFS to be acceptable for smoking prevention education [42]. These promising results led to the development of a larger, controlled trial to investigate the effectiveness of SFS in Liverpool primary schools. The present study evaluated whether SFS, a sport-for-health smoking prevention intervention, is effective in increasing non-smoking intentions in 9-10 year old primary school children from Liverpool, immediately post-intervention and at a follow-up one year later. Secondary aims were to investigate the impact of the intervention on children’s attitudes towards smoking and cigarette refusal self-efficacy, termed smoking-related cognitions hereafter. The study also investigated whether sex moderated the intervention effects as differences in cognitive vulnerability towards smoking have been found between preadolescent boys and girls [14]. In addition, focus groups with children were conducted to produce more complete knowledge to inform interpretations of intervention effectiveness. Since sport-for-health interventions are an emergent area of health promotion research where evaluations are sparse and/or have lacked methodological rigour [26, 43], it is recognised that rigorous evaluations of interventions are needed to inform future practice and procedures [25]. BODY.METHODS: BODY.STUDY DESIGN: A school-based non-randomised controlled trial was conducted to evaluate the effect of a sport-for-health smoking prevention intervention, SmokeFree Sports on children’s intentions (not) to smoke and smoking-related cognitions. Due to funding requirements, SFS was offered to all primary schools within the Liverpool City Council administrative boundaries and therefore an a priori sample size calculation was not undertaken. Schools within Knowsley, another metropolitan borough in Merseyside with similar characteristics to Liverpool in terms of adult smoking rates (Liverpool: 24.2 %; Knowsley: 27.6 %) [44], deprivation levels [45] and ethnic composition [46], were recruited as comparison schools. For logistical reasons, it was not possible to blind the research team to the group-allocation. Schools were clustered into two groups:Intervention group (Liverpool): Schools received their usual smoking-related education plus SFS Comparison group (Knowsley): Schools received their usual smoking education only A schematic overview of the intervention and evaluation components is shown in Fig. 1. Data collection occurred over 20 months with measurements at baseline (T0, September and October 2012) and post-intervention (T1, June 2013) whilst children were in Year 5 of primary school, and at one year after the intervention had finished (T2, June 2014; Year 6 of primary school). Ethical approval for the study was granted by Liverpool John Moores University Research Ethics Committee (12/SPS/038).Fig. 1Schematic overview of SmokeFree Sports intervention and evaluation components BODY.PARTICIPANTS AND RECRUITMENT: In September 2012, all eligible primary schools (mainstream state schools; n = 154), from Liverpool (n = 104) and Knowsley (n = 50), were invited to participate in the study via email, post and telephone. Once schools had given written informed consent to participate in the study, all Year 5 children (aged 9–10 years; n = 1393) were invited to take part. Parents/guardians received a letter containing a parent and child information sheet and opt-out form. Parents/guardians were given contact information for the research team to discuss the project and could opt their child out of the study by telephone or by signing and returning the opt-out form. At data collection, children were given a verbal explanation of the study and asked to give signed assent. Children could withdraw from the research study at any time. BODY.INTERVENTION: The SFS intervention was delivered during school hours in Liverpool primary schools between October 2012 and May 2013. The intervention focused on smoking prevention and therefore Year 5 children (aged 9-10) were identified as an important cohort to target. Further, NICE [35] guidelines postulate that smoking prevention efforts would be most effective if they began in primary school. A detailed description of the SFS intervention has been published elsewhere [47, 48]. Briefly, the socio-ecological model [49] and cognitive theories including the Health Belief Model [50], Theory of Planned Behaviour [9] and Social Cognitive/Learning [51], guided the intervention design. The intervention aimed to implement a programme of smoking prevention actions through fun, participatory and interactive sports activities delivered by teachers and coaches. Intervention components included provision of professional development training, a training manual including 10 session plans, five coaching sessions delivered by SFS coaches, a school assembly with a local sports star, sports equipment as incentives for teachers to deliver and evaluate a minimum of five SFS sessions, a smoke free pledge for children to sign, and incentives for children for participating in the research (SFS water bottle, drawstring bag and pen). Behaviour change techniques [52] used with children included a behavioural contract (smoke free pledge); social support, advice, verbal persuasion and positive reinforcement (from peers, teachers and coaches) on remaining never smokers; information and salience of the social, emotional and health consequences of smoking; an exploration of the pros and cons of smoking; awareness raising of regret children will feel if they smoke; social comparisons with peers to dispel myths that smoking is normative; modelling of never smoking (from elite athletes); cigarette refusal rehearsals, and the promotion of physical activity as a positive distraction to smoking. BODY.COMPARISON GROUP: Children in the comparison group were requested to follow their usual smoking education. It is not mandatory to address smoking education in Key Stage 2 (pupils aged 7–11) of the UK National Curriculum [53], and it is at schools’ discretion to include the topic as part of Personal Social Health and Economic education. On completion of data collection at one year follow-up, comparison schools received a copy of the SFS training manual, and children were given a SFS water bottle, drawstring bag and pen for participating in the research study. BODY.MEASURES: The primary outcome measure was intentions (not) to smoke of the participating children; secondary outcomes included individual smoking-related cognitions (attitudes and refusal self-efficacy). Outcomes were assessed based on a self-reported questionnaire completed by children at T0, T1 and T2, and through focus groups with children, which were conducted at T1 only due to funding restrictions. BODY.SMOKING QUESTIONNAIRE: A questionnaire was constructed using items adapted from questionnaires previously used with this age group [54–57]. Background demographic information has been described in detail elsewhere [14]. Briefly, demographics assessed included age (years), gender (1 = girl, 0 = boy), ethnicity (1 = white British, 0 = other) and SES (home postcodes were used to generate indices of multiple deprivation (IMD) scores [39]. Children’s enjoyment of physical activity was assessed using the 16 item Physical Activity Enjoyment Scale [58]. Parent, sibling and friend smoking behaviour (1 = smokes (parent/sibling/friend) or tried (friend only), 0 = non-smoker) was assessed using an item from the Health Survey for England [57]. Child smoking behaviour (0 = never smoked, 1 = ever smoked) was also measured for descriptive purposes using a single item from the Health Survey for England [57]. As an indicator of smoking status, expired carbon monoxide (CO) concentrations were taken in private and recorded using a piCOsimple Smokerlyzer (Bedfont Scientific UK, England) with a reading above 10 ppm used as cut-off for defining smokers [59]. Intention (not) to smoke was assessed using two items from the Health Survey for England [57], ‘Do you think you will smoke in the next month/year?’, as well as an item designed by the research team ‘Do you think you will smoke in secondary school?’. Responses ranged from ‘definitely yes’ (1) to ‘definitely not’ (4) and were summed to produce a total intention score (range 3–12). A high score on total intention indicated a strong intention not to smoke. Cronbach alpha for total intention showed good internal consistency (α = 0.81). Refusal self-efficacy was measured using three items adapted from a nine-item self-efficacy scale in adolescents [54]. Items assessed the child’s confidence in their ability to be a non-smoker and refuse cigarettes in different situations. Responses consisted of Likert scales ranging from ‘not confident at all’ (1) to ‘very confident’ (5) and were summed to create a total refusal self-efficacy score (range 3–15). Cronbach alpha for the combined scale showed good internal consistency (α = 0.81). A high score on the scale indicated a high level of refusal self-efficacy. Attitude structure includes affective, behavioural and cognitive components [60]. For the purpose of this study, the cognitive component of children’s attitudes was explored through items adapted from the Global Youth Tobacco Survey (GYTS) [56] and the Health Survey for England [57], including ‘Do you think smoking is bad for your health?’, ‘Once someone has started smoking, do you think it will be difficult to quit?’, ‘Do you think that it is safe to smoke for only a year or two as long as you quit after that?’, ‘Do you think the smoke from other people’s cigarettes is harmful to you?’. An additional item ‘Do you think smoking effects sport performance?’ was developed by the research team. Responses ranged from ‘definitely not’ (1) to ‘definitely yes’ (4). A summary scale was created but internal consistency was low (α = .49). Since the data for individual attitude items were positively skewed and distribution was not improved by statistical transformation, responses were collapsed into dichotomous variables for analyses: a definitive negative attitude towards smoking (i.e. ‘definitely yes’) was scored 1; the remaining response categories (i.e. ‘probably yes’, ‘probably not’ and ‘definitely not’) indicated a more favourable attitude towards smoking and thus were collapsed into a single group and scored 0. One attitude item (‘Do you think that it is safe to smoke for only a year or two as long as you quit after that?’) was reverse coded in order to maintain consistent scale direction for all items. An additional attitude item, ‘Do you think smoking makes you gain weight?’ was also included from the Health Survey for England [57]. Whilst it is recognised that smoking is associated with weight loss [61], smoking is widely discouraged by public health professionals for weight control. Therefore, a key message included within the curriculum was that regular physical activity and healthy eating, but not smoking, was important for maintenance of a healthy weight. Thus responses for this item were collapsed into a dichotomous variable for analysis with ‘no difference’ scored 1 and the remaining response categories (i.e., ‘lose weight’ or ‘gain weight’) grouped and scored 0. BODY.FOCUS GROUPS WITH CHILDREN: Eighteen mixed-sex focus groups with children (n = 95; 45 % boys) were facilitated by trained researchers immediately following the intervention [T1]. Focus groups comprised of five to six children, lasted between 30 and 50 min and were audio recorded using a Dictaphone. Children’s perceptions of smoking, appropriateness of the intervention, and improvements for future implementation were explored. Photographs of SFS games were used to help children recall activity type [62, 63]. To aid the credibility of data, facilitators’ reflected interpretations back to children during the focus groups. The present study focuses on children’s perceptions surrounding the impact of SFS on intentions (not) to smoke and individual smoking-related cognitions, thus other findings are discussed in the process evaluation paper, which has been published elsewhere [64]. BODY.ANALYSES: Participants with missing data at either post-intervention [T1] or follow-up [T2] were not considered in the analyses (i.e., a complete case analysis). To describe the demographic characteristics of children at baseline [T0] and differences concerning primary (smoking intentions) and secondary outcomes (attitudes towards smoking and refusal self-efficacy), general descriptive analyses were conducted. Independent t-tests and chi-square tests were used to assess whether the primary and secondary outcomes differed between the study groups at baseline, and to assess differences between those participants included and excluded from the final analysis. Multilevel linear and logistic regression analyses examined intervention effects on the primary and secondary outcomes. To account for the clustering effect among children being nested in schools, a two-level data structure was conducted. Children were defined as the first level unit of analysis and schools the second level unit of analysis [65]. Two analyses were conducted for each of the outcome variables to examine the intervention effects. The first analysis determined the difference between the intervention and comparison group adjusting for baseline value of the outcome measure (‘crude’ analysis). The second analysis determined this effect when the covariates were added to the model (‘adjusted’ analysis); these covariates included age, ethnicity, deprivation level, mother/father/sibling/friend smoking, intentions to smoke and individual smoking-related cognitions, since these variables may influence each other [9, 5166] to avoid overestimation of effects and for ease of interpretation of results. Analyses were performed using IBM SPSS Statistics v.22 and MLwiN 2.30 software (Centre for Multi-level Modelling, University of Bristol, UK). Statistical significance was set at p < 0.05, and at p < 0.10 for the sex interaction term [66]. Child focus groups were transcribed verbatim, imported into NVivo 10 software, and subjected to thematic analysis [67]. This process involved reading and re-reading text and assigning broad thematic codes, some of which were pre-defined from topics covered in the group schedule. Subsequently, broad codes were collapsed into higher and lower order themes and descriptive and interpretive summaries were written based on recursive engagement with the data. A combination of inductive analysis and deductive techniques were used to generate codes. To aid the credibility and trustworthiness of the results, analyses and interpretations of the data were discussed amongst three members (CM, JT and LF) of the research team [67]. BODY.RESULTS: Figure 2 shows the flow of schools and participants through the trial. In total, 43 schools participated in the study (27.9 % response rate), including 32 (31 %) from Liverpool and 11 (22 %) from Knowsley. Schools that declined to participate provided diverse reasons for not taking part (e.g., too busy, key teacher on sick leave, already in receipt of external projects). Of the 1393 potentially eligible children at T0, 1143 completed baseline measures (92 % response rate); 961 children completed assessments at T0, T1, and T2 and were included in the final analyses (84 % participation rate). Participant retention ranged from 80 % (T0) to 79 % (T2) in the comparison group. The intervention group’s retention ranged from 83 % at baseline to 68 % at T2. However, the withdrawal of two intervention schools due to internal staffing issues excluded 68 children. Had the schools not withdrawn, assuming all children would have continued through the study, the retention at follow-up would have been 74 %. Compared with intervention children included in the analyses, a higher proportion of intervention children that were excluded from the analyses had a sibling that smoked (p < 0.01) and a lower proportion believed that smoking is bad for health (p < 0.05). Other baseline values did not differ between those included and excluded.Fig. 2Flow of schools and participants through the study. *Six children were absent at both post-intervention and follow-up Baseline characteristics for the final sample of child participants (mean age 9.6 ± 0.3 years, 50.4 % female, 98.3 % White British) are shown in Table 1. Over seven in ten (71.9 %) participating children lived within areas ranked within the highest 10 % for deprivation in England. The majority of children reported to have never smoked at T0 (97.5 %: comparison group, 96.3 %; intervention group, 97.9 %). CO readings were recorded for 82 % of participants and confirmed self-reported non-smoking status with all readings below 10 ppm (Mean = 1.3 ± 0.7 ppm). Over half of children (57.3 %) reported that at least one family member was a current smoker, whilst almost one in five children reported having a friend who smoked. Children generally had strong non-smoking intentions and high refusal self-efficacy expectations, though 40-58 % of children displayed more favourable attitudes towards smoking on five of the six attitude items. Children in the intervention group were less likely to be White British and were significantly more deprived (p < 0.01) than children in the comparison group. A higher proportion of intervention children, in particular girls, definitely believed that smoking was difficult to quit (p < 0.01) and that smoking affects sports performance (p < 0.05). No other significant group differences were found at T0.Table 1Sample characteristics of children at baseline ComparisonInterventionAll (n = 241) M ± SD or %Boys (n = 114) M ± SD or %Girls (n = 127) M ± SD or %All (n = 720) M ± SD or %Boys (n = 363) M ± SD or %Girls (n = 357) M ± SD or %Demographics  Age (years) 9.6 ± 0.3 9.6 ± 0.3 9.6 ± 0.3 9.6 ± 0.3 9.6 ± 0.3 9.6 ± 0.3  Ethnicity (White British) 98.3 99.1 97.6 82.1 82.6 81.5  Deprivation level (IMD) 50.9 ± 17.9 49.8 ± 17.7 51.9 ± 18.2 55.7 ± 16.4 55.5 ± 16.5 55.8 ± 16.3 Enjoyment of physical activity  PACES enjoyment scale (range 1–5) 4.3 ± 0.7 4.3 ± 0.7 4.3 ± 0.7 4.3 ± 0.7 4.2 ± 0.8 4.4 ± 0.6 Smoking intentions  Total non-smoking intentions (range 4–12) 11.7 ± 1.0 11.6 ± 1.1 11.7 ± 0.9 11.8 ± 0.8 11.7 ± 1.0 11.9 ± 0.5 Self-efficacy  Total refusal self-efficacy (range 3–15) 13.5 ± 3.2 13.5 ± 3.1 13.5 ± 3.3 13.7 ± 3.0 13.4 ± 3.3 14.0 ± 2.7 Attitudes towards smoking  Smoking is bad for health (‘definitely yes’) 88.8 87.7 89.8 90.1 86.2 94.1  Safe to smoke year or two (‘definitely not’) 59.8 60.5 59.1 64.4 63.6 65.3  Difficult to quit once started (‘definitely yes’) 43.2 45.6 40.9 52.8 51.2 54.3  Others smoke harmful to you (‘definitely yes’) 59.3 60.5 58.3 66.1 63.1 69.2  Affects sports performance (‘definitely yes’) 51.0 53.5 48.8 58.3 59.2 57.4  Makes you gain/lose weight (‘no difference’) 42.3 37.7 46.5 42.6 45.2 40.1 Social influences  Mother smoking 40.7 39.5 41.7 34.9 32.2 37.5  Father smoking 43.6 47.4 40.2 38.2 38.0 38.4  Sibling smoking 10.8 10.5 11.0 9.2 8.0 10.4  Friend smokinga 18.7 25.4 12.6 17.1 22.9 11.2 Notes: IMD, Indices of multiple deprivation score; aat least one friend smokes or tried Smoking behaviour data is reported for descriptive purposes. Self-reported smoking prevalence at T1 (comparison group, 97.5 %; intervention group, 98.6 %) and T2 (comparison group: 97.1 %, intervention group, 98.2 %) remained similar to T0, suggesting that low rates of smoking continued over time. BODY.INTERVENTION EFFECTS: BODY.NON-SMOKING INTENTIONS: The adjusted multilevel linear regression analyses indicated no significant intervention effects on non-smoking intentions between baseline and T1 (Table 2), and between baseline and T2 (Table 3).Table 2Multilevel analyses of the effectiveness of the SmokeFree Sports intervention between baseline (T0) and post-intervention (T1) Mean differencea (T1-T0: M ± SD or %)Crude modelb Adjusted modelc Outcome measureβ or RR (95 % CI)Pβ or RR (95 % CI)PSmoking intentions  Total non-smoking intentions I 0.03 ± 0.98 0.03d (−0.07, 0.13) 0.51 0.02d (−0.08, 0.12) 0.71 C 0.07 ± 0.95 Cigarette refusal self-efficacy  Total refusal self-efficacy I 0.29 ± 3.52 0.28d (−0.10, 0.67) 0.15 0.28d (−0.11, 0.67) 0.17 C 0.15 ± 3.77 Attitudes towards smoking  Smoking is bad for health (‘definitely yes’) I 3.8 1.03e (0.99, 1.06) 0.19 1.03e (0.99, 1.06) 0.15 C 2.5  Safe to smoke…year or two (‘definitely not’) I 3.1 1.23e(1.11, 1.36) <0.001* 1.19e (1.07, 1.33) 0.01 C −5.0  Difficult to quit once started (‘definitely yes’) I 15.4 1.58e (1.41, 1.78) <0.001* 1.56e (1.38, 1.76) <0.001* C −0.5  Others smoke is harmful to you (‘definitely yes’) I 2.4 1.19e (1.07, 1.31) <0.01* 1.19e (1.20, 2.08) <0.01* C −2.5  Affects sports performance (‘definitely yes’) I 30.3 1.70e (1.58, 1.85) <0.001* 1.73e (1.59, 1.88) <0.001* C 0.9  Makes you gain or lose weight (‘no difference’) I 26.3 2.11e (1.85, 2.41) <0.001* 2.13e (1.86, 2.44) <0.001* C −9.1 Notes: β = beta coefficient; RR = relative risk; CI = confidence interval, I = intervention group; C = comparison. Values reflect the intervention effects (i.e., between-group differences) between baseline and post-intervention *Significant intervention effect (P < 0.05) aUnadjusted within-group mean difference (post-intervention minus baseline) bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical activity and mother/father/sibling/friend smoking dβ value eRelative risk Table 3Multilevel analyses of the effectiveness of the SmokeFree Sports intervention between baseline (T0) and one year follow-up (T2) Mean differencea (T2-T0: M ± SD or %)Crude modelb Adjusted modelc Outcome measureβ or RR (95 % CI)Pβ or RR (95 % CI)PSmoking intentions  Total non-smoking intentions I 0.06 ± 0.97 0.08d (−0.02,0.18) 0.13 0.08d (−0.02,0.17) 0.14 C 0.06 ± 1.18 Cigarette refusal self-efficacy  Total refusal self-efficacy I 0.56 ± 3.23 0.27d (−0.02,0.56) 0.07 0.23d (−0.07,0.52) 0.13 C 0.45 ± 3.73 Attitudes towards smoking  Smoking is bad for health (‘definitely yes’) I 5.5 0.98e (0.96,1.01) 0.28 0.99e (0.97,1.01) 0.42 C 8.3  Safe to smoke…year or two (‘definitely not’) I −0.6 1.05e (0.95,1.16) 0.42 1.03e (0.93,1.15) 0.65 C 0.4  Difficult to quit once started (‘definitely yes’) I 5.8 1.15e (1.02,1.29) 0.05 1.15e (1.02,1.3) 0.06 C 6.6  Others smoke is harmful to you (‘definitely yes’) I 0.0 1.14e (1.08,1.81) 0.03 1.13e (1.05,1.79) 0.05 C −2.0  Affects sports performance (‘definitely yes’) I 23.2 1.55e (1.42,1.69) <0.001* 1.57e (1.43,1.72) <0.001* C 1.3  Makes you gain or lose weight (‘no difference’) I −2.0 1.05e (0.90,1.22) 0.60 0.98e (0.83,1.16) 0.84 C −3.7 Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Values reflect the intervention effects (i.e., between-group differences) between baseline and post-intervention *Significant intervention effect (P < 0.05) aUnadjusted within-group mean difference (one year follow-up minus baseline) bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical activity and mother/father/sibling/friend smoking dβ value eRelative risk BODY.CIGARETTE REFUSAL SELF-EFFICACY: The adjusted multilevel linear regression analyses showed no significant intervention effects between baseline and T1 (Table 2), and between baseline and T2 (Table 3), for refusal self-efficacy. BODY.ATTITUDES TOWARDS SMOKING: The adjusted multilevel logistic regression analyses indicated small positive intervention effects between baseline and T1 (Table 2) for four attitude towards smoking items. At T1, compared with children in the comparison group, children that participated in the SFS intervention were more likely to ‘definitely’ believe that: ‘it is not safe to smoke for a year or two as long as you quit after that’ (RR = 1.19, 95 % CI 1.07 to 1.33, p < 0.001), ‘it is difficult to quit smoking once started’ (RR = 1.56, 95 % 1.38 to 1.76, p < 0.001), ‘smoke from other peoples’ cigarettes is harmful to you’ (RR = 1.19, 95 % CI 1.20 to 2.08, p < 0.001), and ‘smoking affects sports performance’ (RR = 1.73, 95 % CI 1.59 to 1.88, p < 0.001). In addition, a moderate positive intervention effect was observed on the attitude item: ‘smoking makes ‘no difference’ to weight’ (RR = 2.13, 95 % CI 1.86 to 2.44, p < 0.001). Between baseline and T2 (Table 3), significant between-group differences remained on only one of the six attitude items. Compared with children in the comparison group, children who received the SFS intervention were more likely to ‘definitely’ believe that ‘smoking affects sports performance’ (RR = 1.57, 95 % CI, 1.43, 1.72, p < 0.001), a small positive intervention effect. BODY.SEX INTERACTION EFFECTS: Tables 4 and 5 show the results of the sex interaction analyses between baseline and T1, and between baseline and T2, respectively. Between baseline and T1, sex moderated the intervention effects for cigarette refusal self-efficacy; a significant small positive intervention effect was found in girls (β = 0.72, 95 % CI 0.21 to 1.23, p < 0.01) but not boys (β = -0.18, 95 % CI -0.77 to 0.41, p = 0.54). No other sex interactions were observed.Table 4Multilevel analyses exploring interaction effects by sex between baseline (T0) and post-intervention (T1) Mean differencea (T1-T0: M ± SD or %)Intervention* sexb (crude model)Boysc Girlsc Outcome measureBoysGirlsβ or RR (95 % CI)Pβ or RR (95 % CI)Pβ or RR (95 % CI)PSmoking intentions  Total non-smoking intentions I 0.10 ± 1.2 −0.04 ± 0.7 0.10d (−0.10, 0.30) 0.32 n/a − n/a - C 0.16 ± 1.0 0.00 ± 0.9 Cigarette refusal self-efficacy  Total refusal self-efficacy I 0.32 ± 3.9 0.25 ± 3.2 0.87d (0.09, 1.64) 0.03* −0.18d (−0.77, 0.41) 0.54 0.72d (0.21, 1.23) <0.01* C 0.39 ± 3.6 −0.07 ± 3.8 Attitudes towards smoking  Smoking is bad for health (‘definitely yes’) I 5.5 2.0 1.05e (1.00, 1.11) 0.12 n/a - n/a - C 4.4 0.8  Safe to smoke…year or two (‘definitely not’) I 0.9 5.3 1.16e (0.95, 1.42) 0.23 n/a - n/a - C −4.4 −5.6  Difficult to quit once started (‘definitely yes’) I 17.4 13.5 1.19e (0.90, 1.57) 0.30 n/a - n/a - C 2.6 −3.1  Others smoke harmful to you (‘definitely yes’) I 3.3 1.4 1.14e (0.84, 2.23) 0.29 n/a - n/a - C −0.9 −4.0  Affects sports performance (‘definitely yes’) I 30.3 30.3 1.16e (0.90, 1.50) 0.34 n/a - n/a - C 5.3 −3.1  Makes you gain or lose weight (‘no difference’) I 23.4 29.1 1.21e (0.79, 2.31) 0.36 n/a - n/a - C −2.6 −15.0 Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Where crude analyses were significant, adjusted analyses (i.e., interaction term included in adjusted multilevel models) were conducted with results for each sex shown *Significant intervention effect (P < 0.05) aUnadjusted within-group mean difference (post-intervention minus baseline) bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical activity and mother/father/sibling/friend smoking dβ value eRelative risk Table 5Multilevel analyses exploring interaction effects by sex between baseline (T0) and one year follow-up (T2) Mean differencea (T2-T0: M ± SD or %)Intervention* sexb (crude model)Boysc Girlsc Outcome measureBoysGirlsβ or RR (95 % CI)Pβ or RR (95 % CI)Pβ or RR (95 % CI)PSmoking intentions  Total non-smoking intentions I 0.10 ± 1.2 0.00 ± 0.7 −0.01d (−0.21, 0.19) 0.89 n/a - n/a - C 0.06 ± 1.4 0.06 ± 0.9 Cigarette refusal self-efficacy  Total refusal self-efficacy I 0.84 ± 3.4 0.27 ± 3.0 −0.20d(−0.78, 0.39) 0.51 n/a - n/a - C 0.37 ± 3.8 0.53 ± 3.7 Attitudes towards smoking  Smoking is bad for health (‘definitely yes’) I 8.3 2.5 1.02e (1.00, 1.04) 0.17 n/a - n/a - C 10.5 6.3  Safe to smoke…year or two (‘definitely not’) I −0.5 −0.9 0.94e (0.76, 1.17) 0.64 n/a - n/a - C −2.6 3.1  Difficult to quit once started (‘definitely yes’) I 7.8 4.0 1.03e (0.81, 1.31) 0.84 n/a - n/a - C 6.2 7.1  Others smoke harmful to you (‘definitely yes’) I 1.6 −1.7 1.11e (0.77, 2.15) 0.41 n/a - n/a - C −0.9 −3.2  Affects sports performance (‘definitely yes’) I 25.1 21.3 1.05e (0.83, 1.32) 0.73 n/a - n/a - C 5.3 −2.3  Makes you gain or lose weight (‘no difference’) I −2.5 −1.7 0.99e (0.26, 3.70) 0.99 n/a - n/a - C 1.8 −8.7 Notes: β = beta coefficient; RR = relative risk; CI = confidence interval; I = intervention group; C = comparison. Where crude analyses were significant, adjusted analyses (i.e., interaction term included in adjusted multilevel models) were conducted with results for each sex shown aUnadjusted within-group mean difference (one year follow-up minus baseline) bAdjusted for group and baseline value of the outcome measure cAdditionally adjusted for school and deprivation level, sex, age, ethnicity, other individual smoking-related cognitions at baseline, enjoyment of physical activity and mother/father/sibling/friend smoking dβ value eRelative risk BODY.QUALITATIVE FINDINGS: During focus groups the majority of children articulated that SFS made them more determined not to smoke in the future. Children’s reasons for not smoking surrounded some of the key messages received and or activities played during the intervention (see Table 6 for a summary of children’s reasons for their non-smoking intentions). During group discussions some children expressed a level of uncertainty regarding their future smoking behaviour and verbalised that they may smoke in the future because of social norms, and or using smoking as a coping mechanism for managing stress:“ “I’m not saying I definitely won’t [smoke] because it’s just something that might happen if something stressful happens” (Boy, School 2). Table 6Children’s reasons for their non-smoking intentions Health messagesQuotes Health implications of smoking “I won’t [smoke] because if you smoke you will damage your heart and if you don’t smoke you can live a long and healthy life” (Girl, School 7). “There’s a reason why I wouldn't smoke because your lungs wouldn't be in the best condition” (Boy, School 10). Impact on sports performance “I wouldn’t [smoke] because It’s harder to breathe and harder to do exercise” (Boy, School 9). “Because I like doing lots of sports and if I smoked in the future it would be difficult to do [sports]” (Boy, School 8). Cigarette contents and addiction “Because I’ve learnt [in SFS] how many chemicals are in a cigarette and what goes into them, that’s why I wouldn’t smoke” (Boy, School 8). I’m very confident that I’m not going to smoke cos they’ve [SFS] told us how bad it [smoking] is and there are over four thousand chemicals [in a cigarette] and it can be hard to quit” (Girl, School 1). Financial costs of smoking “I wouldn’t [smoke] because it costs you loads of money”(Girl, School 5). During group discussions children were able to recall the health messages delivered during the intervention, particularly in relation to the health implications associated with smoking, its impact on sport performance, the chemical properties in a cigarette and its addictive nature, and its impact on weight (see Table 7).Table 7Children’s’ understanding of the health messages received during the intervention Health messagesQuotes Health implications of smoking “The [SFS] games show you the damage that [smoking] does to your arteries and lungs” (Boy, School 3). “A smoker would get more phlegm and a non-smoker would get less phlegm” (Boy, School 4, Gp 1). Impact on sports performance “Your heart beats faster when you’re doing exercise when you’re a smoker” (Boy, School 5). “If you smoked you wouldn’t be able to run as long or play as long as other [non-smoking] people” (Boy, School 3). Cigarette contents and addiction “There’s over 4,000 chemicals in a cigarette and they’re not nice, rat poison, nicotine, rocket fuel” (Girl, School 6). “I wouldn’t [smoke] because it’s addictive and you won’t be able to stop cos of the nicotine” (Girl, School 5). Impact on weight “People think when you smoke you lose weight but you really don’t” (Boy, School 5). “It’s healthier not to smoke and there’s no difference in your weight, you’re just better off not smoking”(Boy, School 6). BODY.DISCUSSION: This study examined the short and medium term effects of a sport-for-health intervention (SFS) on 9-10 year old children’s intentions (not) to smoke and smoking-related cognitions (attitudes and refusal self-efficacy) using a controlled trial. The SFS intervention had no effect on children’s ratings of non-smoking intentions, though qualitative data suggested that participation in SFS made children more determined not to smoke. In addition, a small positive short term effect was found for refusal self-efficacy among girls in the intervention group. Participation in SFS also increased the likelihood of having negative attitudes towards smoking immediately after the intervention, with children stating that the intervention reinforced non-smoking opinions, though limited effects were found one year after the intervention. Smoking intentions are precursors to and predictive of smoking initiation in youth [8, 68]. Quantitative data indicated that the SFS intervention did not significantly impact on children’s smoking intentions; children in both the intervention and comparison group reported a strong intention not to smoke in the future and thus a ceiling effect limited our ability to detect between-group differences. These findings are comparable to other smoking prevention programmes targeting primary school aged children [19, 69], but are inconsistent with two school-based interventions that reported a positive effect on intentions to smoke among elementary school children from the USA [70–72]. A further study examined the immediate and long term effects of a smoking education programme implemented in Dutch elementary schools [73]. The study reported no short term effects on intention to smoke during elementary school. However, when children were followed up at secondary school one year after the intervention, children who received the intervention had significantly higher non-smoking intentions and smoked less than the control group [73]. The above mentioned studies might suggest that smoking prevention interventions may be more effective if implemented in secondary school, as children may be more likely to have developed intentions to smoke if they are closer to the actual age of smoking onset (i.e., age 14-16 years [19, 73]. Nevertheless, given that qualitative data suggested that SFS had strengthened children’s non-smoking intentions, a longer term follow-up study is warranted to investigate whether implementing SFS during primary school is effective at reducing smoking behaviour and smoking intentions in adolescence, following the transition to secondary school. Children’s intentions to smoke can be shaped by their attitudes towards smoking and their self-efficacy expectations [9, 10, 74, 75]. Smoking-related knowledge and attitudes are frequently measured and have a propensity to increase following smoking prevention interventions [72, 76–78]. Consistent with these studies, children who participated in the SFS intervention were more likely to develop negative attitudes towards smoking immediately following the intervention than children in the comparison group. Focus group data with children supported these findings and revealed that the SFS games and smoke free messages positively influenced children. Given that many preadolescent children living in socially deprived communities display pro-smoking attitudes [14], these findings are encouraging and suggest that SFS could therefore provide a mechanism for health education to dispel myths that exist among children around smoking harms and challenges. However, it is also worth noting that the majority of the positive intervention effects on attitudes had diminished one year after the intervention. Though not directly comparable due to methodological differences, these results are in accord with Crone and colleagues [72], who also noted a number of short term positive between-group effects on attitudes towards smoking that had reduced by long term follow-up. It is possible, therefore, that additional ‘booster’ sessions may be necessary to sustain attitude changes in preadolescent children, particularly those residing in deprived communities. However, the evidence on the effectiveness of booster sessions is limited and inconsistent [79]. The likelihood of starting to smoke increases in adolescence [80, 81] and so enhancing skills to resist social pressures to smoke is important for smoking prevention [82]. Whilst a small positive effect was observed on cigarette refusal self-efficacy among girls in the intervention group at post-intervention, no group differences were apparent at one-year follow-up. It is possible that short term intervention effects on refusal self-efficacy were not found in boys because efficacy levels increased from baseline to post-intervention among boys in both the intervention and comparison groups. It is also possible that short term effects on girls’ refusal self-efficacy were not maintained at one-year follow-up because girls’ in the comparison groups ratings increased and they appeared to ‘catch up’. These mixed findings likely reflect that self-efficacy is not a static concept [83] and levels of self-efficacy fluctuate over time [80]. The findings are similar to those reported by Isensee et al. [78], who also noted a lack of medium term effects and documented increases in refusal self-efficacy among control group participants. Further, the absence of intervention effect could again be attributed to a ceiling effect; children in the intervention and comparison groups both reported high refusal self-efficacy, reducing the power to detect noticeable effects. It is also worth noting that most children in the current study did not have friends who smoke, and so have yet to be put to the test of resisting social influences to smoke. Given that self-efficacy is subject to change over time, it has been recommended that smoking prevention programmes are implemented annually in preadolescence and throughout adolescence until the completion of secondary school [35, 77, 80]. Long-term research is required to determine if the SFS primary school smoking prevention intervention can facilitate children in making a rational and logical decision not to smoke during a period when smoking is more age-related and considered as accepted behaviour [84]. To the authors knowledge SFS was the first sport-for-health intervention to engage children in smoking prevention. A recently published process evaluation of the intervention suggests that this unique approach was well-received by children, and was considered acceptable to coaches and teachers as intervention deliverers [64]. However, there were variations in intervention fidelity and teachers’ implementation of intervention activities that may have reduced the potency of the intervention and the ability to sustain short term effects one year after the intervention [64]. Nevertheless, the limited intervention effects are more likely attributed to children at this age having strong intentions not smoke in the future, though important lessons have been learned that can inform the design of a randomised controlled trial [64]. The present study has several strengths. First, in accordance with MRC guidance for the development of complex interventions, SFS was designed following extensive formative work, school and community feasibility studies [29, 41, 42]. Second, this study adopted a mixed-methodology approach consistency with the Standard Evaluation Framework for physical activity interventions [85]. Third, this study followed children one year after the end of the intervention. Fourth, the study had a large sample size and reasonably low attrition rates were observed. Fifth, process evaluation measures were used to explore the implementation of SFS [64]. Finally, to the date, the results of this study provide the first globally published evidence for the effectiveness of a large scale school-based sport-for-health smoking prevention intervention. Several limitations should be acknowledged. First, of the 154 schools approached, only 43 agreed to participate. Previous research has shown that some parents and school officials may be concerned that exposing preadolescent children to smoking prevention programmes may stimulate their interest and curiosity about smoking [86]. However, the primary reason given by schools for non-participation was limited time, which might be related to the fact that smoking prevention is not mandatory in Key Stage 2 of the UK National Curriculum [53]. Further, it was encouraging to note that participation in SFS did not increase rates of smoking initiation. Second, the reliance on self-report in the assessment of outcome variables carries a risk of measurement error due to inaccurate recall, literacy issues and social desirability bias [82, 87]. However, self-reports have been demonstrated to be accurate provided confidentially is assured [88]. Third, it was not possible to blind study participants or the research team to the intervention because of the practical nature of the intervention. Fourth, primary and secondary outcomes focused on intentions to smoke and smoking-related cognitions, respectively, which may or may not result in smoking initiation at a later age [13]. Fifth, given that the majority of children were White British and from one of the most deprived local authorities in England, these results may not generalise to other racial and socio-economic child populations. Sixth, focus groups were only conducted with children from intervention schools to inform interpretations of intervention effectiveness and therefore it is unknown if children from comparison schools would have conveyed similar perspectives. Finally, the study did not include a cost-effectiveness evaluation, thus it is unknown whether SFS is a cost-effective smoking prevention initiative. BODY.CONCLUSIONS: In summary, the results of this study indicate that SFS was effective at changing attitudes towards smoking, and increasing the level of individual self-efficacy to refuse cigarettes among girls immediately post-intervention. Although no quantitative intervention effects were observed for non-smoking intentions, children articulated that SFS made them more determined not to smoke. Overall, these findings may suggest that sport-for-health interventions offer a promising strategy for smoking prevention efforts, though a long term follow-up study is needed to determine whether the SFS intervention is effective at preventing smoking in secondary school. Moreover, further evidence is needed from randomised controlled-trials. The fact that almost all children had not developed an intention to smoke might indicate that smoking prevention programmes should target early adolescents (aged 11-13 years), who are closer to the actual age of smoking onset.

TITLE: Minimally access versus conventional hydrocelectomy: a randomized trial ABSTRACT.ABSTRACT: ABSTRACT.OBJECTIVE:: To compare our previously published new minimally access hydrocelectomy versus Jaboulay's procedure regarding operative outcome and patient's satisfaction. ABSTRACT.MATERIALS AND METHODS:: A total of 124 adult patients were divided into two groups: A and B. Group A patients were subjected to conventional surgical hydrocelectomy (Jaboulay's procedure) and group B patients were subjected to the new minimal access hydrocelectomy. The primary endpoint of the study was recurrence defined as a clinically detectable characteristic swelling in the scrotum and diagnosed by the two surgeons and confirmed by ultrasound imaging study. The secondary endpoints were postoperative hematoma, wound sepsis and persistent edema and hardening. ABSTRACT.RESULTS:: The mean operative time in group B was 15.1±4.24 minutes and in group A was 32.5±4.76 minutes (P≤0.02). The mean time to return to work was 8.5±2.1 (7–10) days in group B while in group A was 12.5±3.53 (10–15) days (P=0.0001). The overall complication rate in group B was 12.88% and in group A was 37%. The parameters of the study were postoperative hematoma, degree of scrotal edema, wound infection, patients’ satisfaction and recurrence. ABSTRACT.CONCLUSION:: Hydrocelectomy is considered the gold standard technique for the treatment of hydrocele and the minimally access maneuvers provide the best operative outcomes regarding scrotal edema and hardening and patient's satisfaction when compared to conventional eversion-excision hydrocelectomies. BODY.INTRODUCTION: Hydrocele is the most common benign scrotal swelling with estimated incidence as one percent of the adult male population (1). A controversy exists about the treatment of primary vaginal hydrocele. Aspiration and sclerotherapy have been described; however hydrocelectomy remains the treatment of choice for the management of hydroceles (2). Aspiration and sclerotherapy with doxycycline seems as effective and safe as nonsurgical treatment option for hydrocele where the success rate of a single hydrocele aspiration and sclerotherapy procedure is claimed to have the same success rates involving hydrocelectomy while avoiding the hospital expense and many other complications (3); other studies reported lower success rate and less patient's satisfaction than hydrocelectomy (4, 5). Hydrocelectomy through eversion procedures for hydrocele may cause postoperative discomfort, temporary limitation of normal activities and complications, such as hematoma, infection, persistent swelling, chronic pain and decreased fertility (6, 7). The author in the present study compared his previously published new minimally access hydrocelectomy (8) versus Jaboulay's procedure (9). BODY.OBJECTIVES: The aim of this prospective randomized study was to compare the author's previously published new minimally access hydrocelectomy versus Jaboulay's eversion procedure in adult patients regarding operative outcome and patient's satisfaction. BODY.PATIENTS AND METHODS: BODY.PATIENTS: A total of 124 adult patients, aged 18-56 years within the period April 2006 to October 2011, with diagnosis of hydrocele were enrolled to this prospective randomized study and divided into two equal groups A and B. Group A (N=62) patients were submitted to conventional surgical hydrocelectomy (Jaboulay's procedure) while group B patients (N=62) were submitted to the new minimal access hydrocelectomy. All patients were subjected to either conventional eversion or the new minimal access hydrocelectomy as an ambulatory procedure with general anesthesia or spinal analgesia. The cohort of our patients represents adult males working in Port-Said free industrial zone, Port-Said, Egypt. Patients came from more than 4 governorates; Port-Said, Ismailia, Sharkya and Kafr el-Sheikh. Written consents were obtained from all patients before the study. The steps of both operative interferences were explained to all patients. The local ethics committee had approved all operative procedures. Ethical approval for this study was granted by the ethical review committee under supervision of the general director of Port-Fouad general hospital, Port-Fouad, Port-Said, Egypt. BODY.SAMPLE SIZE: In general, the overall complications rate of conventional surgical hydrocelectomy in previous studies is about 40% (5) and those of minimally access hydrocelectomy is about 14% (8). Calculation of the sample size included the number of participants to be recruited for the study using the mathematical equation. The authors used these two equations to calculate the minimum number required to reliably answer the research question. Using the first equation (10), the number, N=62 patients for each group, was given by: where z (1-a/2) and z (1-β) represent percentage points of the normal distribution for statistical significance level (ά) at 0.05 value is 1.96 and power (1-β) with accepted 95% positive rate is 1.6449, where β, the false-negative rate. Δ represents the standardized difference (i.e. the treatment difference divided by its standard deviation): Standardized difference P¹ represents the overall complications of conventional surgical hydrocelectomy in previous studies=40% (5). P² represents the overall complications of minimally access hydrocelectomy reported in previous studies=17% (8). BODY.RANDOMIZATION: Randomization was performed prior to study commencement as follows: Opaque envelopes were numbered sequentially from 1 to 124. A computer-generated Table of random numbers was used for group assignment; if the last digit of the random number was from 0 to 4, assignment was to Group A (conventional surgical hydrocelectomy), and if the last digit was from 5 to 9, assignment was to Group B (minimally access hydrocelectomy). The assignments were then placed into the opaque envelopes and the envelopes sealed. As eligible participants were entered into the trial, these envelopes were opened in sequential order to give each patient his random group assignment. The envelopes were opened by the operating surgeon after patient consent and just prior to the surgery (10). BODY.PREOPERATIVE WORKUP: The detailed history and full physical examination of each patient were assessed. The diagnosis was confirmed by fluctuation and trans-illumination. Laboratory investigations like hemoglobin, white blood cell count and urine routine examination were done in all. Scrotal ultrasound imaging was done in all patients. BODY.THE SURGICAL TECHNIQUES: Cephradine 1gm IV at the time of induction of anesthesia or just after the administration of spinal anesthesia was given followed by another dose 2 h postoperatively. Patients were seen in the second day and examined for scrotal edema and hematoma. In all of group B patients, drains were removed in the second day while in those of group A drains were removed in the third day. All the excised tissues were sent for pathological examination to rule out any epididymal or vasal structures in the specimen. Jaboulay's procedure: The testis was delivered through an incision in the scrotum, the tunica was opened and everted and most of the hydrocele sac was resected with electrocautery, leaving a reasonable cuff along the borders of the testicle. Bleeding was controlled by a running suture closing the free edges of the hydrocele sac and hemostasis was secured by the aid of electrocautery. Standard two-layer closure was used to close the scrotum with small tube drain (9). The new minimally access hydrocelectomy: A small scrotal incision 2cm long was done and incision of the Dartos muscles in the same line was done with electrocautery (Figures 1A and B). The parietal tunica vaginalis (PTV) was grasped and minimal blunt dissection was made by the aid of the index finger and a small hole was made for aspiration of hydrocele fluid (Figure-2A). Then a disc of tissue was excised of the PTV about double of the skin incision dimension using electrocautery. The edge of the visceral surface tunica vaginalis was sutured to the parietal layer of the tunica vaginalis and then to the Dartos (Figure-2B) and all were sutured to scrotal skin in an everted manner aiming to expose the visceral tunica toward scrotal skin (Figures 3A and B). If the visceral surface of the tunica vaginalis is sutured to the Dartos, eversion will be created. Then when this everted structure is sutured to the scrotal skin, it put in contact the sac with lymph-rich subcutaneous tissues. A drain was left in place and discharge was allowed at the same day (8). Figure 1ABODY.AN OPERATIVE PHOTOGRAPH SHOWING THE LENGTH OF THE SCROTAL SKIN INCISION, 2CM (IT APPEARS LONGER DUE TO STRETCH OF THE SKIN BY THE ASSISTANT).: Figure 1BBODY.AN OPERATIVE PHOTOGRAPH SHOWING DELIVERY OF THE HYDROCELE SAC THROUGH THE SMALL SCROTAL SKIN.: Figure 2ABODY.AN OPERATIVE PHOTOGRAPH SHOWING EVACUATION OF HYDROCELE FLUID THROUGH A SMALL HOLE MADE IN THE TUNICA VAGINALIS.: Figure 2BBODY.AN OPERATIVE PHOTOGRAPH SHOWING IN SITU EXCISION OF THE HYDROCELE SAC USING ELECTROCAUTERY.: Figure 3ABODY.AN OPERATIVE PHOTOGRAPH SHOWING THE BEGINNING OF THE EVERSION TECHNIQUE. BLUE ARROW POINTS TO THE VISCERAL TUNICA VAGINALIS WHILE THE BLACK ONE POINTS TO THE PARIETAL TUNICA.: Figure 3BBODY.AN OPERATIVE PHOTOGRAPH SHOWING COMPLETED EVERSION TECHNIQUE BY SUTURING OF THE EDGE OF THE TUNICA TO THE DARTOS AND SCROTAL SKIN IN AN EVERTED MANNER AIMING TO EXPOSE THE VISCERAL TUNICA TOWARD SCROTAL SKIN.: BODY.END POINTS: The primary endpoint of the study was recurrence defined as a clinically detectable characteristic swelling in the scrotum and diagnosed by the two surgeons and confirmed by ultrasound imaging study. The secondary endpoints were postoperative hematoma, wound sepsis and persistent edema and hardening. BODY.STATISTICAL ANALYSIS: Data were entered and analyzed using SPSS (Statistical Package for Social Sciences) software program version 15.0 for analysis. Values were expressed as means±standard errors of deviation. Student t test was used to compare categorical variables. P value set at <0.05 for significant results. BODY.RESULTS: There was no statistical significant difference between the two groups regarding age, body mass index, duration of symptoms and size of hydroceles. Age ranged between 18–56 years with a mean age of 37±11.4 years. Follow-up included patients’ complaint, if any, clinical examination and ultrasonography. The maximum follow-up period was 96 months and the minimum was 28 months with a mean value of 59.88±24.22 months. None of our patients missed their follow-up program because of the obligatory visit designed by the health organization of the company they work for. The operative time in group B ranged between 12-18 minutes and the mean was 15.1±4.24 minutes and in group A ranged between 25-40 minutes with mean value 32.5±4.76 minutes with significant distribution (P≤0.02). The mean time of hospital stay for group B was 13.48±6.38 hours with 10 hours as a minimum and 30 hours as a maximum value, while in group A was 21.19±11.65 hours with 12 hours as a minimum and 48 hours as a maximum value but this distribution was not significant (P≥0.05). Time off from work was defined as the number of days between the day of surgery and the first day a patient returned to work (10). The mean time to return to work was 8.5±2.1 (7–10) days in group B while in group A was 12.5±3.53 (10–15) days. The mean time off from work in group B was 9±2.35 days and in group A was 13.5±4. (P=0.0001).(Table-1). Table 1BODY.MEAN OPERATIVE TIME, HOSPITAL STAY AND TIME OFF FROM WORK IN BOTH GROUPS.: ItemGroup AGroup BP valueOperative time (minutes) 32.5±4.76 15.1±4.24 ≤0.02 Hospital stay (hours) 21.19±11.65 13.48±6.38 ≥0.05 Time off from work (days) 13.5±4. 9±2.35 =0.0001 We relied on the previously reported data regarding our new minimally invasive hydrocelectomy (8) for the common postoperative findings: postoperative hematoma, degree of scrotal edema, wound infection, patients’ satisfaction and recurrence. The overall complication rate in group B was 12.88% and in group A was 37%. Postoperative hematoma was not observed in any of our patients in group B while mild hematoma treated by conservative measures was detected in three patients in group A (4.8%). Mild and moderate scrotal edema usually subsided within a few days postoperatively (7) while scrotal edema and hardening was considered when pain and swelling interfered with daily activities (11). The present study showed that scrotal edema was inevitable as we observed that mild and moderate scrotal edema and hardening occurred in all patients of both groups with varying proportions. In group A, scrotal edema and hardening represented the higher incidence while mild degree was the least form of scrotal edema. In contrast, in group B mild and moderate degrees formed the majority of patients and scrotal edema and hardening occurred only in three patients (P≤0.05). Persistent edema and hardening were confined to the ipsilateral hemiscrotum and required additional bed rest and anti-inflammatory agents. Mild to moderate cellulitis was seen in four patients in both groups A and B (6.45%). All our patients of group B were completely satisfied with this new minimally invasive procedure by the end of second postoperative week and all over the follow-up periods and only three patients (4.83%) were un-satisfied due to scrotal hardening while in group A, scrotal edema and hardening was observed in 24.2%. Disease recurrence was confirmed by two treating surgeons and by the aid of US study. There was disease recurrence in one patient (1.6%) in both groups A and B (Table-2). Table 2BODY.OVERALL COMPLICATION RATE AND PATIENT'S SATISFACTION IN BOTH GROUPS.: ItemGroup AGroup BP valueOverall complications 37% 12.88% P≤0.05 Postoperative hematoma 3 (4.8%). – NS Edema & hardening 15 (24.2%) 3 (4.8%). P≤0.05 Wound sepsis 4 (6.45%) 4 (6.45%) NS Patient's satisfaction 95.2% 75.8% P≤0.05 Recurrence 1 (1.6%) 1 (1.6%) NS NS = nonsignificant. BODY.DISCUSSION: Hydrocelectomy is considered the gold standard technique for the treatment of hydrocele; aspiration and sclerotherapy have fewer complications and the success rate and patient's satisfaction are inferior to hydrocelectomy (8). Jaboulay's procedure for hydrocelectomy has satisfactory rate of success but with less patient satisfaction due to postoperative scrotal hardening (6, 9, 12). Minimally access hydrocelectomy was performed through fenestration of the tunica (8) and pull-through technique to remove large hydrocele sacs through a small incision and with minimal dissection (7). Our new minimally access technique of hydrocelectomy was previously published (8). The hydrocele sac is treated through smaller scrotal skin incision together with excision of smaller disc of the hydrocele sac. The mean operative time in our study was longer in group A than group B with statistical significant distribution because much time was lost in partial excision of the sac as well as to achieve haemostasis (6, 9, 12). Also, the time off from work was longer for patients of group A than group B with statistical significant distribution and this came in concordance with published data of same interest (4, 13). The overall complication rate in group B was lower than in group A and came in agreement with other reports of previous studies of same interest (5–8). The overall incidence of postoperative complications is significantly lower among patients with less operative trauma (14). In less invasive techniques (7, 8) as well as our minimally invasive maneuver, the overall complications rate was inferior to those in eversion and excision hydrocelectomy (5, 6, 15). Postoperative haematoma and scrotal edema and hardening were observed with higher incidence in group A patients where there was more tissue dissection than those in group B. Excision––eversion technique invites edema and hematoma due excessive handling and wide dissection of the hydrocele sac (5–7). In the current technique a disc of the hydrocele sac is pulled and resected through a small scrotal incision with minimal dissection. So, the hematoma formation was not seen in group B patients as well as patients of the in situ techniques (6) while in conventional hydrocelectomy, the hematoma formation may reach up to 3.3% (4, 14). In scrotal surgery, significant postoperative infection occurred in patients subjected to more operative trauma and ranged from superficial surgical site infection, scrotal abscess formation and pyocele (5, 7, 8) with a rate of incidence between 5-14% (16). In the present study, superficial surgical site infection confined to the scrotal skin as mild to moderate cellulitis was detected in four patients in both groups that needed additional course of Cephradine orally (1gm 12/12hs). Regarding recurrence, there was disease recurrence in one patient (1.6%) in both groups A and B, while others reported 1.3-7% recurrence (8, 17). The assessment of the patient's satisfaction with the treatment procedure depends on physician's instructions for postoperative period, the postoperative outcome, the follow-up period and the success rate (18). Regarding the success rate, it was reported that the level of satisfaction was superior with hydrocelectomy when compared with sclerotherapy due to fewer incidence of recurrence (4). The most common complications following scrotal surgery for hydrocele are persistent scrotal swelling and hardening (6) while with minimally access procedures (7, 8) as in the present study, the scrotal swelling and hardening are much less than the eversion-excision hydrocelectomy (6–8, 14) A total of 95.17% of our patients of group B were completely satisfied with this new minimally invasive procedure by the end of the second postoperative week and all over the follow-up periods and only three patients (4.83%) were unsatisfied due to scrotal hardening. In group A, 24.2% of patients were unsatisfied. BODY.CONCLUSIONS: Hydrocelectomy is considered the gold standard technique for the treatment of hydrocele and the minimally access maneuvers provide the best operative outcome regarding scrotal edema and hardening and patient's satisfaction when compared to conventional eversion-excision hydrocelectomies.

TITLE: Effects of intravenous immunoglobulin therapy in Japanese patients with polymyositis and dermatomyositis resistant to corticosteroids: a randomized double-blind placebo-controlled trial High-dose intravenous immunoglobulin (IVIG) therapy has been effective in treating various autoimmune and systemic inflammatory diseases. Here, we assessed the efficacy and safety of IVIG therapy with polyethylene glycol-treated human IgG (drug code GB-0998) for patients with corticosteroid-refractory polymyositis (PM) and dermatomyositis (DM) by means of a randomized, double-blind, placebo-controlled study. We randomly assigned 26 subjects (16 PM and 10 DM) to receive either GB-0998 or placebo. Intragroup comparison in the GB-0998 group showed statistically significant improvements due to GB-0998 administration in the primary endpoint (manual muscle test score) and secondary endpoints (serum creatine kinase level and activities of daily living score). However, significant improvements were also found in the placebo group, and comparison of the GB-0998 group with the placebo group did not show any significant difference between the groups. We discuss possible reasons for the absence of a clear intergroup difference in efficacy. Nineteen adverse drug reactions were observed in 11 of 26 subjects (42.3%), of which 2 events (decreased muscle strength and increased serum creatine kinase) were assessed as serious; however, they are previously known events. These results indicate that GB-0998 can be safely used with the same precautions as other current IVIG therapy. BODY.INTRODUCTION: Polymyositis (PM) and dermatomyositis (DM) are diffuse inflammatory myopathies of unknown etiology, causing decreased strength in the limb proximal group muscles, cervical muscles, and pharyngeal muscles, and have been designated as targets for disease treatment research by the Japanese Ministry of Health, Labor, and Welfare. Oral corticosteroids are the first-line treatment for patients with PM and DM, and they produce good response in approximately 80% of patients [1]. For patients showing either no response or incomplete response, other therapies such as methylprednisolone pulse therapy and/or concomitant immunosuppressive drug therapy are empirically used, but are not always effective [2]. In addition, long-term and/or high-dose administration of corticosteroids and/or immunosuppressive drugs may cause severe side-effects, such as infection, and therefore new therapies are required. Here, we present the results of a double-blind, placebo-controlled, crossover study designed to evaluate the efficacy and safety of polyethylene glycol-treated human IgG (GB-0998) in patients with corticosteroid-resistant PM or DM. BODY.PATIENTS AND METHODS: BODY.STUDY DESIGN: This randomized, double-blind, placebo-controlled, crossover trial was performed in 47 medical institutions in Japan. The study protocol was approved by the Institutional Review Board of each participating institution, and the trial was carried out in accordance with the Declaration of Helsinki and Good Clinical Practice. Patients gave written informed consent prior to registration for this study. This trial is registered with ClinicalTrials.gov, number NCT00335985. The trial began with a 6-week run-in period, during which corticosteroid resistance was confirmed. Patients who had given informed consent, and had been confirmed to be resistant to corticosteroid treatment during the run-in period, were randomly allocated to receive GB-0998 (Benesis Corporation, Osaka, Japan), a polyethylene glycol-treated human normal immunoglobulin, followed by an indistinguishable placebo (GB-0998 group), or the same two treatments in the reverse order (placebo group), as shown in Fig. 1. GB-0998 at a dose of 400 mg (8 mL)/kg/day or the placebo was administered by intravenous infusion once daily for 5 consecutive days, in a double-blind manner. After an assessment duration of 8 weeks from the start of administration (the first period), subjects receiving the active drug were switched to receive the placebo in the second period, whereas those receiving the placebo in the first period were switched to receive the active drug in the second period. The assessment duration in the second period was 8 weeks, and there was an additional 4-week observation period (follow-up period) after the second period. Because of the seriousness of the disease, the second period was included in the study design to enable all subjects in the study to receive GB-0998. For ethical reasons, patients who did not show disease alleviation within 4 weeks after the start of the first period, rendering continuation of the study difficult, were given an early transition to the second period; i.e., these subjects were transferred to the second period without continuing in the first period for the full 8 weeks. For this reason, the primary evaluation was of efficacy in the first period.Fig. 1Outline of study design. GB-0998 (active drug) was administered to the GB-0998 group in the first period and to the placebo group in the second period. Differences between before and after the 8-week first period in the GB-0998 group were assessed in the primary analysis Increases in corticosteroid and immunosuppressant doses, including methylprednisolone pulse therapy, were not allowed during the period from acquisition of informed consent until completion or termination of the study. Decreases in the dose were allowed when side-effects were elicited by corticosteroid or immunosuppressant during the period prior to the start of test drug administration, and were also allowed depending on the patients’ medical condition following the administration of the test drug. BODY.PATIENTS: Patients with corticosteroid-resistant PM/DM (Japanese people, resident in Japan) who were aged 16–75 years and fulfilled the diagnostic criteria of Bohan and Peter [3] for “definite” PM/DM were considered eligible to participate in this study. Among them, those that met one of the following criteria with respect to their history of corticosteroid therapy were enrolled for this study after obtaining their informed consent:Had received high-dose corticosteroid therapy (≥50 mg/day or ≥1 mg/kg/day for 1 month or more), starting between 6 weeks and 1 year prior to acquisition of informed consent, and continued until the day of acquisition of informed consent. In receipt of ≥50 mg/day or ≥1 mg/kg/day of corticosteroids on the day of acquisition of informed consent. Had received two or more methylprednisolone pulse therapies within 6 weeks before the acquisition of informed consent, and receiving ≥30 mg/day or ≥0.6 mg/kg/day of corticosteroid on the day of acquisition of informed consent. Patients with manual muscle test (MMT) scores of ≤80 points (normal score 90 points), assessed after having obtained their informed consent, and with twice the standard upper limit value of serum creatine kinase (CK) or more were tentatively enrolled, and followed for 6 weeks (run-in period). The patients who did not show any improvement in MMT scores and serum CK levels during these 6 weeks were formally registered, and received the investigational drug. Patients excluded from participation in this study were those with malignant tumors; acute interstitial pneumonia including acute exacerbation of chronic phase; severe muscular atrophy for a long period; severe infectious disease; severe hepatic disorder or severe renal disorder; those who had a previous history of shock or hypersensitivity to the test drug; those who had a history of cerebral infarction or ischemic heart disease, or had symptom of these diseases; those who had a history of IgA deficiency diagnosed; pregnant, lactating, and possibly pregnant patients; patients who wanted to become pregnant; and those who had been administered immunoglobulin within 6 weeks of obtaining consent. BODY.EFFICACY ASSESSMENT: Since muscle weakness is the most common symptom of PM/DM, changes in MMT scores were used as the primary endpoint, because the therapeutic effect could be assessed by observing the degree to which muscular strength was restored. MMT was scored on the following 18 muscles: the right and left of each of the following: deltoid, biceps brachii, brachioradialis, triceps brachii, iliopsoas, gluteus maximus, quadriceps femoris, and hamstring muscle; and the flexor and extensor muscles of the neck area. Each muscle was scored 0–5, with a healthy state thus being a score of 90 [4, 5]. In order to achieve consistency of evaluation, in principle, a single assessor evaluated the MMT for all patients throughout the study. The secondary efficacy endpoints were serum CK level and the activities of daily living (ADL) score. Serum CK measurements and other clinical laboratory tests were carried out at a central laboratory, at Mitsubishi Chemical Medience Corporation (Tokyo, Japan). The upper limits of the normal serum CK range were taken as 270 and 150 IU/L for males and females, respectively. The ADL score was based on the following 15 actions, with each action being scored 0–3, and the normal total score being 45: raising arms, taking off outer clothes, turning on water tap, combing hair, fastening buttons, getting out of bed, lifting up feet, standing up from chair, climbing stairs, walking on flat surface, turning over in bed, holding up head, sitting down on bed, having conversation, and swallowing [4, 5]. In addition to the above endpoints, we considered the time (number of days) to discharge from hospital during the first period. Furthermore, two additional parameters evaluated were dysphagia, which was graded as none, mild, or severe by each attending physician, and the occurrence or nonoccurrence of early transition to the second period. The safety endpoint was the occurrence or nonoccurrence of adverse events. BODY.TARGET NUMBER OF SUBJECTS: The number of patients meeting the criteria for inclusion in this study is very small. It was therefore considered that, with a study duration of 3 years, the number of subjects who could be enrolled would be limited to 20. Therefore, from the viewpoint of the feasibility of carrying out the study, the target number of subjects was set at 10 per group (a total of 20 subjects). BODY.STATISTICAL ANALYSIS: As described above, the number of target patients for this study was very small, and it was not practically possible to set the target number of subjects sufficiently high to permit a clear demonstration of the superiority of GB-0998 over placebo. Therefore, the primary analysis consisted of an intragroup comparison in the GB-0998 group, using the paired t test, of changes in MMT score, serum CK level, and ADL score between before initiation of study drug administration and after administration for 8 weeks in the first period. An intergroup comparison between the GB-0998 and placebo groups was also carried out as a subsidiary analysis. For this analysis, descriptive statistics were calculated separately for the two groups, with respect to the changes in MMT score, serum CK level, and ADL score between before initiation of administration during the first period and after administration for 8 weeks. The serum CK levels were widely scattered, depending upon the severity of disease in each subject, and logarithmic transformation was therefore carried out for efficacy evaluation in relation to change in serum CK level. Then, using the mean changes in these parameters, the point-estimation value and 95% confidence interval were calculated for the difference between the GB-0998 and placebo groups. In addition, the Kaplan–Meier method was used to compare the time (number of days) until improvement of the MMT score in the GB-0998 and placebo groups. On the basis of a report by Dalakas et al. [6], the time at which “improvement” was taken to have occurred was the first time point when the MMT score was increased by 5 or more (and subsequently maintained at the higher level) during the period from before initiation of administration during the first period until transition to the second period. The median value of the time was calculated for each group, and the log-rank test was applied. For the serum CK level, the Kaplan–Meier method was used to compare the time (number of days) until normalization in the GB-0998 and placebo groups. The event taken to constitute “normalization” was the serum CK reaching a level below the upper limit of the normal range for the first time during the period from before initiation of administration during the first period until transition to the second period. The median values for the groups were calculated, and the generalized Wilcoxon test was applied. With respect to parameters other than the primary and secondary endpoints, the Kaplan–Meier method was used to compare the time (number of days) until first discharge from hospital during the first period. The median value was calculated for each of the groups. Several supplementary analyses were also carried out, as follows. For MMT score, the percentage of subjects showing improvement was calculated for each group. In addition, for each of the individual muscles evaluated, an intergroup comparison using the paired t test was carried out for the change during the first period. For ADL score, the paired t test was used for intergroup comparison of the change in each evaluated individual action during the first period. In addition, the change in dysphagia during the first period was calculated, and the Fisher direct probability test was used for intergroup comparison of the elimination or nonelimination of dysphagia. Numbers of subjects undergoing early transition to the second period were also compared in the two groups. Efficacy was assessed using the last observation carried forward (LOCF) approach at 8 weeks after the start of administration in the first period, when premature discontinuation occurred in the first 8-week period, or when the second period was started before the scheduled end of the first period. Similarly, when premature discontinuation occurred in the second 8-week period, the efficacy after 8 weeks for the second period was assessed using the LOCF approach. BODY.RESULTS: BODY.BACKGROUND OF SUBJECTS: Of 63 subjects who gave informed consent to participate in the study, corticosteroid resistance was confirmed in 26 (12 in the GB-0998 group and 14 in the placebo group). Study drug administration and evaluation of safety and efficacy were carried out with these subjects (Fig. 2).Fig. 2Flow diagram of patients participating in this trial. In this study, a total of 26 subjects received GB-0998. Asterisk Premature discontinuation occurred after the sixth week of the first period Table 1 presents the backgrounds of these subjects, separately for each of the two groups. In the GB-0998 group there were 6 subjects with PM and 6 with DM, while in the placebo group, there were 10 with PM and 4 with DM.Table 1Patients’ characteristics CharacteristicGB-0998 group (n = 12)Placebo group (n = 14)Total (n = 26)Gender, n (%)  Male 1 (8.3) 5 (35.7) 6 (23.1)  Female 11 (91.7) 9 (64.3) 20 (76.9) Age (years)  Mean 50.6 48.1 49.3  Standard deviation 13.1 12.1 12.4  Minimum value 27 30 27  Median 49.0 50.5 49.0  Maximum value 69 68 69 Diagnosis, cases (%)  Polymyositis 6 (50.0) 10 (71.4) 16 (61.5)  Dermatomyositis 6 (50.0) 4 (28.6) 10 (38.5) Disease period, cases (%)  <6 months 5 (41.7) 3 (21.4) 8 (30.8)  ≥6 months and <1 year 0 (0.0) 4 (28.6) 4 (15.4)  ≥1 year 7 (58.3) 7 (50.0) 14 (53.8) Interstitial pulmonary disease, cases (%)  Absence 6 (50.0) 11 (78.6) 17 (65.4)  Presence 6 (50.0) 3 (21.4) 9 (34.6) Daily dose of corticosteroid at formal registration (mg)  Mean 34.79 40.71 37.98  Standard deviation 14.56 14.69 14.65  Minimum value 10 7.5 7.5  Median 35.00 47.50 42.50  Maximum value 60 55 60 Therapeutic history with immunosuppressant, cases (%)  Absence 5 (41.7) 7 (50.0) 12 (46.2)  Presence 7 (58.3) 7 (50.0) 14 (53.8) Manual muscle test score at formal registration (points)  0–50 2 1 3  51–60 4 3 7  61–70 4 8 12  71–80 2 2 4  Mean ± SD 61.8 ± 10.6 64.7 ± 9.0 –  Minimum value 42 43 42  Maximum value 77 77 77 Activities of daily living score at formal registration (points)  0–15 3 2 5  16–20 3 1 4  21–25 0 1 1  26–30 2 0 2  31–35 2 5 7  36–40 2 3 5  41–45 0 2 2  Not done 0 0 0  Mean ± SD (points) 23.8 ± 11.1 30.2 ± 10.3 –  Minimum value 4 11 4  Maximum value 40 45 45 BODY.RESULTS OF PRINCIPAL ANALYSES: INTRAGROUP COMPARISON DURING THE FIRST PERIOD: With respect to the principal analyses of the primary and secondary endpoints, Table 2 presents the changes in efficacy endpoints between before initiation of study drug administration, and after administration for 8 weeks during the first period.Table 2Differences of assessment parameters between before administration and at 8 weeks after start of administration for the first phase and corresponding between-group differences No. of subjectsMeanStandard deviationMinimum valueMedianMaximum valueCorresponding t testDifference of mean (GB-0998 group − placebo group)95% confidence intervalMMT  GB-0998 12 11.8 8.0 −4 11.0 25 t = 5.0655p = 0.0004 1.9 (−4.8 to 8.5)  Placebo 14 9.9 8.3 0 8.0 22 t = 4.4334p = 0.0007 CK  GB-0998 12 −1.1633 1.4123 −3.006 −1.4389 1.229 t = −2.8533p = 0.0157 0.1029 (−0.8382 to 1.0439)  Placebo 14 −1.2662 0.8900 −2.797 −1.4353 −0.134 t = −5.3229p = 0.0001 ADL  GB-0998 12 7.3 6.6 −1 6.0 26 t = 3.8169p = 0.0029 3.3 (−1.8 to 8.3)  Placebo 14 4.0 5.8 −4 3.0 18 t = 2.5610p = 0.0237 The primary before–after analysis was performed using t tests to evaluate the differences in MMT scores, serum CK levels, and ADL scores between before the administration of the drug and at 8 weeks after the start of the administration in the first phase. As a subanalysis, descriptive statistics were calculated by group and the before–after differences (between before the drug administration and at 8 weeks after the start of the administration in the first phase) were compared for each assessment parameter. The between-group differences (GB-0998 group versus placebo group) and confidence intervals for means of differences between before and after administration were estimated based on analysis of variance. Patients whose treatment was discontinued or who were transferred to the second phase before 8 weeks after the start of administration in the first phase were assessed after 8 weeks in the first phase using the LOCF approach MMT manual muscle test, CK creatine kinase, ADL activities of daily living The MMT score was the primary endpoint, and the mean (±standard deviation, SD) of the change in this score in the GB-0998 group during the first period was 11.8 ± 8.0, this change being statistically significant according to the paired t test (p  Serum CK level and the ADL score were the secondary endpoints. The mean (±SD) of the change in serum CK level was −1.1633 ± 1.4123 Ln (IU/L) (paired t test, p = 0.0157), and the mean (±SD) of the change in the ADL score was 7.3 ± 6.6 (paired t test, p = 0.0029). The changes in both these parameters were statistically significant. Statistically significant increases during the first period were also found in the intragroup comparison in the placebo group. The mean change in MMT score was 9.9 ± 8.3 (paired t test, p = 0.0007), the mean change in serum CK level was −1.2662 ± 0.8900 Ln (IU/L) (paired t test, p = 0.0001), and the mean change in ADL score was 4.0 ± 5.8 (paired t test, p = 0.0237). BODY.RESULTS OF SUBSIDIARY ANALYSES: INTERGROUP ANALYSES: BODY.CHANGES DURING THE FIRST PERIOD: The results for intergroup comparison of MMT score, serum CK level, and ADL score during the first period are presented in Table 2. For the primary endpoint, the intergroup difference in mean MMT scores during the first period was 1.9, with the 95% confidence interval being −4.8 to 8.5. As for the secondary endpoints, the difference between the mean logarithmic serum CK levels was 0.1029 Ln (IU/L), with the 95% confidence interval being −0.8382 to 1.0439, and the mean difference between the ADL scores was 3.3, with the 95% confidence interval being −1.8 to 8.3. The GB-0998 group showed numerically greater changes in the MMT and ADL scores than did the placebo group, but the differences were not statistically significant. In addition, the placebo group showed a greater change of serum CK level than did the GB-0998 group, but this difference was not statistically significant. BODY.TIME TO IMPROVEMENT IN MMT SCORE: The time (number of days) until the improvement in the MMT score reached 5 or more was compared between the two groups. The Kaplan–Meier survival curves for the time until improvement are shown in Fig. 3. The median times until improvement were 16.5 days in the GB-0998 group, and 29.0 days in the placebo group, but the difference between the two groups was not statistically significant (log-rank test, p = 0.1154).Fig. 3Kaplan–Meier curve of patients for whom the MMT scores increased by 5 points or more in the first period. Event occurrence was defined as the time point when MMT scores initially increased by 5 points or more, and subsequently remained at the higher level, during the period from before drug administration until transfer to the second period. When the score was increased by less than 5 points upon transfer to the second period, the test was discontinued on day 56 BODY.TIME UNTIL NORMALIZATION OF SERUM CK LEVEL: The Kaplan–Meier survival curves for time (number of days) until the first normalization of serum CK level after initiation of study drug administration during the first period are shown in Fig. 4. The median time until normalization of the serum CK level was significantly less in the GB-0998 group, at 22.0 days, than in the placebo group, in which it was 57.5 days (generalized Wilcoxon test, p = 0.0301).Fig. 4Kaplan–Meier curve of patients in whom serum CK level was normalized in the first period. Event occurrence was defined as the first decline of the serum CK level to below the upper limit of the normal range during the first phase. In one subject whose serum CK level was not normalized on day 57, the test was discontinued on day 58 BODY.TIME UNTIL DISCHARGE FROM HOSPITAL: The time until the first discharge from hospital after initiation of administration during the first period was analyzed using the Kaplan–Meier method, as shown in Fig. 5. The median time (number of days) until discharge was 11.5 days in the GB-0998 group, and 41.5 days in the placebo group. In the GB-0998 group, all 12 subjects were discharged from hospital during the first period, whereas only 8 of 14 subjects in the placebo group were discharged during the first period.Fig. 5Kaplan–Meier curve of patients discharged from hospital in the first period. Event occurrence was defined as the first discharge from hospital after test drug administration in the first period BODY.RESULTS OF SUPPLEMENTARY ANALYSES AND INVESTIGATIONS: BODY.PERCENTAGE OF SUBJECTS SHOWING IMPROVEMENTS IN MMT SCORE: The time-courses of percentage of subjects showing improvement in MMT score are shown in Fig. 6a. Even after week 8 of the first period, no statistically significant intergroup difference in the percentage of subjects showing improvement was found (Fisher direct probability test, p = 0.0809). However, in the GB-0998 group the percentage showing improvement at week 8 was 91.7% (11 of 12 subjects); further, the percentage showing improvement increased up to week 6, and was then maintained even after the week-8 time-point. In the placebo group, on the other hand, the percentage showing improvement at week 8 was 57.1% (8 of 14 subjects), this percentage having shown no increase from week 4. In addition, at week 8 of the second period, the percentage of the GB-0998 group (administered placebo during the second period) that showed improvement was 81.8% (9 of 11 subjects), and the percentage of the placebo group (administered GB-0998 during the second period) that showed improvement was 84.6% (11 of 13 subjects). In the GB-0998 group, the percentage that showed improvement at week 8 of the second period was approximately the same as that at week 8 of the first period. However, in the placebo group the percentage that showed improvement increased during the second period, when they were receiving GB-0998, and became approximately the same as the percentage in the GB-0998 group during the first period (when they were receiving GB-0998) (Fig. 6b).Fig. 6a Time-dependent changes in the rate of improved MMT scores in the first period. When MTT score increased by 5 points or more compared with that before administration of the drug, this was defined as an improvement. The time-dependent changes in the rate of improvement are shown by group. For cases discontinued or transferred to the second period before 8 weeks after the start of administration in the first period, the assessment in each assessment period was performed using the LOCF approach. Fisher’s exact test was used to compare the rates of improved MMT scores between the two groups at the end of the first 8-week period. b An MTT score that increased by 5 points or more compared with before drug administration in the first period was considered as an improvement, and improvement rates after 8 weeks in the first period and after 8 weeks in the second period were compared by group. For subjects discontinued or transferred to the second period before the end of the 8-week first period, the assessment after the first period was performed using the LOCF approach. For subjects discontinued before the end of the 8-week second period, the assessment after the second period was performed using the same method We found that 42.9% (6 of 14 subjects) of the placebo group had no improvement in MMT score during the first period. These subjects did not show improved muscle strength at the initiation of the second period, despite having undergone corticosteroid therapy by that time, but four of them did show improvements in MMT score with GB-0998 administration during the second period. BODY.MMT SCORES CLASSIFIED BY EVALUATED MUSCLE: The intergroup differences in changes of individual MMT scores in the 18 muscles evaluated in this study, during the first period, are shown in Fig. 7. The point-estimation values were greater in the GB-0998 group than in the placebo group for 14 of the 18 muscles, although the differences were small for all individual muscles.Fig. 7Forest plots of before–after differences in MMT scores for the 8-week first period. Black circles and lines, respectively, represent point estimates and 95% confidence intervals of between-group differences (GB-0998 group versus placebo group) concerning MMT scores for each assessed muscle BODY.ADL SCORES CLASSIFIED BY EVALUATED PARAMETER: The intergroup differences in changes of ADL scores for the 15 actions evaluated in this study, during the first period, are shown in Fig. 8. The point-estimation values show that the changes were greater in the GB-0998 group than in the placebo group for 12 of the 15 actions, although the differences for individual actions were small. Among these actions, the difference (value 1.17) with respect to swallowing action at week 8 of the first period between an increase of 1.00 in the GB-0998 group and a decrease of −0.17 in the placebo group (the normal score is 3) was statistically significant (t test, p = 0.0222).Fig. 8Forest plots of before–after differences in ADL scores for the 8-week first period. Black circles and lines, respectively, represent point estimates and 95% confidence intervals of between-group differences (GB-0998 group versus placebo group) in the ADL scores for each activity BODY.DYSPHAGIA: The number of subjects showing symptoms of dysphagia in the GB-0998 group before initiation of study drug administration in the first period was 7 of 12, whereas the number in the placebo group was 2 of 14. At week 8 of the first period, the symptoms had ceased in 5 of the 7 subjects in the GB-0998 group, whereas they had not ceased with either of the two subjects in the placebo group (Fisher direct probability test, p = 0.1667). BODY.TRANSITION TO THE SECOND PERIOD: From week 4 of the first period, it was permissible to make an early transition to the second period if alleviation of the disease was not seen on the basis of the time-courses of MMT score and serum CK level, if continuation of the first period was judged to be inappropriate, or if it was considered that treatment intensification would have been necessary. The numbers of subjects experiencing such early period transitions were 1 of 12 in the GB-0998 group and 4 of 14 in the placebo group. BODY.TIME-COURSES OF MMT SCORES AND SERUM CK LEVELS OF INDIVIDUAL PATIENTS FOR 8 WEEKS FROM INITIAL ADMINISTRATION: The time-courses of MMT scores and serum CK levels in individual patients for 8 weeks after the initial administration are shown in Figs. 9, 10, respectively, for both the GB-0998 and placebo groups. The broken lines indicate the time-courses in patients after early transition to the second period.Fig. 9Time-courses of MMT scores of individual subjects for 8 weeks after initiation of first-period administration (a GB-0998 group, b placebo group). Broken lines indicate the time-courses after transfer to the second period in the cases of subjects who were transferred before the full 8 weeks in the first period Fig. 10Time-courses of serum CK levels of individual subjects for 8 weeks after initiation of first-period administration (a GB-0998 group, b placebo group). Broken lines indicate the time-courses after transfer to the second period in the cases of subjects who were transferred before the full 8 weeks in the first period BODY.SAFETY: Total numbers of adverse events occurring after initiation of GB-0998 administration were obtained for the 26 subjects receiving the study drug. One hundred one adverse events occurred in 23 of 26 subjects (88.5%) after initiation of the first period. This total does not include 91 cases of abnormal changes in clinical laboratory test results that occurred in 18 subjects. In terms of classification by severity, 89 adverse events in 15 subjects were mild, 11 events in 7 subjects were moderate, and 1 event in 1 subject was severe. The severe adverse event was cerebral infarction, but it was considered that there was no causal relationship between this and GB-0998 administration. Nineteen adverse drug reactions occurred in 11 of 26 subjects (42.3%). These included gingivitis, hyperkalemia, disorders of glucose tolerance, diarrhea, dry skin, perspiration disorder, oral herpes, eructation, nausea, increased serum CK, decreased muscle strength, hot sensation, purpura, chest pain, headache, increased serum β-d-glucan, increased blood pressure, bronchitis, and fatigue. All these adverse drug reactions were mild to moderate in severity, and none occurred more than once. Four serious adverse events occurred in 3 subjects. Of these, one case of cerebral infarction in a 36-year-old female occurred during the first period in the GB-0998 group, 50 days after initiation of first-period administration, and continued for 6 days until recovery. This event was thought to have been due to long-term corticosteroid administration, and a causal relationship to GB-0998 administration was considered unlikely. A second serious adverse event, Prinzmetal’s angina in a 56-year-old male, occurred during the second period, 55 days after initiation of second-period administration, and continued for 84 days until improvement. This patient was in the placebo group, and a considerable time had elapsed since completion of study drug administration, so a causal relationship with GB-0998 was considered unlikely. In addition, two adverse drug reactions in one 47-year-old female in the GB-0998 group occurred during the second period, 56 days after initiation of first-period administration. They were decreased muscle strength and increased serum CK level, which continued for 149 days until recovery. A causal relationship between these events and GB-0998 was judged to be probable. These are all known adverse events. BODY.DISCUSSION: Since the first report of the use of immunoglobulin formulations to treat PM by Roifman et al. in 1987 [7], numerous clinical studies have confirmed the efficacy of intravenous immunoglobulin for treating PM, DM, and/or juvenile DM resistant to existing treatments. In addition, various textbooks [8] and guidelines [9, 10] present immunoglobulin formulations as treatment options for treatment-resistant PM and DM. However, the only published report of a placebo-controlled study relates to a double-blind, crossover study carried out by Dalakas et al. in 1993 [6]. Furthermore, the target disease for that study was treatment-resistant DM, and PM subjects were excluded. The present authors have previously carried out two clinical studies on the use of GB-0998 to treat corticosteroid-resistant multiple PM and DM. As in the present study, the efficacy endpoints in the previous studies were MMT score, ADL score, and serum CK level, and in those studies, GB-0998 was found to be effective against corticosteroid-resistant multiple PM and DM [4, 5]. However, both of the previous studies were uncontrolled, and therefore the present study was designed as a placebo-controlled, double-blind trial in order to obtain a more objective efficacy evaluation. In addition, the protocol for the present study was made stricter, in that the criteria for resistance to corticosteroid treatment were more clearly defined, including an increase of the prestudy run-in period for confirmation of resistance to 6 weeks from the 4 weeks used in the two previous studies. With respect to the intragroup comparison in the GB-0998 group, which was the principal analysis, we found statistically significant improvements due to GB-0998 administration in the primary endpoint (MMT score) and the secondary endpoints (ADL score and serum CK level). Further, the GB-0998 group was statistically significantly superior to the placebo group with respect to the time to normalization of serum CK level and score of swallowing action. These results suggest that early normalization of serum CK level, which is an index of inflammation and increases during the active stage of PM or DM, can be achieved by administration of GB-0998. Normalization of serum CK is important for achieving improvement in muscle strength, which is the ultimate goal of treatment. In addition, dysphagia, which is related to swallowing, occurs in an active and advanced stage of PM and DM, and is often refractory to corticosteroid treatment; this may result in aspiration pneumonia, which significantly affects the prognosis [11–13]. It is therefore noteworthy that GB-0998 also appeared to improve dysphagia, a life-threatening complication of PM and DM. With respect to safety, no severe adverse drug reactions occurred, and all serious adverse drug reactions were previously known effects. It is therefore considered that GB-0998 can be administered safely, with the same precautions that are applied in current IVIG treatments. However, a case of cerebral infarction did occur in the present study, although a causal relationship with administration of the study drug was considered unlikely. In addition, asymptomatic myocardial infarction was found as an adverse event in a previous study by the authors [4]. Furthermore, there have been reports of cerebrovascular and cardiovascular disorders, thought to be due to circulatory disturbances associated with increased blood viscosity, when high doses of immunoglobulins were administered to patients at risk of cerebrovascular and cardiovascular events [14, 15]. In the treatment of patients who have, or have a history of, cardiovascular and/or cerebrovascular disorders, and patients who are at high risk of developing thromboembolism, careful consideration should be given before administration of GB-0998, and careful monitoring is needed after administration. Thus, the safety and efficacy results in the present study indicate that GB-0998 has the potential to be a useful drug for treatment of PM and DM subjects in whom corticosteroid treatment has not been effective. However, the intragroup comparison in the placebo group also revealed significant improvements, and intergroup comparison of the changes in efficacy endpoints between before initiation of study drug administration and after administration for 8 weeks during the first period did not show a significant difference between the groups. In order to clarify the reasons for this, we tried to identify features of the subjects’ backgrounds that might have led to significant improvement being achieved even with placebo administration. The results suggested that the inequality of changes in MMT scores during the prestudy run-in period may have been the factor that had the most effect on intergroup difference. The number of subjects whose MMT scores increased by 1 or more during the prestudy run-in period was 7 of 26 in the two groups (26.9%). However, a breakdown of these subjects showed that they consisted of 1 of 12 subjects in the GB-0998 group, and 6 of 14 in the placebo group, so subjects who were already showing a tendency toward recovery thus tended to be more numerous in the placebo group. When these 7 subjects were excluded, an intergroup comparison of MMT scores during the first period among the other 19 subjects, who underwent either no change or deterioration during the prestudy run-in period, gave an increased intergroup difference, 3.8, with a 95% confidence interval of −4.4 to 11.9, although this is still not statistically significant. A detailed assessment of the above seven subjects indicated that they can be considered as subjects in whom corticosteroid treatment showed some, albeit minor, efficacy, and therefore it is conceivable there might have been a carry-over effect from the corticosteroid treatment in these subjects. In addition to steroid carry-over effect, the possibility of steroid myopathy must be considered. The patients in the present study had been administered steroids at high doses and/or for extended periods, and steroid administration was continued during the study period. Therefore, development of steroid myopathy is a potential issue [8], and one patient did in fact develop this condition. However, because it is difficult to distinguish muscle weakness due to steroid myopathy and that due to the primary disease, PM or DM, no evaluation of steroid myopathy was carried out in this study. Therefore, the possibility that evaluation of the primary endpoint, MMT score, was biased by the effect of steroid myopathy cannot be ruled out. Perhaps the greatest limitation of the present study is the small number of patients included, and it is considered that the resulting intergroup bias in inclusion of subjects had a major impact on evaluation of intergroup difference. This highlights the need for great care in subject allocation to eliminate intergroup bias in future studies. However, PM and DM are rare diseases, and clinical studies with extremely limited numbers of patients are inevitably problematic. GB-0998 was approved last year for cover by the Japanese National Health Insurance system for the indication of decreased muscle strength due to corticosteroid-resistant PM or DM. However, in the light of the present results, we suggest it would be desirable to carry out a postmarketing study with all patients administered GB-0998, in order to obtain clear evidence as to its efficacy. It will be necessary to evaluate the efficacy in accordance with the methods used in clinical studies, in order to assure comparability with the clinical study results. We think that such continuing evaluation will be important to fully characterize the efficacy of this drug in corticosteroid-resistant PM and DM, and also to ensure that the benefit for patients can be maximized.

TITLE: Safety and Efficacy of Deep Sedation with Propofol Alone or Combined with Midazolam Administrated by Nonanesthesiologist for Gastric Endoscopic Submucosal Dissection ABSTRACT.BACKGROUND/AIMS: Endoscopic submucosal dissection (ESD) is accepted as a treatment for gastric neoplasms and usually requires deep sedation. The aim of this study was to evaluate the safety and efficacy profiles of deep sedation induced by continuous propofol infusion with or without midazolam during ESD. ABSTRACT.METHODS: A total of 135 patients scheduled for ESDs between December 2008 and June 2010 were included in this prospective study and were randomly assigned to one of two groups: the propofol group or the combination group (propofol plus midazolam). ABSTRACT.RESULTS: The propofol group reported only one case of severe hypoxemia with no need of mask ventilation or intubation. Additionally, 18 cases of mild hypotension were observed in the propofol group, and 11 cases were observed in the combination group. The combination group had a lower mean total propofol dose (378 mg vs 466 mg, p<0.012), a longer mean recovery time (10.5 minutes vs 7.9 minutes, p=0.027), and a lower frequency of overall adverse events (32.8% vs 17.6%, p=0.042). ABSTRACT.CONCLUSIONS: Deep sedation induced by continuous propofol infusion was shown to be safe during ESD. The combination of continuous propofol infusion and intermittent midazolam injection can decrease the total dose and infusion rate of propofol and the overall occurrence of adverse events. BODY.INTRODUCTION: The number of gastrointestinal endoscopic procedures has increased substantially over the last decade. Survey data from the United States suggest that more than 98% of gastroscopies and colonoscopies were performed with sedation.1 In recent years, more complex therapeutic endoscopic procedures have been developed. Endoscopic submucosal dissection (ESD), one of the most difficult endoscopic procedures, is a new and minimally invasive method of treating early gastrointestinal epithelial neoplasms.2 A lengthy and potentially uncomfortable endoscopic procedure like ESD usually requires moderate to deep sedation using a method to maintain a stable sedation level.3 Deep sedation during ESD with careful monitoring minimizes patient movement, allowing the endoscope to be manipulated precisely with little interruption.4 Several sedation protocols have been proposed to improve the acceptability and tolerability of ESD; the propofol sedation protocol is one example. Propofol is a short-acting sedative with a rapid recovery profile. The use of propofol has additional advantages, including the relative ease of safely maintaining a proper sedation level and a good amnesic effect.3 These advantages have resulted in increased use of propofol for endoscopic procedures around the world. However, there is an increased risk of cardiopulmonary complications due to the narrow therapeutic range of the drug.4 Elderly patients are often candidates for ESD and have a higher risk of complications associated with sedation. Recently, a number of studies for propofol sedation during ESD by nonanesthesiologist have proven that propofol sedation has similar safety profile compared to conventional sedation with midazolam.3,5,6 In these studies, propofol was administrated in bolus or by continuous infusion, and target sedation level was mostly moderate sedation. However, intermittent bolus injection of propofol may be theoretically associated with fluctuation of plasma concentration. This variation of plasma level could lead to more frequent complications or delay of procedure due to unsteady sedation level. We hypothesized that continuous infusion of propofol may be associated with a more consistent level of plasma concentration so that maintaining a stable deep sedation level during ESD could be more easily and safely achieved. This study was designed to assess the efficacy and safety profile of deep sedation by continuous propofol infusion in patients underwent ESD, and also aimed to determine the additional advantage of propofol continuous infusion in combination with midazolam. BODY.MATERIALS AND METHODS: BODY.1. PATIENTS AND STUDY DESIGN: This prospective, randomized trial included 135 patients who were scheduled for ESD to remove gastric neoplasms between December 2008 and June 2010 at the Hallym University Sacred Heart Hospital. Adult patients who were 20 to 80 years of age, were classified by the American Society of Anesthesiologists (ASA)7 as categories I to III, had gastric adenoma or early gastric cancer (EGC) that had been documented on a previous biopsy, and had a gastric submucosal tumor (SMT) were included. Patients were excluded if they were less than 20 years of age or more than 80 years of age; had an ASA classification of IV to V; had received antiplatelet agents or anticoagulation therapy; or had a history of complications during sedative endoscopy, adverse reactions to propofol and/or midazolam injection, severe obstructive sleep apnea, or allergy to eggs or soybeans. Using computer-generated random sequencing, all study subjects were randomly assigned to one of two groups: the propofol continuous infusion group (propofol group) or the propofol continuous infusion plus bolus midazolam group (combination group). Verbal and written informed consent was obtained for ESD and sedation. The study was conducted according to the Declaration of Helsinki and was approved by the Institutional Review Board of Hallym University Sacred Heart Hospital. At least one physician with advanced training in basic and cardiac life support was present during each ESD procedure. BODY.2. SEDATION AND MONITORING PROTOCOLS: The patients in the propofol group received a continuous 0.3 mg/kg/min intravenous propofol infusion. The infusion rate was titrated according to the level of sedation. The patients in the combination group received an initial bolus of 2 mg of midazolam and a continuous infusion of 0.3 mg/kg/min of intravenous propofol. Trained registered nurses administered all medications under the supervision of the physician performing the endoscopy. The level of sedation was defined according to the ASA classifications: minimal, moderate, and deep sedation.7 Minimal sedation is the state in which the patient can respond normally to verbal stimulation. Under moderate sedation, the patient can respond purposefully to verbal commands, either alone or accompanied by light tactile stimulation. Under deep sedation, the patient cannot be aroused easily but responds purposefully following repeated or painful stimulation. In this study, the target level of sedation was deep sedation. Resuscitation equipment was available at all times within the endoscopy unit, and an on-call anesthesiologist was in the building during the procedure. The patients received supplemental oxygen (2 L/min) via a nasal cannula in the endoscopy room, and their vital signs and oxygen saturation were continuously monitored every 5 to 10 minutes using a standard three-lead electrocardiogram, pulse oximetry, and an automatic blood pressure cuff. To assess the accurate sedation level during the ESD procedure, an observer who didn't involve the ESD procedure evaluated the sedation level every 5 minutes using the Modified Observer's Assessment of Awareness/Sedation (MOAA/S) scale.8 If MOAA/S score showed the responsiveness score over three or a patient became agitated or produced involuntary movements without any stimulation, the sedation level was considered inappropriate (either moderate or minimal sedation), and the propofol infusion rate was titrated. BODY.3. OUTCOME MEASUREMENT AND DEFINITIONS: Sedation-related adverse events included hypoxemia and hypotension. Mild hypoxemia was defined as a decline in the oxygen saturation to <90% for less than 30 seconds that is corrected with supplemental oxygen and a decrease in the propofol infusion rate. Severe hypoxemia was defined as a decline in the oxygen saturation to <90% for more than 30 seconds that was not corrected with the aforementioned method. Severe hypoxemia was managed by discontinuing the propofol infusion and interrupting the procedure to secure the airway. If there was no response, bag-mask ventilation and/or endotracheal intubation were considered. Hypotension was defined as a decline in blood pressure to <90/50 mm Hg or a decrease in the baseline blood pressure by >20%. Mild hypotension was corrected with a saline infusion or a decrease in the propofol infusion rate. Severe hypotension was defined as hypotension that could not be corrected with the aforementioned method and lasted for more than 5 minutes. It was managed by discontinuing the propofol infusion, administering large volumes of intravenous fluid, and using the Trendelenburg position. The procedure time was the time from the entry of the sedatives to the withdrawal of endoscope following submucosal dissection. The recovery state was defined as a condition in which the patient can respond to verbal orders. The recovery time was the time from the cessation of the drug infusion to the recovery state. After the procedure, the degree of satisfaction of both the endoscopist and the patient was evaluated using an endoscopist score and a patient score, respectively. The endoscopist score was a scale from one (unsatisfactory) to four points (very satisfactory). The patient score was a scale from zero (very severe discomfort) to 10 points (no discomfort). BODY.4. STATISTICAL ANALYSES: The χ2 test or Fisher's exact test was used for comparisons of categorical data, and the t-test was used for continuous data. A p-value of <0.05 was considered to be statistically significant. All data were analyzed using the SPSS version 16.0 for Windows (SPSS Korea, Seoul, Korea). BODY.RESULTS: A total of 135 patients were enrolled in this prospective study; 67 patients were randomly assigned to the propofol group, and 68 were assigned to the combination group. The patient characteristics of both groups are shown in Table 1. There were no significant differences between the two groups in terms of age, gender, body weight, baseline blood pressure, underlying diseases (diabetes mellitus, hypertension, cardiovascular disease, or pulmonary disease), or ASA score. The combination group was more likely to consume higher volumes of alcohol than the propofol group, but there was no significant difference between the groups. The characteristics of the gastric lesions are shown in Table 2. The diagnostic pathology results were adenoma, EGC, or SMT. In the propofol group, there were 44 adenomas, 17 EGCs, and six SMTs. In the combination group, there were 39 adenomas, 23 EGCs, and six SMTs. The lesion was more commonly located in the lower third of the stomach in both groups. The average specimen size was 34.74±13.65 mm in the propofol group and 42.74±16.86 mm in the combination group (p=0.003). Table 3 shows that the mean procedure time was not significantly different between the two groups (57 minutes vs 53 minutes, p=0.528). The mean total dose of propofol administered was lower in the combination group than in the propofol group (378±220 mg vs 466±200 mg, p=0.012). The mean propofol infusion rate was lower in the combination group (0.118±0.03 mg/kg/min vs 0.160±0.06 mg/kg/min, p<0.001), but the combination group had a longer mean recovery time (7.9±3.78 minutes vs 10.5±8.74 minutes, p=0.027) (Fig. 1). Based on a 10-point visual analogue scale, the patients in both groups showed similar degrees of overall satisfaction. The adverse events are shown in Table 4. There were three cases of mild hypoxemia in the propofol group and one in the combination group (p=0.362), and there was one case of severe hypoxemia in the propofol group. Mild hypotension was observed in 18 cases in the propofol group and 11 cases in the combination group (p=0.144). There was no severe hypotension in either group, and no patients required mask ventilation with an Ambu bag or intubation. Frequency of each adverse event was similar between two groups. But total adverse event rate of combination group was significantly less than that of propofol alone group (32.8% vs 17.6%, p=0.042). Table 5 compares the study participants who had an adverse event (adverse event group) with those who did not (no-adverse-event group). The mean total propofol dose was 394 mg in the adverse event group and 431 mg in the no-adverse-event group. The propofol infusion rate was 0.136 mg/kg/min in the adverse event group and 0.140 mg/kg/min in the no-adverse-event group. The study results indicate that there was no correlation between the propofol dose or infusion rate with adverse events. The total procedure time was longer in the adverse event group than in the no-adverse-event group, but did not reach significance (59 minutes vs 54 minutes, p=0.414). The mean age was higher in the adverse event group (68±10.48 years vs 62±10.32 years, p=0.012), and cases with an ASA class of more than II were more frequent in the adverse event group (p=0.032). BODY.DISCUSSION: Our results show that propofol was successfully administrated by continuous infusion for induction and maintenance of deep sedation during ESD. The use of propofol for endoscopic sedation by nonanesthesiologist providers has increased markedly during the last decade,1 and recent survey data suggest that propofol sedation is associated with a lower risk of complications and serious adverse events during standard endoscopy compared to conventional sedation. The safety and efficacy of propofol for other complex therapeutic endoscopic procedures, such as endoscopic retrograde cholangiopancreatography and endoscopic ultrasound, has already been established by several studies.9-11 Nevertheless, the most effective and safest sedative agent and sedation protocol for ESD has not yet been clearly established. Propofol is a potent sedative agent with a narrow therapeutic window. It has a rapid onset of action and short recovery time. Propofol also provided significantly better patient cooperation than midazolam.12,13 However, this agent occasionally decreases systemic vascular resistance, cardiac contractility, and ultimately cardiac output without a concomitant change in heart rate, and respiratory depression can also occur.5,14,15 Recently, several studies have revealed most adverse events associated with propofol sedation were mild and often transient during advanced interventional endoscopic procedure.12,16-18 A randomized study in Japan, which compared continuous propofol infusion with intermittent midazolam injection during ESD for EGC, found that propofol is a safe and effective sedative agent and that patients treated with propofol had a quicker recovery than those who were treated with midazolam.3 In those studies, however, sedation was mostly targeted to a moderate level and did not monitor an accurate sedation level during the procedure. Moderate sedation with propofol, especially in bolus injection, may frequently move into lighter or deeper sedation due to inconstant plasma concentration of propofol, disturbing the ESD procedure. In our studies, an assessment of an accurate sedation level was performed using a MOAA/S scale every 5 minutes during ESD procedure. Similar to other studies for propofol sedation targeting a moderate sedation, our results showed that deep sedation with continuous propofol infusion during ESD had a relatively low incidence of cardiopulmonary complications. There was only one case of severe hypoxemia, and that case did not require mask ventilation or intubation. Both patients and endoscopists gave high satisfaction scores after ESD, and there was no interruption or delay of the procedure. Propofol may be administered alone, but in clinical practice, it is commonly administered in combination with low doses of midazolam or a narcotic.19 Because of the synergic effects of propofol, its dose can be reduced significantly when it is used in combination with midazolam and narcotics, possibly improving its safety.20,21 Recent studies have assessed the safety and efficacy of sedation with co-administration of propofol and opiates or midazolam to maintain a moderate sedation level. Similar to the results for propofol alone, the pooled analysis showed that the combination protocol resulted in a low incidence of hypoxemia (10%) but a relatively high incidence of nausea (26%) and memory of the procedure (66%).22 Our study demonstrated that the deep sedation using combination protocol for ESD was as safe and effective as sedation by continuous infusion of propofol alone. Furthermore, sedation could be maintained with a significantly lower dose of propofol in the combination group. A significantly slower mean infusion rate in the combination group was also observed. Because propofol is a potent respiratory depressant, a significant reduction in the amount of propofol required for deep sedation could theoretically decrease the risk of respiratory depression. In our study, each of hypotension and hypoxemia showed no difference between two groups, but total adverse event rate of the combination group was significantly lower than propofol infusion alone group. Low doses and slow infusion rate of propofol may lead to a low incidence of adverse events. However, the combination protocol had a longer recovery time even though we only administered midazolam as a bolus at the start of the procedure to minimize the delay in recovery. A recent study showed that midazolam has a pharmacodynamic interaction during prolonged propofol infusion.23 In this study, the results indicated that midazolam, at sedative levels, increases blood propofol concentrations by 25% and that propofol reduces the distribution and clearance of midazolam in a concentration-dependent manner. This synergetic effect of these two drugs could explain a prolonged recovery time in the combination group. Additionally, one point of clinical importance in our study is that trained nurses administered the propofol under the supervision of an experienced endoscopist. Recent large-scale trials of nurse-administered propofol sedation (NAPS) have suggested that appropriately trained nurses could assist the endoscopist in propofol administration and sedation monitoring, clearly saving resources.24-27 Our study showed that the cardiopulmonary complication rate from sedation was 25.2% and that all of the complications were transient and clinically insignificant. However, NAPS is still a subject of debate, and we cannot completely exclude the potential risk of cardiopulmonary complications of NAPS during ESD, even though we did not observe any major complications. Previous studies have suggested that NAPS is a safe method of sedation, at least in a tertiary hospital gastroenterology department, provided that training and the use of emergency measures to counteract its adverse effects is adequate.25 There are several limitations in our study. First, older and high-risk patients (ASA category IV) were excluded, and the proportion of ASA category III patients was small. This could be a cause of the relatively low incidence of cardiopulmonary complications. Second, we could not apply a double blind design; the endoscopist could not be blinded, which might have biased the satisfaction assessment. Third, the sample size was too small to exclude rare adverse events or to adequately power the study to demonstrate the safety and efficacy of deep sedation by propofol. Finally, we did not directly compare the continuous propofol infusion and intermittent bolus injection of propofol, so further studies to compare the continuous infusion and intermittent bolus injection of propofol are needed to investigate the most effective and safe sedation protocol for ESD. The aim of this study was to evaluate and compare the safety and efficacy of deep sedation induced by the continuous infusion of propofol administrated by a nonanesthesiologist during ESD. Using an adequate monitoring system, safe and efficient endoscopic procedures were performed with no serious adverse events using propofol. The combination protocol of continuous propofol infusion and intermittent intravenous midazolam injection can decrease the adverse events. However, further studies are needed on the specific adverse effects of propofol or other sedative agents used for deep sedation during complicated endoscopic procedures.

TITLE: Daily Pomegranate Intake Has No Impact on PSA Levels in Patients with Advanced Prostate Cancer - Results of a Phase IIb Randomized Controlled Trial Pomegranate has been shown to prolong PSA doubling time in early prostate cancer, but no data from a placebo controlled trial has been published yet. The objective of this study was to prospectively evaluate the impact of pomegranate juice in patients with prostate cancer. We conducted a phase IIb, double blinded, randomized placebo controlled trial in patients with histologically confirmed prostate cancer. Only patients with a PSA value ≥ 5ng/ml were included. The subjects consumed 500 ml of pomegranate juice or 500 ml of placebo beverage every day for a 4 week period. Thereafter, all patients received 250 ml of the pomegranate juice daily for another 4 weeks. PSA values were taken at baseline, day 14, 28 and on day 56. The primary endpoint was the detection of a significant difference in PSA serum levels between the groups after one month of treatment. Pain scores and adherence to intervention were recorded using patient diaries. 102 patients were enrolled. The majority of patients had castration resistant prostate cancer (68%). 98 received either pomegranate juice or placebo between October 2008 and May 2011. Adherence to protocol was good, with 94 patients (96%) completing the first period and 87 patients (89%) completing both periods. No grade 3 or higher toxicities occurred within the study. No differences were detected between the two groups with regard to PSA kinetics and pain scores. Consumption of pomegranate juice as an adjunct intervention in men with advanced prostate cancer does not result in significant PSA declines compared to placebo. BODY.INTRODUCTION: Prostate cancer is the most commonly diagnosed cancer in men accounting for 29% of all male cancers and is the second leading cause of cancer-related death in the United States (American Cancer Society: Cancer Facts and Figures 2011. Atlanta, Ga: American Cancer Society, 2011). Therefore, the identification of effective preventive strategies for prostate cancer is of particular clinical importance. Since free radicals seem to play a pivotal role in the development of prostate cancer reduction of intracellular free radicals by antioxidants hold promise for disease prevention. However, in a large randomized trial (SELECT) the anti-oxidant vitamin E has failed to show any benefit in the prevention of prostate cancer 1. Polyphenols are anti-oxidants that have been shown to positively influence inflammation and cancer 2, 3. A study utilizing capsules containing polyphenols equivalent to 12 cups of green tea found a significant reduction in serum levels of PSA, hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) in men with prostate cancer after a short treatment period 4. Pomegranate juice (PJ) is a natural source of bioavailable 5 ellagitannins and has been demonstrated to reduce redox activity 6. As compared to red wine or green tea, pomegranate juice from commercial sources has an antioxidant capacity that is 3 times greater 7. Pomegranate extracts contain ellagic acid, caffeic acid, luteolin and punicic acid, all of which have been associated with anti-cancer effects in preclinical models 6, 8. Ellagic acids are considered the most active component of PJ, accounting for more than 50% of its antioxidant effects 7. In a carcinogenic mouse model (TRAMP) oral pomegranate extract supplementation results in significant tumor control by inhibiton of IGF-I/Akt/mTOR pathways in the prostate tissues and tumors 9. Pantuck and colleagues performed a single center, open-label study, examining the effect of PJ in men with rising PSA levels after surgery or radiation 10. The investigators found an impressive increase in the PSA doubling time (PSADT) from 15 to 54 months under the treatment (p< 0.001). Another study 11 tested pomegranate juice extract capsules in men with rising PSA levels after local therapy for prostate cancer (NCT01220817). Again, a statistically significant increase of the PSADT for one or three capsules daily (overall 11.9 to 18.5 months, p<0.001) was observed. In both studies a relevant proportion of men even showed a decrease of PSA serum levels due to consumption of PJ (15% and 13%). An on-going phase III study (NCT00413530) is examining whether the pomegranate liquid extract prolongs PSADT in patients who have rising serum PSA levels after primary therapy for localized prostate cancer. That randomized, double-blind, placebo-controlled study with an estimated enrollment of 200 patients was scheduled to close on December 2012. All of the above studies have focused on the role of PJ in patients with early biochemical failure after initial treatment. No data about a therapeutic impact of PJ in patients with more advanced or metastatic prostate cancer has been published yet. A standard procedure for patients having failed hormone ablation therapy would be the initiation of chemotherapy 12, 13. In the absence of symptoms the time point of starting chemotherapy is debatable hence patients and physicians often prefer to delay the start of this rather aggressive treatment. In our trial we report the influence of PJ compared to placebo in a cohort of asymptomatic or oligosymptomatic patients, the majority (68%) of which had castration resistant prostate cancer. BODY.PATIENTS AND METHODS: BODY.ELIGIBILITY CRITERIA: Patients with histologically proven prostate cancer and a pathologic serum PSA (entry level at least 5 ng/ml) were eligible for the study. While patients were on study they had to continue their baseline treatment (e.g. androgen deprivation, zoledronic acid). The initiation of any new potential active treatment was not allowed. Exclusion criteria were any planned therapeutic intervention such as surgery, irradiation or any alteration of the ongoing therapy, e.g. withdrawal of medication or introduction of new drugs during the study period. BODY.DESIGN AND ASSESSMENTS: The study was conducted at two Swiss centers (University Hospital Zurich and KS Graubünden) and designed as a phase IIb, double-blind, placebo-controlled randomized trial. Consenting patients with a documented PSA above 5 ng/ml and histologically confirmed prostate cancer were randomized into two groups. Group A received 500 ml placebo juice per day and Group B 500 ml of PJ (equivalent to 1147 mg polyphenol gallic acid) per day. PSA serum levels were measured the day before treatment started, and on days 14 and 28. Patients were given a diary to document their daily juice intake, any adverse events and their pain score with time and date. The intensity of pain was self-documented by the previously instructed patients by choosing a value from a non-linear scale (range 0-10). At day 29 patients entered the open label phase of the trial. In this second period of the study, 250 ml of PJ (equivalent to 573 mg polyphenol gallic acid) was consumed by all patients (bottles labelled C) and the diaries were continued as before. A final PSA serum value was taken on day 56. While on study, especially in the first period, patients were not informed about their current PSA values. No other diagnostics, e.g. CT-scans or laboratory values were part of this study. The relatively short observational period of 8 weeks was chosen because the projected patient collective often converts from watchful waiting to intervention due to disease progression. BODY.TREATMENT: Block randomization (block size 4) was used to assign patients to the treatment groups 500 ml PJ or placebo daily. The patients were instructed to drink one bottle of 500 ml per day; the timing was left to the patient´s discretion. Consumption was self- monitored by daily recording using diaries. In addition, episodes of pain and usage of analgesics were also to be recorded by the patients in their diary. The producer of the beverage (Biotta AG, Egnach, Switzerland) provided the pomegranate juice and the placebo. Both beverages had a very similar taste and color (Supplementary figure 1). The juices contained the same basic ingredients, pear purée, white tea, agave concentrate and aronia berry juice. In the placebo drink the 27.5% pomegranate extract was replaced with an artificial pomegranate flavoring substance. The pomegranate juice contained 2294 mg/l polyphenol gallic acid. The daily intake in the intervention arm was 1147 mg/day polyphenol gallic acid during the first study period and 573 mg/day polyphenol gallic acid in the second period of the study. The company supplied PJ and placebo and gave limited financial support. The company had neither influence on the acquisition of the data nor on the statistical analysis. The investigational sites received identical bottles labelled with A, B and C. Bottles A and B were distributed to the patients according to the predefined randomization scheme in the first study period. Bottles labelled C were given to all patients in the second study period. Information which letter corresponded to pomegranate or placebo was kept secret at the company and was not revealed to the investigators and the study personnel before the end of the study. Unblinding was not done before all statistical analysis had been performed. BODY.PSA TESTING: The majority of patients´ PSA serum levels (75%) was determined by a third generation assay (chemiluminescence-enzyme-immunoassay, third generation Immulite 2500, Siemens healthcare diagnostics, Eschborn, Germany) at the Institute of Clinical Chemistry, University Hospital Zurich, Switzerland. All patient sera from Chur (n=23) were tested with an assay according to the ECLIA principle (ElectroChemiLuminescenceImmunoAssay, cobas e411, Roche diagnostics, Mannheim, Germany) at the central laboratory of Kantonsspital Graubünden, Chur, Switzerland. For the report of this study PSA progression was defined as PSA increase ≥25% 14 and PSA response was defined as a decline of PSA ≥50% from baseline according to the recommendations from the PSA working group 15. Patients with a PSA decline >30% are also reported here, as a PSA decrease of >30% under active treatment within the SWOG 99-16 study has been associated with a 50% reduction in the risk of death from prostate cancer 16. Following the recommendation of the Prostate Cancer Clinical Trials Working Group PSA responses of the individual patients are shown in waterfall plots 17. BODY.STATISTICAL ANALYSES: Fisher's exact test was used to compare categorical data between groups and Mann-Whitney test for continuous data. Applying Wilcoxon signed rank test PSA levels between follow-up and baseline in the same group were evaluated. The aim was to show a difference of change scores before and after intervention as well as before and in between the treatment. With an estimation of 47 patients in each arm the study was calculated to have a power of 85% to detect a difference of 5ng/ml (the difference of a group A mean of 0 ng/ml and group B of - 5 ng/ml) under the assumption of a standard deviation of 8 ng/ml, if a two group t-test with a double sided significance of 0.05 was used. Statistical comparisons of patient characteristics were performed with Graphpad Prism 5 software (Graphpad Inc. La Jolla, USA). BODY.ETHICS AND REGISTRATION: This study was approved by the local Ethics Committee (# EK-1545) and registered at “Deutsches Krebsstudienregister der Deutschen Krebsgesellschaft” (www.studien.de) under the study ID No.555. BODY.RESULTS: BODY.PATIENT CHARACTERISTICS: Between October 2008 and May 2011 a total of 102 patients with prostate cancer gave written consent and were enrolled. Of those, 5 patients were found ineligible as they did not fulfill the inclusion criteria. The trials profile is shown in Figure 1. Baseline characteristics of the 97 patients in the full analysis population are summarized in Table 1. With respect to ongoing treatment, 38% (18/47) of patients in the placebo group and 46% (22/48) in the verum group were under anti-hormonal treatment (LHRH analogues). Some patients received bicalutamid in combination or as a sole antihormonal treatment (22% in group A and 27% in group B). Low dose steroids (prednisone 5-10mg) were recorded in 4% of all patients (2% vs. 6%). 27% (25/94) study patients received no treatment but were under a watch and wait strategy. No patient received chemotherapy or underwent surgery or irradiation while on study. Statistically significant differences in tumor size (T3; 31% in the control vs. 51% in the intervention group, p=.036) and distant tumor spread (M1; 18% in controls vs. 44%, p=.032) at the time of initial diagnosis were observed (Table 1). Apart from these two variables the study was well balanced. BODY.SAFETY: Pomegranate juice was generally well tolerated with no grade 3 or 4 toxicities. Bowel disturbances were the most frequent adverse event reported. Obstipation was observed in both groups: 2/46 (4%) in the placebo group and 1/48 (2%) in group B. One patient on placebo withdrew his consent due to a CTC grade 2 obstipation. One patient in the PJ group reported a CTC grade 1 diarrhea (1/48; 2%) but no diarrhea was reported from patients in the placebo arm. The adherence to the protocol was good, with 94 patients (96%) completing the first period (days 1-28) and 87 patients (89%) completing both periods (days 1-56). Three patients dropped out within the first 28 days due to unrelated medical problems. The other seven patients were unable or unwilling to complete the second period. Thus, 94 patients were eligible for analysis of the randomized part of the study and 87 patients (42 patients in the placebo group A or 45 in the PJ group B) completed the full trial protocol. 45 patients of group B could be analyzed for both dosages of the pomegranate juice. BODY.EFFICACY: The number of patients experiencing a PSA decrease during the placebo-controlled period of the study was not statistically significant between the two arms (table 2). PSA progression within the first four weeks was observed in 41% in the control group compared to 38% in the pomegranate group (p=0.83). There were no responses with PSA decline >50% in either group, but 1 patient in the placebo group and 3 patients in the pomegranate group showed a decline ≥30% (Table 2A). In the second period of the study, when all patients consumed 250 ml of PJ, PSA progression was observed at a rate of 24% in the former placebo group and 29% in those patients that had been given 500 ml PJ in the first period (group B), (p=0.63, Table 2A). Despite the nominal reduction of patients with PSA progression in the placebo group the difference between the non-interventional period (day 1-28) and the pomegranate consumption period (day 29-57) was not significant (p=0.11). To address the question whether heterogeneity within the patient populations may have had an influence subgroup analysis were performed (Table 2B and 2C). Patients were pooled according to their status of disease at study entry e.g. castration resistant prostate cancer (CCRPC) (n=61) and non CCRPC (n=33). These analyses as shown in table 2B and 2C revealed no difference for PJ or placebo in patients with regard to their hormonal response. The corresponding waterfall plots depicting individual PSA changes are shown in figure 2. A combined PSA level analysis of both study periods showed PSA stabilization in 74% of patients starting with placebo versus 71% of those patients that had been under PJ continuously. The patients reported pain values in the range from 0-5 at study entry without significant group differences (mean pain score A=0.97 and B=0.98; p=0.49). After the randomized phase of the study, at day 28, no relevant pain decrease was found in either group. A non-significant trend toward lower pain scores was seen in group B (mean day 1: 0.98 ±1.25 versus mean day 28: 0.62 ±1.13; p=0.092). BODY.DISCUSSION: The objective of this study was to assess the impact of pomegranate juice on PSA levels in patients with advanced prostate cancer. There were no significant differences with regard to PSA levels or pain intensity between the observed groups in this trial. The study design included two treatment periods: Initially, patients were randomized to placebo (group A) or pomegranate juice consumption (group B). The results from the double-blinded, randomized controlled part of the study show no differences with regard to PSA level rise or decline, pain, frequency of dropout or adverse effects. The fact that 35% of men in the control group and 25% in the intervention group had a decline in their PSA value during the first phase of the study is remarkable. A possible explanation for this phenomenon could be a natural fluctuation of serum PSA levels in a four-week period in men with advanced prostate cancer that has not been recorded to this extend until now. The latter is particularly important when interpreting results from earlier and on-going studies that report on declining PSA levels due to specific interventions. An alternative explanation for the high percentage of men experiencing a PSA decline in both groups could be an unanticipated PSA test-to-test variability. With regard to this, an intermediate PSA level measurement had been predefined in the study at day 14. The majority of PSA levels obtained at day 14 and at day 28 were in accordance with the individual PSA course, e.g. if day 14 and 28 values were not pointing in the same direction, no more than a 10% difference of the day 14 value versus one of the other values was observed. Furthermore, test variability was controlled by processing all individual blood samples at the enrolling institution. Thus, there was no variability regarding the test used or the laboratory where the set of four patient's samples was analyzed. We conclude that test instability did not influence the final results. Another factor in this study that might have had an impact on patients with declining PSA values regardless of their group was the controlled intake of 500 ml of fluids (either pomegranate juice or placebo) in addition to other daily fluids. In the absence of a non-interventional control this influence cannot be ruled out. Regarding the value of pomegranate in the setting of advanced prostate cancer this variable plays no role as it appeared in both groups. Due to the design of a verum-containing treatment period for all patients (second part of the trial) we were able to analyze PSA levels of all men who started with placebo in comparison to the PSA levels after a subsequent four-week consumption phase of 250 ml of PJ, equivalent to 573 mg/ml phenol gallic acids (Table 2A placebo day 1-28 versus placebo day 28-56 panel). Here, no difference between the placebo and the verum period in group A was detected (41% vs. 24% progress, 57% vs. 74% stabilization, 2% vs. 2% response; p=0.08) indicating that the pomegranate ingredients of the drink were not responsible for any change. The absolute PSA level declines observed in this study did not reach clinical significance in either cohort when a PSA level decrease of ≥ 50% is defined as objective response. Overall, 5 responses of PSA level decline ≥ 30%, with a non-significant difference between the groups, were recorded at the end of the randomized part of the trial. With respect to the impact of pomegranate juice in early prostate cancer patients there have been two reports 11 and a phase III study is currently underway with reports expected in 2012 (NCT00413530). Table 3 illustrates the differences between the clinical data available to date. The major difference of the studies is that the two previously reported studies in early prostate cancer patients focussed on PSADT and had longer observational periods. Within the context of low PSA values (<5 ng/ml, median 2.2) and minimal active disease this is an adequate approach. Our study included patients with more advanced disease and higher PSA values (median 19.7 ng/ml). These patients are often to start their next therapy within a short period of time. Thus, these patients cannot be observed for an extended time period. Hence, PSADT would not have been a good end point. However, with a half-life of PSA of 2.2 days (18) we calculated that any meaningful changes in the PSA course of these patients should be readily detectable by 4 independent measurements at least 14 days apart and within a follow-up of two months. An unexpected limitation of our study is a certain heterogeneity of the included patient cohort. For example the recorded number of metastases in our study seems to indicate an imbalance in favor of the placebo arm. In this regard it has to be considered that no imaging diagnostics were performed at study entry. Baseline diagnostics included clinical examination and PSA level determination. Metastatic status was derived from previous clinical reports. Data concerning initial metastases (M1 at operation) was available in 58% (A) and 69% (B) of patients. The apparent imbalance could be due to under-diagnosing or under-reporting in the placebo group. Further supporting this notion is the fact that the average entry PSA level showed no difference with a median of 19 ng/ml (mean 90) in the placebo group and a median of 21 ng/ml (mean 60) in the interventional arm. When we examined responses of those men with castration resistant prostate cancer only (68% of all study participants), no difference between the groups was noted (Table 2B). Taken together, this imbalance, if present, should have had no impact on the results. The molecular pathway of pivotal importance in prostate cancer is the IGF-1/AKT/mTOR pathway. Bi-allelic loss of PTEN in about 50% of metastatic prostate cancers 19 results in over activation of this pathway, rendering targeted therapy an attractive concept. Everolimus like pomegranate is an inhibitor of the IGF-1/AKT/mTOR pathway (9). Single agent everolimus in a patient collective comparable to the one in this study has yielded PSA response rates in the range of 3% (PSA decrease ≥50%) and 11% (≥30%) 20. This result indicates some activity but also the need of refining the therapeutic approaches directed against this pathway. In a preclinical model IGF-1/AKT/mTOR stimulation drives cancer cells into hormone insensitivity and leads to neuroendocrine differentiation 21. This could explain why everolimus and pomegranate as inhibitors of this pathway may not be very effective in patients that have failed anti-hormonal therapy and should rather be considered earlier in a treatment algorithm. The analysis of our prospective study shows that pomegranate in the form of a daily beverage does not alter the course of PSA in patients with advanced prostate cancer regardless of the dosage. The maximum dose used here was twice as high as in the previously published study 10. However, the dosage of pomegranate does not seem to be of utmost importance. Two studies 10, 11 have shown no differences in the increase of PSADT when comparing 8 oz to a higher dose of 24 oz (6.9 months versus 5.3 months, p=0.92). In conclusion, pomegranate beverage had no significant impact on PSA progression in patients with recurrent and advanced prostate cancer when compared to placebo. The hitherto published benefit of pomegranate juice seems to be restricted to early and well differentiated prostate cancer. Our results provide important information for the design and patient selection within further trials with pomegranate. BODY.SUPPLEMENTARY MATERIAL: Supplementary figure 1Click here for additional data file.

TITLE: Association between miR-31-3p expression and cetuximab efficacy in patients with KRAS wild-type metastatic colorectal cancer: a post-hoc analysis of the New EPOC trial ABSTRACT.BACKGROUND: High miR-31-3p expression is associated with inferior outcomes in KRAS wild-type (WT) advanced colorectal cancer patients treated with anti-EGFR therapy. This study evaluated miR-31-3p expression in patients with operable colorectal liver metastases (LM) enrolled in the New EPOC study. ABSTRACT.METHODS: MiR-31-3p expression was measured in primary tumors (PT) from 149 KRAS WT patients including 71 receiving chemotherapy alone (CT) and 78 receiving chemotherapy plus cetuximab (CTX). Each treatment arm was split into tertiles based on miR-31-3p expression levels. MiR-31-3p expression was also measured in LM from 94 patients with tumor tissue available. ABSTRACT.RESULTS: The median progression-free survival for the combined populations with mid or high miR-31-3p expression was shorter in the CTX versus the CT arm (26.7 months versus 12.3 months, HR=2.28 95%CI 1.27; 4.09 p=0.006). Low miR-31-3p expressers had similar outcomes irrespective of treatment (HR=1.06 95%CI 0.43; 2.61 p=0.9). MiR-31-3p expression was correlated between paired PT and LM samples in the CT group but not in the CTX group. ABSTRACT.CONCLUSIONS: Patients with low miR-31-3p expression in the New EPOC study were not harmed by the addition of cetuximab. This supports miR-31-3p as a promising predictive biomarker for anti-EGFR therapy in KRAS WT advanced colorectal cancer. BODY.INTRODUCTION: There have been significant improvements in the management of colorectal cancer over the last decade involving increasingly sophisticated combinations of chemotherapy, targeted agents, and surgery. This has included the use of anti-epidermal growth factor receptor (EGFR) antibodies such as cetuximab. Following several studies demonstrating the benefit of cetuximab as a single agent or in combination with chemotherapy for advanced colorectal cancer, [1-4] its role was evaluated as an adjunct to chemotherapy and surgery for operable colorectal liver metastases in the New EPOC study. [5] Unexpectedly, this resulted in a shorter progression-free survival for patients treated with cetuximab in contrast to previous data associated with its use in inoperable disease. This effect persisted despite restriction of patients to an all RAS subgroup, suggesting that additional biomarkers linked with treatment efficacy have yet to be identified [6]. MicroRNAs (miRNA) are small non-coding RNA molecules that play a key role in the regulation of intracellular processes through post-transcriptional regulation of gene expression. The stable presence of miRNAs in formalin-fixed paraffin-embedded (FFPE) tissues and their involvement in multiple physiological pathways and pathologies suggests an opportunity to identify new biomarkers for multiple diseases. [7-11] MicroRNAs controlling expression of oncogenes and tumor suppressor genes have been shown to be frequently deregulated in cancer cells [7-9] suggesting potential biomarker candidates for precision oncology. A miRNA which has both oncogenic and tumor suppressive functional roles frequently deregulated in a variety of cancers is mir-31. [12] In colorectal cancer, mir-31 is typically overexpressed with high expression correlating with advanced disease. [13-15] Functional studies have shown mir-31 has pleiotropic activity, promoting colorectal cancer progression through enhanced migration and invasion. [16, 17] While the most frequently identified mature sequence of mir-31 identified in the literature is miR-31-5p, the other mature sequence of mir-31, miR-31-3p, has been reported to be associated with outcomes for patients treated with anti-EGFR therapy. [18-20] As a follow-up to these findings, the present study sought to evaluate whether expression of miR-31-3p could predict outcomes in the setting of operable colorectal liver metastasis in patients treated with chemotherapy plus cetuximab enrolled in the New EPOC study. BODY.RESULTS: The modified ITT population (mITT) was composed of 149 patients, 78 patients in a chemotherapy plus cetuximab arm and 71 patients in a chemotherapy alone arm (Figure 1). One hundred and thirteen of these patients were confirmed all RAS WT. Thirty-six patients in the mITT population had incomplete RAS mutational status by NGS but were known to be KRAS WT (codons 12, 13, and 61) based on pyrosequencing results obtained during the original New EPOC study. Figure 1BODY.FLOW DIAGRAM FOR PATIENTS INCLUDED IN STUDY: BODY.PATIENT BASELINE CHARACTERISTICS IN MITT AND ASSOCIATION WITH MIR-31-3P EXPRESSION: In the mITT population, baseline characteristics were well balanced between treatment arms (Table 1) and between miR-31-3p expression tertiles (Table 2) with the exception of BRAF mutational status, which was more frequent in the high tertile group (6/7 BRAF mutations were in patients with high miR-31-3p expression, p=0.017). Table 1BODY.BASELINE CHARACTERISTICS IN THE MITT POPULATION FOR EACH TREATMENT ARM: Chemotherapyn = 71Chemotherapy plus cetuximabn = 78Sex  F 25 (35.2%) 22 (28.2%)  M 46 (64.8%) 56 (71.8%) Age  [0-60] 20 (28.2%) 19 (24.4%)  [60-70] 34 (47.9%) 39 (50%)  [70+] 17 (23.9%) 20 (25.6%) Chemotherapy regimen  CapOx 15 (21.4%) 18 (23.4%)  FOLFIRI 6 (8.6%) 12 (15.6%)  FOLFOX 49 (70%) 47 (61%) BRAF status  WT/Unknown 68 (95.8%) 73 (94.8%)  Mutated 3 (4.2%) 4 (5.2%) Primary tumor location  Left sided 59 (83.1%) 59 (75.6%)  Right sided 12 (16.9%) 19 (24.4%) Presentation of metastases  Non-synchronous 33 (46.5%) 24 (30.8%)  Synchronous 38 (53.5%) 54 (69.2%) Number of liver metastases  < 4 57 (81.4%) 58 (74.4%)  ≥ 4 13 (18.6%) 20 (25.6%) ECOG performance status  0 51 (71.8%) 54 (69.2%)  1 20 (28.2%) 22 (28.2%)  2 0 (0%) 2 (2.6%) miR tertiles  Low 23 (32.4%) 27 (34.6%)  Intermediate 20 (28.2%) 29 (37.2%)  High 28 (39.4%) 22 (28.2%) Table 2BODY.BASELINE CHARACTERISTICS IN THE MITT POPULATION FOR EACH MIR-31-3P TERTILE: Low n = 50Intermediate n = 49High n = 50p-valueSex  F 13 (26%) 19 (38.8%) 15 (30%) 0.401  M 37 (74%) 30 (61.2%) 35 (70%) Age  [0-60] 11 (22%) 12 (24.5%) 16 (32%) 0.815  [60-70] 27 (54%) 24 (49%) 22 (44%)  [70+] 12 (24%) 13 (26.5%) 12 (24%) Chemotherapy regimen  CapOx 10 (20.4%) 10 (20.4%) 13 (26.5%) 0.439  FOLFIRI 3 (6.1%) 8 (16.3%) 7 (14.3%)  FOLFOX 36 (73.5%) 31 (63.3%) 29 (59.2%) BRAF status  WT/Unknown 49 (98%) 48 (100%) 44 (88%) 0.017  Mutated 1 (2%) 0 (0%) 6 (12%) Primary tumor location  Left+rectum 43 (86%) 39 (79.6%) 36 (72%) 0.229  Right 7 (14%) 10 (20.4%) 14 (28%) Presentation of metastases  Non-synchronous 18 (36%) 19 (38.8%) 20 (40%) 0.933  Synchronous 32 (64%) 30 (61.2%) 30 (60%) Number of liver metastases  < 4 37 (75.5%) 38 (77.6%) 40 (80%) 0.855  ≥ 4 12 (24.5%) 11 (22.5%) 10 (20%) ECOG performance status  0 34 (68%) 34 (69.4%) 37 (74%) 0.850  1 15 (30%) 15 (30.6%) 12 (24%)  2 1 (2%) 0 (0%) 1 (2%) BODY.PATIENT CLINICAL OUTCOMES IN MITT POPULATION: Despite a high percentage of censored patients (52% for progression-free survival and 82% for overall survival) progression-free survival was significantly shorter in the chemotherapy plus cetuximab arm versus the chemotherapy alone arm with a median of 14.5 months versus 24.2 months (HR=1.8 [1.1 ; 2.9], p=0.02). The same was observed for overall survival (HR=2.2 [1.0 ; 4.9], p=0.047). The above aligns with previously published results from the New EPOC interim analysis. [20] BODY.MIR-31-3P AS A PROGNOSTIC MARKER IN PATIENTS TREATED WITH CHEMOTHERAPY PLUS CETUXIMAB: Progression-free survival was not significantly different between the miR-31-3p expression subgroups in the whole mITT population (Figure 2A) with a median progression free survival of 18.0 months in the low expression subgroup versus 15.9 months in the combined mid plus high expression subgroup (HR=1.4 [0.8 ; 2.3] p=0.25). In the chemotherapy alone arm (Figure 2B), progression-free survival was not statistically different between the miR-31-3p expression subgroups (HR=0.9 [0.4 ; 2.0], p=0.79). In contrast, patients in the chemotherapy plus cetuximab arm (Figure 2C) with low miR-31-3p expression had a longer progression-free survival compared to patients in the combined mid plus high expression subgroup (median of 20.3 months versus 12.3 months, HR=2.0 [1.0 ; 4.2], p=0.049). Overall survival was not significantly different between the miR-31-3p expression groups in the whole mITT population (HR=0.9 [0.4 ; 2.1], p=0.86), in the chemotherapy alone arm (HR=0.7 [0.2 ; 2.9], p=0.62), or in the chemotherapy plus cetuximab arm (HR=1.1 [0.4 ; 3.0], p=0.85). Figure 2BODY.KAPLAN-MEIER CURVES OF PROGRESSION-FREE SURVIVAL (PFS) ACCORDING TO MIR-31-3P EXPRESSION LEVEL GROUPS: (A) PFS for the entire miTT population; (B) PFS for the chemotherapy alone treatment arm (C) PFS for the chemotherapy plus cetuximab treatment arm. Objective response rates did not significantly vary according to miR-31-3p expression in the whole mITT population with 34 patients (68%) achieving complete or partial response out of the 50 patients with low miR-31-3p expression and 62 patients (63%) out of the 99 in the mid plus high miR-31-3p expression subgroup, (p=0.59). There was also no difference in objective response rate when analyzed by treatment arm. For the chemotherapy alone arm, 15 of the 23 patients (65%) with low miR-31-3p expression and 27 of the 48 patients (56%) in the mid plus high miR-31-3p expression subgroup achieved complete or partial responses (p=0.61). For the chemotherapy plus cetuximab arm, 19 of the 27 patients (70%) with low miR-31-3p expression and 35 of the 51 (69%) with mid or high miR-31-3p expression had a complete or partial response (p=1.0). BODY.MIR-31-3P AS PREDICTIVE MARKER OF CETUXIMAB EFFICACY ON SURVIVAL OUTCOMES: Patients in the mid plus high miR-31-3p expression group treated with cetuximab had a shorter progression free survival compared to patients treated with chemotherapy alone (median progression free survival 12.3 versus 26.7 months respectively; HR=2.3 [1.3 ; 4.1], p=0.005). This difference was not observed in low expressers (median progression free survival 20.3 months versus 18.9 months respectively; HR=1.1 [0.4 ; 2.6] p=0.91) (Figure 34). The treatment-miR-31-3p expression group interaction test was not significant (p=0.16). Figure 3BODY.KAPLAN-MEIER CURVES OF PROGRESSION-FREE SURVIVAL (PFS) BY TREATMENT ARM: (A) OS for the mid plus high miR-31-3p expressers; (B) OS for low miR-31-3p expressers. Figure 4BODY.KAPLAN-MEIER CURVES OF OVERALL SURVIVAL (OS) BY TREATMENT ARM: (A) OS for the mid plus high miR-31-3p expressers; (B) OS for low miR-31-3p expressers. Objective response rates were not significantly different according to treatment arm. For the mid plus high miR-31-3p expression subgroup, 35 of the 51 patients (68%) in the chemotherapy plus cetuximab arm, and 27 of the 48 patients (56%) in the chemotherapy alone arm achieved either a partial or complete response; p=0.22. For the low expressers subgroup, 19 of the 27 patients (70%) in the chemotherapy plus cetuximab arm and 15 of the 23 patients (65%) in the chemotherapy alone arm achieved either a partial or complete response; p=0.77. BODY.MULTIVARIATE ANALYSES: Consistent with the univariate analysis results, multivariate analyses adjusted for confounding factors showed that miR-31-3p expression group was not a significant predictive factor for progression free survival in the overall population or in the chemotherapy alone arm, but remained significant in the cetuximab treated population (low versus mid-high, HR = 2.1 [1.0; 4.4], p=0.05). BODY.COMPARISON OF MIR-31-3P EXPRESSION BETWEEN METASTASES AND PRIMARY TUMORS: An analysis of miR-31-3p expression in liver metastases collected post neoadjuvant treatment versus primary tumors collected at baseline was performed using 94 paired samples of primary tumors and liver metastases. MiR-31-3p expression was significantly lower in metastases compared to primary tumors with a mean (std) of log-fold of -0.66 (1.18) for primary tumors versus -1.79 (1.37) for liver metastases, p<0.001. Expression levels did not significantly differ between treatment arms either in primary tumors (-0.81 [1.11] for chemotherapy plus cetuximab versus -0.87 [1.18] for chemotherapy alone, p=0.45) or in liver metastases (-1.68 [1.28] for chemotherapy plus cetuximab versus -1.96 [1.43] for chemotherapy alone, p=0.76). The absolute change of expression between treatment arms was not significantly different (0.87 [1.32] for chemotherapy plus cetuximab versus 1.08 [1.62] for chemotherapy alone, p=0.36) (Figure 5). Figure 5BODY.DISTRIBUTION OF MIR-31-3P EXPRESSION (LOGARITHMIC SCALE) BY TREATMENT ARM, IN PRIMARY TUMOR AND LIVER METASTASES: (CT = chemotherapy alone treatment arm; CTX = chemotherapy + chemotherapy treatment arm). In the 47 paired samples from the chemotherapy alone arm, miR-31-3p expression levels in primary tumors and corresponding metastases were highly correlated with a correlation coefficient of 0.42; p=0.0031. In contrast, for the 47 paired samples from the chemotherapy plus cetuximab arm, a lower, non-statistically significant correlation was observed (correlation coefficient = 0.22, p=0.14) (Figure 6A and 6B). Figure 6Correlation of miR-31-3p expression (log-transformed) between paired primary tumors and liver metastases (A) chemotherapy alone treatment arm; (B) chemotherapy + cetuximab treatment arm. BODY.DISCUSSION: The New EPOC study demonstrated a shorter progression-free survival with the addition of cetuximab to chemotherapy for patients with operable colorectal liver metastases. [5] While it is a reminder that the consequences of combining treatment strategies can be unpredictable, it does afford a unique opportunity to increase our understanding of tumor responses to treatment with anti-EGFR therapy. Not only can putative predictive biomarkers be assessed in the “pre-treatment” primary tumor tissue, but the additional availability of “post-treatment” resected liver metastases enables paired analyses to aid a mechanistic dissection of the effect of cetuximab. In our study we observed that in the subgroup of patients treated with cetuximab, patients in the combined mid plus high miR-31-3p expression group had a shorter progression free survival in comparison to patients treated with low miR-31-3p expression. By contrast in the chemotherapy alone arm, progression free survival was similar regardless of miR-31-3p expression level. We also observed that patients in the mid plus high miR-31-3p expression group had a shorter progression free survival and shorter overall survival when treated with cetuximab compared to patients treated with chemotherapy alone, whereas patients with low miR-31-3p expression had similar clinical outcomes for both study treatment arms. Although we could not demonstrate the predictivity of miR-31-3p (homogeneity test p-value p=0.16), this is likely due to the low number of patients and lack of maturity of the data (52% censored patients for progression free survival and 82% for overall survival). The power of this study to demonstrate predictivity, computed a posteriori, was only 65.3%. [21] Similarly, the absence of demonstration of a prognostic effect of miR-31-3p on overall survival in the cetuximab-treated population is likely to be related to the limited number of events. We failed to observe a clear association between miR-31-3p expression and response to cetuximab. This could be due to several reasons including the high objective response rate observed both in the chemotherapy alone (42/71 59%) and cetuximab (54/78 69%) arms suggesting that cetuximab impact on the tumor response was relatively marginal, limiting the capacity to demonstrate an interaction between miR-31-3p expression and response in this setting. While analysis of miR-31-3p expression and its association to clinical outcomes did not enable the ability to identify a patient subgroup that benefited from the addition of cetuximab to chemotherapy, this needs to be interpreted in the context of the results of the New EPOC study in which the addition of cetuximab to chemotherapy produced a detrimental effect. However, our results show that the poorer outcomes associated with the addition of cetuximab to chemotherapy were limited to patients who had middle to high miR-31-3p expression and that this detrimental effect was not observed in patients with low miR-31-3p expression levels. As such, the predictive value of miR-31-3p expression is consistent with the primary outcome of the New EPOC study. The potential interest of miR-31-3p expression as a predictive biomarker of the response to anti-EGFR therapy in metastatic colorectal cancer has been previously reported in several papers. [19, 20] Preliminary results of studies assessing the association with miR-31-3p expression and therapeutic benefit from anti-EGFR therapy have also been reported for the PICCOLO and FIRE-3 studies. [22, 23] The present analysis was performed prior to the determination of the optimal cut-off value for miR-31-3p that has recently been reported based on the analysis of tumors samples from patients enrolled in the FIRE-3 trial. [23] As a result, the present study in which the patient population was separated into tertiles represents an initial exploration of potential cut-off points for miR-31-3p expression as it relates to differential outcomes. Although the specific context and results of the study as well as the lack of maturity of the data, prevent the generalization of the conclusions to other clinical settings, we believe the present study contributes to the accumulation of data supporting the clinical and biological rationale of miR-31-3p expression as a predictive biomarker of response to anti-EGFR therapy in metastatic colorectal cancer. The loss of correlation of miR-31-3p expression level for the cetuximab group between primary tumors, which were collected prior to any treatment, and liver metastasis, which were collected after treatment, suggests that treatment with cetuximab may alter the expression of this microRNA. This finding suggests the regulation of miR-31-3p expression as a part of the EGFR pathway and is consistent with a previous report where maturation of mir31 in the miR-31-5p form was found to be regulated via EGFR in a tumor hypoxia context. [24] It is also important to note that the selection of miR-31-3p for the present study was done following a broad screening of 1,145 miRNAs, [19] and as a result of previous findings that pre-mir-31 leads to maturation of the highly correlated mature forms of mir-31, miR-31-3p and miR-31-5p. [20] Despite this high correlation between the two mature forms of mir-31, prediction of response to anti-EGFR therapy differs between miR-31-3p and miR-31-5p, suggesting the existence of fine regulation mechanisms involving specifically miR-31-3p or genes targeted by miR-31-3p, beyond regulation of mir-31 expression. [18] In summary, analysis of miR-31-3p expression enabled the identification of a subgroup of patients with operable colorectal liver metastases who have a poorer outcome when treated with chemotherapy plus cetuximab. While the mechanism of this differential effect relative to miR-31-3p expression requires investigation, these data support a biological explanation for the detrimental effect observed with the use of cetuximab in patients in the New EPOC study, rather than, for example, inadequate surgery being responsible. Further work is ongoing to validate the use of this promising predictive biomarker in advanced colorectal cancer. BODY.MATERIALS AND METHODS: BODY.PATIENTS AND SAMPLES: The New EPOC trial has previously been reported [5] and the full protocol can be found on the Southampton Clinical Trials Unit website (www.ctu.soton.ac.uk). Patients with resectable or suboptimally resectable liver metastases from colorectal cancer were randomly allocated to two treatment arms; chemotherapy (oxaliplatin/irinotecan plus fluorouracil) with or without cetuximab given before and after liver resection. The study was closed by the Trial Steering Committee on advice from the Data Monitoring and Ethics Committee (DMEC) in November 2012 when the protocol defined futility criteria were met (the lower limit of the 95% Confidence Interval (CI) for progression free survival HR was >1). [25] Primary tumor and liver metastasis samples collected from patients enrolled in the New EPOC trial were analyzed for the present study. As per the New EPOC study protocol, primary tumor samples were collected at baseline and samples for liver metastases were collected following neoadjuvant treatment and liver resection. Primary tumors from 167 of the 236 patients in the original primary analysis population [5] were available for extended RAS mutational testing and miR-31-3p expression in the current analysis. The modified intention to treat (mITT) population was defined as those patients included in the original New EPOC primary analysis population (e.g. those who were KRAS wild-type for codons 12, 13 and 61 at trial entry) in whom further testing did not demonstrate any RAS mutation. This resulted in a mITT population comprised of 149 patients. Liver metastasis samples were available for 147 patients enrolled in the original New EPOC study with paired samples of both primary tumor and metastasis available for a total of 94 included in the mITT population. BODY.MUTATIONAL ANALYSES: Primary tumors and metastases analyzed for the present study were sequenced for KRAS codons 12, 13, 61, 117 and 146, NRAS codons 12, 13, 61, 117 146, and BRAF codon 600 using Sequencing By Synthesis (SBS) on a MiSeq platform (Illumina, San Diego, California). Mutations were identified with respect to two known non-mutated controls for all mutations considered and the three positions of the codon were analyzed when needed. A 99% confidence interval for the minor allele frequency was calculated, taking into account the total number of bases read for a nucleotide in a particular codon and the number of “new” bases read for the same nucleotide of the same codon. Mutations were considered actual results when the same base was identified regarding the 2 controls. A frequency below 5% was considered as non-mutated. Samples which had indeterminate results were resequenced using Ion Torrent™ Technology (Thermo Fisher Scientific, Carlsbad, CA). For this analysis libraries were prepared according the manufacturer’s instruction (Ion AmpliSeq™ DNA and RNA Library Preparation Rev.B, Ion Torrent, Thermo Fisher Scientific, Carlsbad, CA). Samples were amplified with the Ion AmpliSeq™ Cancer HotSpot Panel v2 and the concentration of each library was determined with Experion™ Automated Electrophoresis system (Bio-Rad Laboratories Inc., Hercules, CA). Emulsion PCR was done according to the manufacturer’s user guide (Ion PGM™ Template OT2 200 Kit Revision A.0). Sequencing was performed using two Ion 318TM Chip V2 on an Ion PGM System™. Data analysis, including alignment to the hg19 human reference genome and variant calling, was done using the Torrent Suite Software v4.0. Alignments were visually verified and annotated with the ALAMUT software v2.2 (Interactive Biosoftware, Rouen, France). BODY.MIR-31-3P EXPRESSION ANALYSES: A pathologist reviewed all samples and the tumor area was marked for subsequent macrodissection. Only samples with more than 20% tumor cell content were selected for nucleic acid extraction. For each tumor sample, 5 FFPE slides of 5μm thickness were scratched in the tumor area and total RNA was extracted using the FFPE miRNeasy extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Specific quantification of expression level of miRNA hsa-miR-31-3p was performed on retrotranscribed RNA using specific TaqMan pre-designed assays on ABI7900HT Real-Time PCR System. Expression levels were normalized to a reference miRNA using the ΔΔCt method. BODY.STATISTICAL ANALYSIS: The primary endpoint was progression free survival defined as the time from randomization to recurrence, disease progression, or death, whichever occurred first. Secondary endpoints included overall survival (time from randomization to death; patients still alive at the date of last follow-up were censored) and preoperative response (using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.0). All analyses reported here include data up to November 1, 2012 to match the time frame presented in the interim report to the DMEC. Analyses were performed for the modified Intent-to-Treat (mITT) population which consisted of 149 KRAS WT patients whose RAS status was either WT or indeterminate. Patients whose tumors were confirmed to be KRAS or RAS mutated were excluded from the analysis. In line with the study’s pre-specified statistical analysis plan, patients were divided into three subgroups defined as either low, medium (mid) or high miR-31-3p expression levels based on tertiles of miR-31-3p expression distribution. Progression free survival and overall survival comparisons according to miR-31-3p expression tertile and between each treatment arm were performed using Kaplan-Meier (KM) methodology and two-sided log-rank test. Objective Response Rate according to tertiles and across treatment arms were compared using Fisher exact test, considering non-evaluable patients as non-responders. Because similar results were observed in the mid and high tertile subgroups (Supplementary Figure 1), these subgroups were merged into a single “high” miR-31-3p expression subgroup and subsequent analyses compared the “low” miR-31-3p expression subgroup (low tertile) with the “high” subgroup (mid + high tertiles). Impact of covariates on progression free survival or overall survival (i.e. age, gender, ECOG status, tumor location, BRAF mutational status, chemotherapy backbone, number of liver metastases) was first analyzed in separate univariate Cox models and then in a multivariate stepwise Cox model. Potential predictors with univariate p-value below 0.20 were selected to enter in the model and were kept in the multivariate model if their multivariate p-value was below 0.10. MiR-31-3p predictivity was explored in this multivariate model using an interaction term of treatment with miR-31-3p subgroup. Expression levels of miR-31-3p in LM vs PT were compared using the log of miR-31-3p expression fold and a paired Student t test. A Pearson test was performed to evaluate correlation of miR-31-3p expression in primary tumors and their paired liver metastases. Two-way analysis of variance was used to test paired primary tumor-liver metastases and treatment effects on miR-31-3p expression. BODY.SUPPLEMENTARY MATERIALS FIGURE:

TITLE: Permission Form Synopses to Improve Parents' Understanding of Research: A Randomized Trial ABSTRACT.OBJECTIVE: We hypothesized that, among parents of potential neonatal research subjects, an accompanying cover sheet added to the permission form (intervention) would increase understanding of the research, when compared to a standard form (control). ABSTRACT.STUDY DESIGN: This pilot study enrolled parents approached for one of two index studies: one randomized trial and one observational study. A one-page cover sheet described critical study information. Families were randomized 1:1 to receive the cover sheet or not. Objective and subjective understanding and satisfaction were measured. ABSTRACT.RESULT: Thirty-two parents completed all measures (17 control, 15 intervention). There were no differences in comprehension score (16.8 ± 5.7 v. 16.3 ± 3.5), subjective understanding (median 6.0 v. 6.5), or overall satisfaction with consent (median 7.0 v. 6.5) between control and intervention groups (all p>0.50). ABSTRACT.CONCLUSION: A simplified permission form cover sheet had no effect on parents' understanding of studies for which their newborns were being recruited. BODY.INTRODUCTION: The current research informed consent process often fails to fulfill its goal. Several studies of both adult subjects giving consent and of parents giving permission for their children to participate in research have found their understanding of the research to be unsatisfactorily low [1-3]. This comprehension may be even lower in high-stress populations, such as the parents of neonatal or pediatric oncology patients [4-7]. One potential contributor to the problem of poor subject understanding of research studies is the written consent/permission form. These forms are often long, complex and difficult to read [8, 9]. Attempts at modifying the written forms have yielded conflicting results of the effect on comprehension [5, 6, 10-14]. One approach, a condensed informed consent document, has been associated with higher comprehension in some studies [11, 12, 15-18], but not all [13, 14, 19]. However, few studies of modified forms have used subjects considering participation in an actual (as opposed to hypothetical) research protocol [6, 11, 15, 17, 20, 21]. In the current regulatory environment, shortened consent/permission forms that omit or abbreviate portions required by federal or local institutional review board (IRB) rules are likely to face opposition. In addition, many parents of children with leukemia completing interviews about the permission process express the wish for more information about the study, rather than less [22]. In a recent commentary, experienced pediatric oncology investigators proposed a preamble to the informed permission document that briefly explained the nature of clinical research and the clinical protocol, and referred the parents to specific portions of the complete document [23]. This approach might allow the complete consent/permission form to remain unabridged, but provide the simplified language that has been shown to be associated with better comprehension. This study tested a similar mechanism, an abbreviated cover sheet, in a pilot study among a population (parents of premature infants) at high risk for poor consent comprehension. We hypothesized that, among parents of potential neonatal subjects, an accompanying, one page, cover sheet in addition to the standard permission form (intervention) would increase parent understanding of the nature and risks of the research, when compared to parents receiving only a standard permission form (control). BODY.SUBJECTS AND METHODS: This was a randomized, controlled, single-blind, pilot study. The study was approved by the University of Rochester's Institutional Review Board. Parents of premature infants under 35 weeks' gestation hospitalized in the neonatal intensive care unit (NICU) at Golisano Children's Hospital at the University of Rochester were recruited if their infants were being approached for permission for one of two greater-than-minimal-risk studies. The first of these was a trial of hydrocortisone to decrease bronchopulmonary dysplasia (BPD) (“Hydrocortisone”) and the second was a local, observational protocol including in-hospital and outpatient evaluations, conducted by Rochester site of the Prematurity and Respiratory Outcome Program (“PROP”). The studies (referred to hereafter as the “index” studies) were chosen for entailing greater than minimal risk, but not having significant time pressure for the permission decision. Time from identification of eligibility to enrollment ranged from 7 (PROP) to 14 (Hydrocortisone) days. Inclusion criteria included being a parent of an infant eligible for one of the index studies, permission being sought for one of the studies and the ability to speak, read and understand English. Parents were excluded if the infant's attending physician felt it would be inappropriate to include them. If parents were approached for both index studies, they were eligible for this study only during the approach for PROP, for which approaches occurred earlier in the hospitalization. BODY.INTERVENTION: The intervention was a one-page cover sheet affixed to the front of the standard permission form for the trial for which permission was being sought (Supplemental Figure 1). The form identified the study as research and contained sections that included investigator contact information, the purpose of the study, a brief description of the study procedures, the alternatives, the voluntary nature, the risks, the potential benefits, privacy and confidentiality, the costs, and the payments. Each section contained a reference to the page in the permission form containing the full description of the topic. The cover sheet was designed to have a reading level no higher than 6th to 8th grade. The Hydrocortisone cover sheet had a Flesch-Kincaid Grade Level of 7.1 and the PROP cover sheet, 6.6. The cover sheet was pre-tested for layout and comprehensibility among the investigators, but not pre-tested among parents. Parents also received the full, standard permission form for the relevant index study. Control parents received the standard, full permission form, without the cover sheet. The full forms' Flesch-Kincaid Grade Levels were 12.0 (Hydrocortisone) and 10.7 (PROP). All staff (study investigators or research coordinators) obtaining permission for index studies were trained on the use of the cover sheet. The person presenting the permission form for the index study described the study and answered questions from both groups of parents using the standard procedures for each study, which included verbal review of salient points with all parents and page-by-page review of the permission form with parents interested in participation. The only difference in the presentation of the index study between the two study arms was the inclusion of the cover sheet in the intervention group. All interventions and measures occurred in the NICU or maternity ward. BODY.MEASURES: The main study measure was the Deaconess Informed Consent Comprehension Test (DICCT), a 14-item questionnaire covering study purpose, procedures (2 items), risks, benefits, alternatives, contact information (2 items), compensation for injury (2 items), voluntary nature, freedom to withdraw, lack of participation decision's effect on other care decisions and confidentiality [24]. The DICCT is scored on a 28 point scale, with each of the 14 parameters being scored as 0 = incorrect, 1 = partially correct, 2 = correct, and had face validity, high reliability and preliminary external validity in a previous study [24]. The DICCT was modified as appropriate to each index study, specifically with changes to questions about study procedures (Supplemental Figure 2). The DICCT was administered in an oral format, without a time limit, with a research staff member recording the parent's answers. The participant was allowed to refer to the permission form (and cover sheet, if present) to answer the questions. The participant then completed a short survey that included self-reported age, gender, race/ethnicity and level of education. The survey also contained two, self-administered, 7-point Likert scales, for satisfaction with the consent/permission process and for perceived understanding of the index study. BODY.STUDY PROCEDURES: Eligible parents were asked for verbal permission to participate in a study of consent comprehension before initiation of the permission process for the index study. The initial verbal description did not include mention of the cover sheet or the randomization. Parents who refused verbal permission were not enrolled in the cover sheet study and were given a standard (control) permission form for the index study. Parents who gave verbal permission received either the intervention or control index study permission form. The form was taken, in order, from a sealed envelope in a folder in which the order of intervention and control forms had previously been computer randomized (1:1) by the study statistician. Randomization was stratified by index trial. Parents, but not staff, were masked to study group assignment (by virtue of being unaware of the nature of the intervention). If both parents were present for the approach for permission and both had agreed to participate in the cover sheet study, each parent was considered a subject. Parent pairs were randomized together, as there was no practical way to randomize them separately. Following the completion of the approach for permission (and their decision regarding allowing their infant to participate in the index study), parents were given an information sheet explaining the study and the surveys (but omitting information about the cover sheet and randomization) and, if they agreed, completed the DICCT and associated survey. This was done as soon as possible after the completion of the consent approach for the index study, but not always the same day. Parents were asked to complete the DICCT and survey whether or not they have agreed to give permission for the trial for their infant. When the parents had completed the DICCT and survey, they were given a post-survey debriefing form that explained the purpose of the cover sheet study and the randomization, and were asked for permission to use their data. Staff administering the measures were specifically trained in their use. No demographic or other information was collected on parents who did not initially provide verbal agreement, failed to complete the DICCT and/or survey, or opted out after receiving the post-survey information sheet. Parents who initially agreed, but did not complete the DICCT or opted out were replaced until the planned sample size was reached. BODY.ANALYSIS: The DICCT was reviewed by a single reader (CDA), who scored the DICCT using index-study-specific templates based on published criteria [24]. The central reader was unaware of the study group, demographic survey information and whether the parent had given or withheld permission for the index study. The primary outcome was the score on the post-permission DICCT. The study was powered, with a planned 16 subjects per group, to detect an effect size of 1.0, using α = 0.05 and a power of 0.8 – in this case an increase in the mean DICCT score from 20 (the published mean) to 24, assuming the published standard deviation of 4 points [24]. Secondary outcomes included subjective comprehension, satisfaction and multivariate analysis of primary and secondary outcomes adjusted for covariates. The DICCT scores were expressed as means and analyzed by Mann-Whitney U test. Likert scale scores were expressed as medians and analyzed by Mann-Whitney U test. Other bivariate analyses used the Mann-Whitney U test or Pearson correlation, as appropriate. ANCOVA tested cover sheet effect on outcomes, after adjusting for index study (PROP vs. Hydrocortisone), consent to index study and parent education. All tests were 2-sided. No a priori corrections were made for co-linearity of parent pairs. Analysis was by intention-to-treat. BODY.RESULTS: Study enrollment occurred from March 2012 – April 2013. Sixty-five parents provided verbal permission to participate in this study (Supplemental Figure 3). Thirty-three parents, of whom 23 had refused to have their infants participate in the index study, were unavailable or unwilling to complete the study questionnaires when approached to do so. Of parents who gave a reason, most cited lack of interest in the index study as the reason for declining to complete the questionnaires. Thirty-two of the refusing parents were being approached for the PROP index study; 1 was being approached for Hydrocortisone. Thirty-two parents from 24 families (8 couples included) completed the study questionnaires. Their characteristics are described in Table 1. No participant withdrew consent upon debriefing. Of families with only one parent participating, all participants were women. There were no adverse events. Use of a cover sheet had no effect on objective understanding of the research permission process, as measured by the DICCT, subjective understanding of research permission or satisfaction with the research permission process (Table 2). Participants nearly uniformly rated their subjective understanding of the study as high, despite their being able, on average, to score about 60% the points possible on the objective measure. When the scores were limited to the elements that other parents of premature infants [25] had indicated were most important to the research permission process (study purpose, procedures and risks, defined as questions 1-4 on the DICCT, which would yield a possible high score of 8), subjects were not able to answer a higher proportion of questions correctly, and there was no difference between groups (No Cover Sheet 5.3 ± 1.7 vs. Cover Sheet 5.1 ± 1.8, p = 0.91). Multivariate analysis using linear regression confirmed no effect of the cover sheet when other variables were controlled (Table 3). Being approached for participation in the Hydrocortisone study and higher educational attainment were each independently correlated with higher DICCT scores, with each accounting for about 4 additional points on the DICCT. Post hoc evaluation to assess the independence of family members' scores showed an intra-class correlation coefficient of 0.38 for DICCT scores, consistent with moderate within-class correlation. Repeating the multivariate linear regression for DICCT scores adjusting for within-family correlation showed no difference from the unadjusted analysis (data not shown). Multivariate linear regression incorporating subjective understanding showed no association of any of the variables with subjective understanding (Supplemental Figure 4). Using a similar regression model, only agreeing to have a child participate in the index study was associated with greater satisfaction with the permission process (mean difference 0.83, 95% CI [0.04, 1.63], p = 0.04) (Supplemental Figure 4). Subjective understanding and satisfaction with the consent process were modestly correlated with one another (ρ = 0.38, 95% CI [0.03, 0.64], p = 0.03), but neither showed any significant correlation with DICCT score. BODY.DISCUSSION: In this study of parents of premature infants being approached to have their children participate in actual clinical protocols, we found that adding a simplified cover sheet containing crucial information about the trial did not improve their measured comprehension of the research studies. Overall comprehension in both the experimental and control groups was low, when compared to the norms established for the DICCT in the adult population [24]. The cover sheet also did not affect participants' subjective impression of their understanding of the index study or their satisfaction with the study permission process. As others have reported [26], educational level was positively associated with measured understanding of the study permission documents. However, it is not possible from our data to ascertain whether education might also affect the general test-taking ability of more highly educated participants, leading them to perform better on the DICCT regardless of their actual level of understanding. Overall, parents in the current study could demonstrate understanding of only 55-60% of the information tested by the DICCT. This is lower than the 60-90% understanding recorded among adult patients being approached for actual studies reported in a recent metaanalysis of randomized controlled trials of consent interventions [11]. The finding is in keeping with the reports of others that recent parents, including NICU parents, may be at particular risk for poor comprehension of research permission [4, 7, 17]. However, the tools for measurement of understanding, including the DICCT, vary widely, are used infrequently, and have not been standardized, making it difficult to compare studies [27]. True cross-population and cross-intervention comparisons await the advent of standardized, validated, widely-accepted measures of understanding. Our data raise the possibility that more complex study design and/or presentation (both the PROP study and the PROP consent form were more complex than Hydrocortisone) may impede subjects' understanding of information. Although previous studies suggest that less complex permission forms may improve overall understanding [11, 15-17], it is not clear whether our finding is a result of the information itself or of the definitions we used to quantify understanding. For instance, for a participant to achieve a score of 4 on the DICCT components measuring understanding of study procedures, a participant would have been required to identify at least 6 of 12 procedures and the correct length of the study for PROP, but only the presence of randomization and the correct length of the study for Hydrocortisone. Simplified presentation may not overcome the problems of complex information. Any factor that decreases understanding of the consent process may consequently diminish potential subjects' autonomous decision making capacity. One recent study among adult cancer patients found that consent forms with higher reading levels were associated with higher likelihood of providing consent to enter a clinical trial [28]. Similarly, a study with adult dental patients found that patients with lower educational levels (who would be expected to have lower understanding) were paradoxically less likely to report consent forms to be confusing [29]. This study has unique strengths. Unlike some previous studies among parents of premature infants [16], the study was performed among parents considering actual (rather than hypothetical) index studies. Parents were also masked as to the nature of the consent intervention until after the study was complete. To our knowledge, such a masked design has never been previously used in this population; it may have decreased bias in our outcomes. The study used a previously published, internally-validated measure [24]. The study also has several weaknesses. It was designed as a pilot study, and thus enrolled a small number of participants, increasing the chance of Type 2 error. Low numbers also hampered subgroup and multivariate analyses. The measures also had potential weaknesses, raising the possibility that the findings represent a failure of the measures rather than failure of the intervention. There is no universally-accepted measure of research participant understanding. Although the DICCT has undergone face, internal and preliminary external validation, there are no standard instruments against which to perform full external validation [24]. In our case, unlike the initial report of the DICCT [24], participants' subjective impression of their understanding was not correlated to the objective measure. Qualitative methods such as cognitive interviewing might have yielded further insight into subjects' understanding. The Likert scales used to measure subjective understanding and satisfaction are prone to an “acquiescence bias” that results in a skew toward high values [30]. The population in this study was relatively highly educated. There were chance variations between study groups in baseline characteristics (e.g. education, index study) that we found to be associated with measured understanding, but this did not seem to favor one study group over the other. Only half of the parents who initially agreed to the study completed the study questionnaires. Parents who did not complete the questionnaires were less likely to have agreed to have their children participate in the index study, were more likely to have been approached for PROP, and may have differed in other ways from those who did not complete the questionnaires. The low study completion rate may thus limit the generalizability of the findings. Finally, it was not possible to mask the research staff members to group assignment. However, there was no evidence of a trend toward higher scores in the Cover Sheet group that might have been expected due to unconscious researcher bias. In summary, the use of an abbreviated synopsis of critical elements of the research permission form, in the form of a cover sheet, did not improve the measured or subjective understanding of the research or the satisfaction with the permission process among parents of premature infants being approached to give permission for one of two clinical studies. Our findings are consistent with those of others who have shown that attempts to modify consent documents (without necessarily simplifying them) by changing reading levels, format or organization do not consistently promote understanding of the documents [11, 15]. Specifically, others who have used leaflet or booklet formats, perhaps most similar to this intervention, have reported mixed results [31, 32]. Although this study wasn't designed to assess the complexity of the standard consent form, our data suggest that the overall simplicity of information may improve understanding. This is in keeping with the results of others that suggest shorter, simpler consent forms may promote comprehension [11, 15-17]. Simpler presentation, along with a focus on parental input and the use of other sensory inputs shown to affect comprehension, including verbal presentation, infographics and/or interactive apps on a tablet device, may hold promise as methods for improving understanding in future [7, 11, 12, 16, 25, 33-35]. BODY.SUPPLEMENTARY MATERIAL: 1234

TITLE: Effect of nalbuphine and pentazocine on attenuation of hemodynamic changes during laryngoscopy and endotracheal intubation: A clinical study ABSTRACT.BACKGROUND:: Narcotic drugs have been used to attenuate laryngoscopy and intubation induced circulatory responses, but are not always available due to tough narcotics laws. Nalbuphine a synthetic opioid, free from restrictions has been in use for post-operative pain relief. Hence, we decided to compare nalbuphine and pentazocine for attenuation of hemodynamic effects during larygoscopy and endotracheal intubation in a randomized, double-blind clinical study. ABSTRACT.MATERIALS AND METHODS:: A total of 60 patients (ASA I and II) of either sex, between 18 years and 50 years were given either nalbuphine 0.2 mg/kg (group N, n = 30) or pentazocine 0.5 mg/kg (group P, n = 30) 5 min before induction of general anesthesia. After, induction with thiopentone and endotracheal intubation with succinylcholine balanced anesthesia was maintained with O2:N2O, 0.2% halothane and non-depolarizing relaxants for surgical duration. Changes in heart rate (HR), systolic blood pressure (SBP), diastolic pressure, mean arterial pressure, and rate pressure product calculated by HR × SBP were recorded at various time intervals. ABSTRACT.RESULTS:: A non-significant fall (P > 0.05) up to 3 min and thereafter a significant rise (P < 0.05) in all the parameters were observed throughout the remaining study period with nalbuphine, whereas a continuous and significant (P < 0.05) rise in these parameters were observed with pentazocine. Maximum rise in both the group was observed immediately after larygoscopy and intubation and these started to return toward the basal values at the end of the study period, but remained above the initial values. (pentazocine > nalbuphine P = <0.01). ABSTRACT.CONCLUSION:: Nalbuphine effectively reduces the tachycardia, hypertension, and cardiac workload associated with laryngoscopy and endotracheal intubation. BODY.INTRODUCTION: Hemodynamic changes due to stress responses caused by laryngoscopy and intubation are variable, transient and well-tolerated in American Society of Anesthesiologist I and II patients, but are of great concern for anesthesiologist especially in patients with pre-existing hypertension, myocardial ischemia, and cerebral pathologies with raised intracranial pressure as sudden rise in the heart rate (HR) and the blood pressure (BP) may cause high incidence of arrhythmias, myocardial ischemia, and cerebral hemorrhage.[12] Numerous drugs including topical and/or intravenous lignocaine,[34] short acting beta blockers,[5] Ca++ channel blockers,[6] vasodilators,[78] magnesium sulphate,[9] α2-agonists,[1011] and opiates drugs[121314] have been used to attenuate these responses. Opiate like fentanyl not only produces intense analgesia, but effectively attenuates these cardiovascular responses to stress, but associated problems such as post-operative nausea and vomiting, respiratory depression and unavailability owing to tough narcotic laws make its use difficult in routine. Pentazocine, a synthetically prepared prototypical mixed agonist-antagonist narcotic (opioid analgesic) drug of the benzomorphan class of opioids is widely used in perioperative period as this is free from narcotic laws, but hypertension and tachycardia limits it use during anesthesia, especially in hypertensive and cardiac patients. Recently, nalbuphine a synthetic opioid agonist-antagonist analgesic of the phenanthrene series free from tough narcotics laws is being marketed in India as peri-operative analgesic. This drug causes a reduction in HR and BP, but is sparingly evaluated for attenuation of stress responses during intubation. This has made us to evaluate and compare the effects of nalbuphine and pentazocine, on hemodynamic parameters during laryngoscopy and endotracheal intubation in a randomized double-blind clinical study. BODY.MATERIALS AND METHODS: After obtaining approval from Institutional Ethical Committee, and well-informed written consent, 60 patients (ASA grade I and II) of either sex and between the ages of 18 years and 50 years posted for various surgical procedures under general anesthesia were divided into two groups of small, fixed and equal size (n = 30 each group) by simple random sampling or unrestricted sampling (lottery method) according to drugs used as below: Any patient with extreme of ages, major organ dysfunction, hypertension, myocardial ischemia, arrhythmias, cerebral pathology, and on medication such as Ca++ channel blocker, hypnotic or narcotic analgesic were not included in this study. All patients received an uniform premedication with Inj. glycopyrrolate 0.2 mg I.M., 30 min before the start of anesthesia. Upon arrival in operative room, baseline HR, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MAP) were recorded by non invasive blood pressure automatic apparatus (model infinity vista by Drager) thrice at an interval of 1 min and mean of these three values were recorded as basal value. Other monitoring including Electrocardiogram and SpO2 were also recorded to observe any complications. After securing an intravenous line the study drug was injected slowly over a period of 2 min by a resident doctor who was not involved in the study and did not know the contents of the syringe. After 3 min of pre-oxygenation, the general anesthesia was induced with thiopentone sodium 5 mg/kg followed by succinylcholine 1.5 mg/kg. Endotracheal intubation with appropriate size tube was done and lungs were ventilated with O2:N2O (33%:66%) and 0.2% halothane using Bain's circuit with a fresh gas flow of 110 ml/kg. Muscle relaxation was provided by Inj. vecuronium 0.08 mg/kg (loading dose) and 0.02 mg/kg (incremental doses). All the patients were reversed with Inj. glycopyrrolate 0.4 mg and Inj. Neostigmine 2.5 mg at the end of the procedure. HR, SBP, DBP, and MAP were recorded at 0 min (basal value), 1, 3, 5 min after the study drug, immediately after intubation and again at 1, 3, 5 min after the intubation. Rate pressure product (RPP) was calculated by formula: HR × SBP. Our study was terminated at 5 min after intubation, but all vitals were monitored throughout the anesthesia period as compulsory monitoring schedule. The observations recorded in both groups were tabulated and statistical analysis carried out by using appropriate statistical software (Epi Calc 2000 v1.02 version). Student t test was used for intra and inter group comparison. P > 0.05 was taken to be statistically insignificant, P < 0.05 taken statistically significant and P < 0.01 taken to be statistically highly significant. BODY.RESULTS: Both groups were comparable for their demographic data [Table 1] and baseline variables [Table 2]. Table 1Demographic data Table 2Statistical analysis of various hemodynamic parameters BODY.HR: In group N, after an initial decrease in mean HR, clinically significant increase (+19.56%) was observed immediately after endotracheal intubation (IAETI). Thereafter, HR again started to decrease, but remained above the mean basal values. In contrast, a continuous increase in mean HR was observed maximum being at IAETI (+24.11%) in group P, which started to decrease thereafter but it, remained well above the mean basal values [Table 2]. On intergroup comparison these changes were statistically insignificant (P > 0.05) until IAETI, but became statistically highly significant (P < 0.01) towards the end of the study period [Table 3]. Table 3Inter-group comparison of various hemodynamic parameters BODY.BP: All the three parameters of BP namely SBP, DBP and MAP showed a similar pattern. In group N, mean values of BP after an initial decrease until 5 min after the study drug showed a clinically significant rise at IAETI (maximum) and until 3 min thereafter, but fell below the mean basal values at the end of the study period. In group P, a continuous increase from mean basal values of BP were recorded throughout the study period maximum being at IAETI and remained well above the mean basal values at the end of the study period [Table 2]. On intergroup comparison, these changes were statistically insignificant (P > 0.05) until 5 min after study drugs, but became highly statistically significant a (P < 0.01) at IAETI and statistically significant (P < 0.05) toward the end of the study period [Table 3]. BODY.RPP: In group N, after an initial fall in mean RPP a clinically significant rise (+28.42%) was observed at IAETI which decreased to + 2.72% at the end of the study period. In group P, a continuous increase in the mean RPP above the basal value was observed throughout the study period maximum being at IAETI (+42.0%). Although mean RPP decreased thereafter, but remained well above the basal value (17.7%) [Table 2]. On intergroup comparison, except for the initial 1 min after the study drugs all the changes were statistically highly significant (P < 0.01) [Table 3]. BODY.DISCUSSION: In this comparative study, we evaluated the changes in hemodynamic parameter after the injection of the study drugs, and laryngoscopy and endotracheal intubation. A non-significant fall (P > 0.05) in HR and all the three parameters of BP (SBP, DBP, MAP) till 3 min was observed with nalbuphine, which could be attributed to strong and predominant k agonistic action.[15] Further a non-significant (P > 0.05) rise in HR and fall in BP at 5 min of injection could be attributed to effect of thiopentone sodium used as an induction agent for general anesthesia, a drug very well known for producing hypotension and associated reflex induced tachycardia.[1617] Both the drugs could not abolish the tachycardia associated with laryngocopy and intubation. A clinically significant rise in HR was observed with pentazocine (+24.11%) as compared to nalbuphine (+19.56%), although these values were statistically insignificant on intergroup comparison (P > 0.05). A statistically highly significant rise (P < 0.01) from basal mean values in all the three parameters of BP was observed immediately after intubation in both groups, but clinically this rise was more apparent with pentazocine. Ahsan-ul-Haq et al.[18] reported an increase in HR (+15.5%) and MAP (+10.5%) with nalbuphine immediately after intubation. Adachi et al.[13] reported that pentazocine 0.5 mg/kg failed to diminish the circulatory response to intubation. RPP is a very sensitive index for myocardial work load and increased myocardial oxygen consumption.[19] In our study it is evident that nalbuphine is able to produce significant decrease in RPP as compared to pentazocine immediately after intubation (−28.42% and −42.0% respectively). Toward the end of the study period RPP returned very near to basal values in group N as against to group P (+2.12% and +17.7% respectively). Both the drugs used in our study are mixed opioid agonist antagonist. Nalbuphine binds to μ, κ, and δ opioid receptors, and is primarily κ agonist and μ antagonist.[15] In group N, the initial fall in all the hemodynamic parameters is because of its strong and predominant κ agonistic action. Rise in hemodynamic parameters after intubation are due to sympathoadrenal stimulation.[20] Pentazocine is a partial κ agonist and μ antagonist[21] and is known to cause an increase in arterial BP, HR, systemic vascular resistance and blood catecholamine levels while depressing the myocardial contractility and increasing the cardiac workload, which may be the cause of the steady rise in all the hemodynamic parameters in group P. BODY.CONCLUSION: We conclude that nalbuphine effectively reduces the tachycardia, hypertension, and cardiac workload associated with laryngoscopy and endotracheal intubation.

TITLE: Efficacy and safety of etomidate–midazolam for screening colonoscopy in the elderlyA prospective double-blinded randomized controlled study ABSTRACT.ABSTRACT: ABSTRACT.BACKGROUND AND AIMS:: Recent studies have shown that etomidate is associated with fewer serious adverse events than propofol and has a noninferior sedative effect. We investigated whether etomidate–midazolam is associated with fewer cardiopulmonary adverse events and has noninferior efficacy compared to propofol–midazolam for screening colonoscopy in the elderly. ABSTRACT.METHODS:: A prospective, single-center, double-blinded, randomized controlled trial was performed. Patients aged over 65 years who were scheduled to undergo screening colonoscopy were randomized to receive either etomidate or propofol based on midazolam. The primary outcome was all cardiopulmonary adverse events. The secondary outcomes were vital sign fluctuation (VSF), adverse events disturbing the procedure, and sedation-related outcomes. ABSTRACT.RESULTS:: The incidence of cardiopulmonary adverse events was higher in the propofol group (72.6%) than in the etomidate group (54.8%) (P = .040). VSF was detected in 17 (27.4%) and 31 (50.0%) patients in the etomidate and propofol groups, respectively (P = .010). The incidence rate of adverse events disturbing the procedure was significantly higher in the etomidate group (25.8%) than in the propofol group (8.1%) (P = .008). Moreover, the incidence rate of myoclonus was significantly higher in the etomidate group (16.1%) than in the propofol group (1.6%) (P = .004). There was no statistical significance between the 2 groups with respect to sedation times and sedation-related outcomes including patients’ and endoscopist's satisfaction. In the multivariate analysis, the etomidate group had significantly low odds ratio (OR) associated with VSF (OR: 0.407, confidence interval: 0.179–0.926, P = .032). ABSTRACT.CONCLUSIONS:: We recommend using etomidate–midazolam in patients with high ASA score or vulnerable to risk factors; propofol–midazolam may be used as a guideline in patients with low ASA score. 1BODY.INTRODUCTION: Endoscopic sedation is widely used to relieve patients’ anxiety and discomfort.[1–3] One of the most common sedatives is propofol owing to its convenience and fast effect.[4,5] However, propofol is associated with several serious adverse events including hypoxia, hypotension, arrhythmia, and respiratory depression.[6–8] Etomidate is a nonbarbiturate sedative with rapid onset (5–15 seconds) and recovery (5–15 minutes).[9,10] Because it does not inhibit the sympathetic tone or myocardial function, it is used for critically ill patients or patients with cardiovascular diseases.[10,11] In addition, it can be used in patients with bronchospasms or those who cannot have hypotension because of intracranial problems.[12,13] It is also known that histamine release, apnea, and allergic reactions are less commonly associated with barbiturate or propofol use.[11] Recently, etomidate has been reported to be noninferior in stability and efficacy during endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic ultrasound (EUS) compared to propofol.[14,15] Cardiopulmonary adverse events such as hypotension, hypoxia, arrhythmias, and aspiration were more common in the elderly aged over 65 years than in young, healthy patients.[16,17] However, there were only a few studies on etomidate for screening endoscopy in the elderly. In this study, we compared etomidate and propofol groups based on midazolam in the elderly for screening colonoscopy with respect to cardiopulmonary adverse events. Our aim was to evaluate the safety and efficacy of etomidate–midazolam for screening colonoscopy in the elderly. 2BODY.METHODS: 2.1BODY.STUDY DESIGN: A single-center, prospective, double-blinded, randomized controlled trial was performed from November 2017 to January 2018 at the Department of Gastroenterology and Digestive Endoscopy at Korea University Anam Hospital (Seoul, Korea). At the time of registration, subjects were randomly allocated to either group. They were randomized using a computer-generated list and were provided with written instructions. All patients provided written informed consent. This study was approved by the International Clinical Trials Registry Platform (KCT0002638). 2.2BODY.PATIENTS: All patients aged over 65 years old with American Society of Anesthesiologists (ASA) scores from I to III who were scheduled to undergo screening colonoscopy and/or gastroscopy were included in this study. Patients were excluded if they had a known or suspected history of adverse events with previous sedation; had known hypersensitivity to egg products, soy beans, etomidate, and propofol; had known adrenocortical insufficiency, or porphyria or received chronic corticoid therapy were pregnant or breastfeeding; desired to undergo endoscopy without sedation; and could not provide informed consent. If there was hemodynamic instability (systolic blood pressure [SBP] < 90 mm Hg or peripheral oxygen saturation [SpO2] of 90% on room air or <95% on 2 L/min oxygen) at baseline measurement before the procedure, the patient was withdrawn from the study. 2.3BODY.PROTOCOL: All procedures were performed by one experienced faculty level endoscopist (ESK). Two well-trained nurses who were trained in advanced cardiac life support and completed a training course operated by the Korean Society of Gastrointestinal Endoscopy participated in all procedures. All patients were monitored by the endoscopist and 2 nurses for non-invasive blood pressure, SpO2, electrocardiographic recordings, and respiratory activity before and during endoscopy. They received nasal oxygen therapy at a rate of 2 L/min during the procedure according to the sedation guideline.[16,18] One nurse assisted with the procedure and another nurse checked and recorded the vital signs and patient status while injecting the sedative. Non-invasive blood pressure was automatically measured at every 5 minutes. We used 70% of the usual dose considering the elderly.[16] In both groups, 0.035 mg/kg intravenous midazolam was initially administered. In the etomidate group, 0.07 mg/kg (0.035 mL/kg) bolus injection of etomidate (Etomidate Lipuro, 20 mg/10 mL/A, B. Braun Korea, Seoul, Korea) was administered. After that, titration with 0.035 mg/kg (0.018 mL/kg) etomidate was performed while assessing the patients’ consciousness if the Modified Observer's Assessment of Alertness/Sedation (MOAA/S) scale score was 0 to 2.[19,20] In the propofol group, 0.35 mg/kg (0.035 mL/kg) bolus injection of propofol (Freefol-MCT, 120 mg/12 mL, Daewon Pharm. Co., Ltd., Seoul, Korea) was initially administered. As with etomidate, if the MOAA/S scale score was > 3 to maintain appropriate sedation, additional injections were required after at least 60 s of observation. The MOAA/S scale score ranges from 0 to 5 (0 = general anesthesia and 5 = fully awake state),[19] and the adequate target range of the MOAA/S score is less than 3 (patient responds after his or her name is loudly or repeatedly called) during endoscopy.[14,19] 2.4BODY.ASSESSMENT OF PATIENT SAFETY AND ADVERSE EVENTS: To reduce deviation between endoscopists, one experienced faculty-level endoscopist performed all endoscopic procedures and used a conventional endoscope (Olympus 290, Olympus Corporation, Tokyo, Japan). In patients without glaucoma or benign prostate hyperplasia who underwent colonoscopy, 5 mg of cimetropium bromide (Bropium, 5 mg/1 mL, Bukwang Pharm Co., Ltd., Seoul, Korea) was administered at the start of colonoscopy to reduce colonic motor response. One nurse checked the baseline vital signs before the start of the procedure and injected sedatives after the endoscopist confirmed the Mallampati class.[21] When the patient had a MOAA/S scale score below 2, the vital signs were rechecked and, thereafter, were recorded every 5 minutes. Both the induction time and total procedure time were recorded by the endoscopist. The 2 nurses and endoscopist recorded the adverse events during the procedure, as well as the duration, onset time, and severity of myoclonus. After the procedure, the endoscopist recorded the physicians’ satisfaction, sedation level during the procedure, and any adverse events. After the patient was sent to the recovery room, the observer other than the nurses or endoscopist recorded the patients’ satisfaction, frequency of recall, abdominal pain, and post-procedural nausea or vomiting. 2.5BODY.STUDY ENDPOINT AND DEFINITIONS: The primary outcome was all cardiopulmonary adverse events including tachycardia, bradycardia, hypertension, transient hypotension, respiratory depression, oxygen desaturation, and fatal arrhythmia. The secondary outcomes were the following: vital signs fluctuation (VSF), which was defined as transient hypotension (SBP < 90 mm Hg or at least 20 mm Hg less than the baseline value, even at least once during the procedure) and oxygen desaturation (SpO2 < 90% on room air or < 95% on 2L/min oxygen) [22]; adverse events disturbing the procedure such as belching, severe coughing, needs for restraint, and myoclonus; and sedation-related outcomes including induction time, total procedure time, awake time, patients’ and endoscopist's satisfaction scores, and frequency of recall. We defined a major adverse event as endotracheal intubation, permanent neurological impairment, and death.[14,15] Cardiovascular events included tachycardia (heart rate [HR] > 110 beats/min), bradycardia (HR < 50 beats/min), and fatal arrhythmia (sustained ventricular tachycardia or ventricular fibrillation). Respiratory depression was defined as the need for efforts to secure the airway by chin lift and jaw thrust In addition, we defined induction time as the time interval from sedative injection to endoscope insertion. Total procedure time was defined as the time interval from endoscope insertion to endoscopic removal. Awake time was defined as the time interval from endoscope removal to full-recovery of the patient (Aldrete score of 10).[23] Moreover, total sedation time was defined as the time interval from administration of sedatives to full recovery of the patient.[14] 2.6BODY.STATISTICAL ANALYSIS: To calculate the sample size, cardiopulmonary adverse events according to the presence of etomidate should be first determined using the PASS 12 (NCSS software, Utah) program and their proportions was 28% using the results of Riphaus et al.[24] When the power was set at 80% to detect a moderate effect size (20% of absolute difference between the 2 groups) at an alpha level of 0.05, 57 patients per group were necessary, and the final sample size of 62 patients per group fulfilled the condition as 62 patients per group considering 10% drop-out rate. Continuous variables were expressed as mean ± standard deviation whereas discontinuous variables were expressed as counts and percentages. SPSS 24.0 for Windows (SPSS Inc., Chicago, IL) was used for data entry and statistical analyses. For the analyses between the 2 treatment groups, Student's t-test was used as appropriate to compare the continuous variables, and the chi-square or Fisher's exact test was used for categorical data. Binary logistic regression tests were used for multivariate analysis. P-values < .05 were considered statistically significant. 3BODY.RESULTS: 3.1BODY.DEMOGRAPHIC AND BASELINE CHARACTERISTICS: Initially, 130 patients aged over 65 years were assessed. Among them, six patients were excluded because of adrenocortical insufficiency (n = 1), desire to undergo endoscopy without sedation (n = 3), hypersensitivity to the drug (n = 1), or previous history of adverse events with sedation (n = 1) (Fig. 1). Finally, a total of 124 patients aged over 65 years were enrolled, analyzed, and randomly assigned to 2 groups: propofol group (n = 62) and etomidate group (n = 62). The baseline characteristics including age, sex, procedure type, body mass index, smoking and alcohol history, outpatient status, anticoagulant use, ASA score, modified Mallampati score, and underlying diseases were not significantly different in the 2 groups (Table 1). Additionally, there was no difference in interventional procedures such as polypectomy (P = .773), biopsy (P = .930), and epinephrine injection (P = .934). Sedation-related outcomes such as induction time (P = .833), total procedure time (P = .111) and awake time (P = .070) were not different (Table 2). Figure 1Assembly of patients. Flow chart showed the recruitment of the study patients. Table 1Baseline characteristics. Table 2Procedure and sedation-related outcomes. 3.2BODY.SAFETY-RELATED OUTCOMES: The incidence rate of all cardiopulmonary adverse events was significantly higher in the propofol group (72.6%) than in the etomidate group (54.8%) (P = .040) (Fig. 2). In both groups, there were no major adverse events including endotracheal intubation, permanent neurological impairment, and death. The incidence rate of VSF was significantly higher in the propofol group (50.0%) than in the etomidate group (27.4%) (P = .010). In addition, the incidence rate of transient hypotension was significantly higher in the propofol group (41.9%) than in the etomidate group (24.2%) (P = .036). In contrast, the incidence rate of adverse events disturbing the procedure was significantly higher in the etomidate group (25.8%) than in the propofol group (8.1%) (P = .008). The incidence rate of myoclonus was significantly higher in the etomidate group (16.1%) than in the propofol group (1.6%) (P = .004) (Table 3). Figure 2Assessment of adverse events. ∗Statistical significance between the 2 groups was tested by chi-square analysis for the excellent group. Table 3Major and minor adverse events. When vital sign changes over time were diagrammed on a scatter plot up to 35 minutes, there was a significant difference in change in median SBP with passing time between the 2 groups (P = .020) (Fig. 3A). Median HR significantly increased over time, but there was no significant difference between the 2 groups (Fig. 3B). Median SpO2 was sustained above 98% on 2 L/min oxygen during the procedure. There was no significant difference over time between the 2 groups (Fig. 3C). Figure 3Time course of vital signs fluctuation. (A) Systolic blood pressure (mm Hg), (B) heart rate (beats/min), (C) peripheral oxygen saturation (as a percentage). SBP = systolic blood pressure, HR = heart rate, SpO2 = peripheral oxygen saturation. Multivariate analysis of VSF with logistic regression showed that the etomidate group had significantly low odds ratio (OR) associated with VSF (OR: 0.407, confidence interval: 0.179–0.926, P = .032) (Table 4). Table 4Multivariate analysis for vital signs fluctuation. 3.3BODY.EFFICACY-RELATED OUTCOMES: Outcomes related to the efficacy of sedation including patients’ satisfaction, endoscopist's satisfaction, postprocedural abdominal pain, and postprocedural nausea or vomiting are shown in Table 2. There was no significant difference in patients’ satisfaction (P = .130) and endoscopist's satisfaction (P = .743) between the 2 groups. In addition, differences in patients’ postprocedural abdominal pain (P = .763) and nausea or vomiting (P = .075) between the 2 groups were not statistically significant. Moreover, the frequency of recall was not significantly different in both the groups (propofol group: 2/62 [3.2%], etomidate group: 5/62 [8.1%], P = .439). 4BODY.DISCUSSION: Recently, endoscopic sedation has been widely used.[4,18,25] Although propofol is commonly used as sedative for endoscopic procedures, respiratory depression is a major problem.[5,8,26] Etomidate has emerged as a new sedative that does not affect cardiopulmonary function.[27–30] Recent studies have demonstrated that etomidate for ERCP and EUS is associated with fewer serious adverse events, especially cardiopulmonary adverse events and that its sedative effect is noninferior in normal, healthy patients.[14,15] Hemodynamic instability is more common during sedation in the elderly aged over 65 years than in young, healthy patients.[16,31,32] In addition, cardiopulmonary adverse events during sedation in the elderly can cause more fatal outcomes than in young patients.[32–34] A previous study demonstrated that etomidate-remifentanil was associated with more stable hemodynamic responses and fewer adverse events than propofol-remifentanil during gastroscopy in the elderly.[35] In addition, another study showed that a combination of etomidate and propofol improved hemodynamic stability and minimal respiratory depression during gastroscopy in the elderly.[36] However, there is no study on etomidate for colonoscopy in the elderly. For these reasons, we designed a comparison study in which etomidate can be used alternatively even in elderly patients during screening colonoscopy. Similar to previous studies, we assumed that the etomidate group had fewer patients with oxygen desaturation and respiratory depression. The incidence of all cardiopulmonary adverse events and VSF was significantly higher in the propofol group. Our study has demonstrated that etomidate–midazolam is associated with fewer cardiopulmonary adverse events and its efficacy is noninferior to that of propofol–midazolam in the elderly similar to young patients. However, our experience indicates that patients on etomidate–midazolam more often move and have more adverse events disturbing the procedure than those on propofol–midazolam, which makes the procedure more difficult for the assistant or nurse than for the endoscopist. Therefore, propofol–midazolam may be used as a guideline in patients with low ASA score; however, we recommend using etomidate–midazolam in patients with high ASA score or vulnerable to risk factors, as shown by our study results. In this study, we included not only healthy patients but also patients with an ASA III score and patients with Mallampati scores of 3 and 4. We demonstrated the sedative efficacy and safety of etomidate–midazolam for screening colonoscopy in the elderly and showed that there were more VSF and cardiopulmonary adverse events in the propofol group. This indicated that etomidate–midazolam was hemodynamically stable compared to propofol–midazolam. In addition, we showed that the etomidate group had lower OR associated with VSF than the propofol group in the multivariate analysis. Our study has limitations. First, although the effect of a single injection of etomidate can last up to 72 hours,[37] studies on adrenal insufficiency have not been conducted. Second, we did not study oversedation and undersedation using bispectral index. Third, this randomized controlled trial was conducted at a single center and included a small number of patients. Multicenter randomized trials are required for further confirmation of the results. Fourth, although we intended to assess the efficacy and safety of etomidate–midazolam for colonoscopy, about 66% patients underwent both colonoscopy and gastroscopy in this study. The rate of patients who underwent both colonoscopy and gastroscopy was not significantly different between the 2 groups. However, the inclusion of patients who underwent both colonoscopy and gastroscopy may have led to bias. In conclusion, we recommend using etomidate–midazolam in patients with high ASA score or vulnerable to risk factors; propofol–midazolam may be used as a guideline in patients with low ASA score. BODY.AUTHOR CONTRIBUTIONS: Conceptualization: Hoon Jai Chun. Data curation: Jung Min Lee, Geeho Min, Hoon Jai Chun. Formal analysis: Jung Min Lee, Geeho Min, Eun Sun Kim. Funding acquisition: Eun Sun Kim. Investigation: Seung Han Kim, Bora Keum, Woojung Kim. Methodology: Jae Min Lee, Seung Han Kim, Bora Keum, Hong Sik Lee, Beom Jae Lee, Seong Ji Choi, Woojung Kim. Project administration: Jae Min Lee, Hyuk Soon Choi, Hoon Jai Chun, Seong Ji Choi. Resources: Bora Keum, Beom Jae Lee. Software: Jae Min Lee, Hyuk Soon Choi, Jong-Jae Park, Woojung Kim. Supervision: Bora Keum, Yoon Tae Jeen, Hoon Jai Chun, Hong Sik Lee, Jong-Jae Park, Beom Jae Lee. Validation: Yoon Tae Jeen, Hoon Jai Chun, Seong Ji Choi. Visualization: Yoon Tae Jeen. Writing – original draft: Jung Min Lee. Writing – review & editing: Eun Sun Kim, Hong Sik Lee, Chang Duck Kim, Jong-Jae Park.

TITLE: Serum testosterone changes in patients treated with radiation therapy alone for prostate cancer on NRG oncology RTOG 9408 ABSTRACT.OBJECTIVES: We reviewed testosterone changes for patients who were treated with radiation therapy (RT) alone on NRG oncology RTOG 9408. ABSTRACT.METHODS AND MATERIALS: Patients (T1b-T2b, prostate-specific antigen <20 ng/mL) were randomized between RT alone and RT plus 4 months of androgen ablation. Serum testosterone (ST) levels were investigated at enrollment, RT completion, and the first follow-up 3 months after RT. The Wilcoxon signed rank test was used to compare pre- and post-treatment ST levels in patients who were randomized to the RT-alone arm. ABSTRACT.RESULTS: Of 2028 patients enrolled, 992 patients were randomized to receive RT alone and 917 (92.4%) had baseline ST values available and completed RT. Of these 917 patients, immediate and 3-month post-RT testosterone levels were available for 447 and 373 patients, respectively. Excluding 2 patients who received hormonal therapy off protocol after RT, 447 and 371 patients, respectively, were analyzed. For all patients, the median change in ST values at completion of RT and at 3-month follow-up were −30.0 ng/dL (p5-p95; −270.0 to 162.0; P < .001) and −34.0 ng/dL (p5-p95, −228.0 to 160.0; P < .01), respectively. ABSTRACT.CONCLUSION: RT for prostate cancer was associated with a median 9.2% decline in ST at completion of RT and a median 9.3% decline 3 months after RT. These changes were statistically significant. BODY.SUMMARY: Patients with non-metastatic prostate cancer who were enrolled in the XXXX trial were randomized to radiation therapy (RT) alone and RT combined with 4 months of total androgen ablation. The current study analyzed whether patients who were treated with RT alone experienced testosterone suppression, presumably due to scatter radiation to the testicular Leydig cells. We found a 9.2% decline in serum testosterone levels at completion of RT and a 9.3% decline 3 months after RT. Alt-text: Unlabelled box BODY.INTRODUCTION: Various studies in the contemporary radiation therapy (RT) literature have evaluated changes in serum testosterone (ST) levels for patients receiving external beam RT for prostate cancer1, 2, 3, 4, 5, 6, 7, 8, 9 and other pelvic malignancies.10, 11, 12, 13 The majority of these studies have indicated that these patients experience a decline in ST after RT. Low-dose scatter radiation to the testicular Leydig cells is believed to be the most likely explanation for this phenomenon. The current study evaluates the changes in ST observed in patients who were enrolled and treated in the NRG oncology RTOG 9408 study. BODY.METHODS AND MATERIALS: From 1994 through 2001, 1979 eligible patients with stage T1b, T1c, T2a, or T2b prostate adenocarcinoma and a prostate-specific antigen (PSA) level of 20 ng/mL or less were randomly assigned to RT alone or RT with 4 months of total androgen suppression starting 2 months before RT. The primary endpoint for the study was overall survival. The secondary endpoints included disease-specific mortality, distant metastases, biochemical failure (eg, an increasing PSA level), and the rate of positive findings on the planned repeat prostate biopsy at 2 years. The details of the study design and outcomes are well described in a prior publication.14 RT consisted of either whole pelvic RT (WPRT) to 46.8 Gy plus a 19.8 Gy prostate boost for a total dose of 66.6 Gy or RT to the prostate only (PORT) for a total dose of 68.4 Gy. Typical field arrangements are shown in Figure 1a and 1b.Figure 1Regional lymphatics target volumes. Shown are typical whole pelvis (a) and prostate boost (b) fields used to treat patients enrolled on NRG oncology RTOG 9408. The testicles (in yellow) are shown to be well outside of the radiation therapy beam paths. Figure 1 Most patients received WPRT. Only patients with the lowest-risk features (PSA <10 ng/mL and Gleason score ≤5 or a negative lymph node dissection) were assigned to receive PORT. RT was delivered at 1.8 Gy per fraction. For this analysis, ST levels were investigated at the following collection periods: study enrollment; completion of RT; and first follow-up 3 months after completion of RT. The Wilcoxon signed-rank test was used to compare the change in pre- and post-treatment ST levels (at completion of RT and at the 3-month follow-up visit) in patients who were randomized to the RT-alone arm. The same paired differences were further compared between WPRT and PORT, and a Wilcoxon signed-rank test was used to detect any statistically significant differences. Two different follow-up periods were analyzed: end of RT and first post-RT follow-up. All statistical tests were carried out at the 0.05 significance level. Although it is well recognized that ST levels are subject to diurnal variation, given the large number of patients evaluated, it is unlikely that the results were skewed by any systematic bias as a result of the time of day blood samples were drawn. All laboratory testing was performed at laboratories that were chosen by the institution that enrolled the patient in the study. As such, there was no standardization of the results. Institutional review board approval of the protocol and consent documentation were obtained at each participating institution. All patients signed informed consent prior to study enrollment. BODY.RESULTS: Of the 1979 eligible patients enrolled in the NRG oncology RTOG 9408 study, 992 were randomized to receive RT alone and 925 (93%) had baseline ST values available. Of these 925 patients, 917 (99%) completed RT. Of these 917, 447 had ST information available at the end of RT. None of the 447 patients received hormonal therapy off protocol before the study evaluation. A total of 373 patients had ST values available 3 months after completion of RT. Two of the 373 patients had received hormonal therapy off protocol prior to this evaluation, leaving 371 patients for analysis at that time point. The outliers were included in all analyses. A breakdown of the patients evaluated is shown in Table 1. The pretreatment characteristics of the analyzable patients are shown in Table 2.Table 1Availability of serum testosterone levels for patients in the radiation therapy alone arm of NRG oncology RTOG 9408 (n = 992) Table 1GroupNo. of patientsTotal sample 992 With baseline testosterone 925 With baseline testosterone and completed RT 917 With baseline and end of RT testosterone 447 With baseline and 3-month post-RT testosterone 371a RT, radiation therapy. aTwo patients were excluded because of hormone initiation. Table 2Pretreatment characteristics of eligible patients Table 2WPRT(n = 807)PORT(n = 110)Total(n = 917)Age (y)  Median 71 70 71  Range 47-84 51-88 47-88  Q1-Q3 66-75 67-73 66-74 Karnofsky performance status  70-80 56 (6.9%) 9 (8.2%) 65 (7.1%)  90-100 751 (93.1%) 101 (91.8%) 852 (92.9%) Tumor stage  T1 383 (47.5%) 52 (47.3%) 435 (47.4%)  T2 424 (52.5%) 58 (52.7%) 482 (52.6%) Node stage  N0 16 (2.0%) 16 (14.5%) 32 (3.5%)  NX 791 (98.0%) 94 (85.5%) 885 (96.5%) Differentiation  Well 96 (11.9%) 44 (40.0%) 140 (15.3%)  Moderately 516 (63.9%) 61 (55.5%) 577 (62.9%)  Poor/undifferentiated 195 (24.2%) 5 (4.5%) 200 (21.8%) Prostate-specific antigen levels  <4 75 (9.3%) 14 (12.7%) 89 (9.7%)  4-20 732 (90.7%) 96 (87.3%) 828 (90.3%) Intercurrent disease  Absent 220 (27.3%) 32 (29.1%) 252 (27.5%)  Present 585 (72.5%) 77 (70.0%) 662 (72.2%)  Unknown 2 (0.2%) 1 (0.9%) 3 (0.3%) Gleason score  2-6 448 (55.5%) 103 (93.6%) 551 (60.1%)  7 257 (31.8%) 4 (3.6%) 261 (28.5%)  8-10 77 (9.5%) 2 (1.8%) 79 (8.6%)  Unknown 25 (3.1%) 1 (0.9%) 26 (2.8%) Testosterone level (ng/dL)  Median 370.00 352.29 367.00  Range 42.00-1380.40 76.00-800.00 42.00-1380.40  Q1-Q3 280.00-475.00 274.00-454.00 279.00-466.00 PORT, prostate-only radiation therapy; Q1, first quartile; Q3, third quartile; WPRT, whole pelvis radiation therapy. For the entire group of patients who completed RT and had no prior hormonal therapy, the median change in ST was −30.0 ng/dL at the end of RT (P < .001) and −34.0 ng/dL 3 months post-RT (P < .001). The distribution is shown in Table 3.Table 3Absolute changes in serum testosterone levels Table 3Baseline to end of RTBaseline to 3 months post-RTAll(n = 447)WPRT(n = 386)PORT(n = 61)All(n = 371)WPRT(n = 320)PORT(n = 51)Minimum −608.07 −608.07 −293.95 −478.39 −478.39 −315.85 5th percentile −270.00 −288.18 −178.67 −228.00 −240.50 −182.00 Q1 −109.00 −110.00 −57.64 −99.00 −100.50 −86.46 Median −30.00 −35.29 −11.00 −34.00 −31.35 −41.00 Q3 38.62 40.00 29.00 32.00 41.61 15.00 95th percentile 162.00 172.00 125.00 160.00 169.00 65.00 Maximum 593.66 593.66 158.50 691.00 691.00 86.46 P-valuea < .001 < .001 .128 < .001 < .001 < .001 P-valueb .216 .277 PORT, prostate-only radiation therapy; Q1, first quartile; Q3, third quartile; RT, radiation therapy; WPRT, whole pelvis radiation therapy. aP-value from Wilcoxon signed-rank test comparing to 0. bP -value comparing WPRT vs. PORT from Wilcoxon test. For the subgroup of patients who were treated with WPRT and completed RT and who had no prior hormonal therapy, the median change in ST was −35.3 ng/dL (n = 386) at the end of RT (P < .001) and −31.4 ng/dL (n = 320) 3 months post-RT (P < .001). The distribution is shown in Table 3, and the relative changes are shown in Table 4.Table 4Relative changes in serum testosterone levels Table 4Baseline to end of RTBaseline to 3 months post-RTAll(n = 447)WPRT(n = 386)PORT(n = 61)All(n = 371)WPRT(n = 320)PORT(n = 51)Minimum −96.5% −96.5% −53.4% −96.8% −96.8% −69.7% 5th percentile −51.1% −52.6% −43.5% −46.4% −46.6% −45.1% Q1 −27.3% −27.8% −14.4% −25.8% −26.1% −24.0% Median −9.2% −10.0% −2.5% −9.3% −9.1% −13.5% Q3 12.8% 13.0% 12.4% 9.5% 13.6% 4.8% 95th percentile 59.1% 59.1% 53.0% 53.1% 57.9% 22.2% Maximum 173.1% 173.1% 78.6% 179.2% 179.2% 28.6% P-valuea < .001 < .001 .354 < .001 .001 .001 P valueb .172 .348 Q1, first quartile; Q3, third quartile; PORT, prostate-only radiation therapy; RT, radiation therapy; WPRT, whole-pelvis radiation therapy. aP-value from Wilcoxon Signed-Rank test comparing to 0. bP-value comparing WPRT vs. PORT from Wilcoxon test. For the subgroup of patients who were treated with PORT and completed RT and who had no prior hormonal therapy, the median change in ST was −11.0 ng/dL (n = 61) at the end of RT (P = .13) and −41.0 ng/dL (n = 51) 3 months post-RT (P < .001). The distribution is shown in Table 3 and the relative changes are shown in Table 4. The difference in the median change in ST between the WRPT and PORT groups was not statistically significant at either the end of RT (P = .216) or 3 months after RT (P = .277). An effort was made to correlate change in ST with change in sexual function. A total of 337 patients in the RT-alone group completed an initial Sexual Adjustment Questionnaire (SAQ), and 270 patients completed the SAQ 12 months after treatment. However, from the group of patients who completed both SAQs, we identified only 73 patients who also had baseline and 3-month ST data available. This small number of evaluable patients presented problems on 2 levels. First, there was the unlikelihood that our analysis would have the statistical power to correlate ST change with a change in sexual function. Second, because these 73 patients only represented 22% of the 337 patients who answered the initial SAQ, from a missing data analysis perspective, conclusions could be unreliable and misleading. As such, we are unable to offer a meaningful correlation between changes in ST and change in sexual function. BODY.DISCUSSION: The current study represents the second largest series in the RT literature to evaluate changes in ST in patients receiving RT for localized prostate cancer and the largest study based on data collected as part of a large-scale prospective multi-institutional trial. Although a large share of the posttreatment testosterone data were not collected (only 447 patients had ST levels drawn at the end of RT and 371 at 3 months after RT), we believe that this was a function of the decentralized nature of data collection. We do not believe that this introduces systemic bias into the analysis. Although only short-term data were collected, the findings are consistent with most previously published studies that demonstrate a decline in ST after photon-based RT. These studies stand in contrast to recently published studies that fail to demonstrate such testosterone changes after proton-based RT or brachytherapy. Table 5 lists the 7 studies that were published after 1990, alongside the current study.Table 5Literature review Table 5LiteratureNo. of patientsModalityDoseSerum testosterone changeZagars et al, 19974 85 2D EBRT 68 Gy (median) 9% decline in mean at 3 months Daniell et al, 20013 33 2D EBRT 70 Gy (approximate) 27.3% decline at 3 to 8 years versus prostatectomy patients Pickles et al, 20022 666 3D CRT 65 Gy (range, 52.5-70 Gy) 17% decline in median at 6 months Oermann et al, 20111 26 Robotic Radiosurgery SBRT 36.25 Gy 23.7% decline in median at 12 months (P < .013) Nichols et al, 20129 150 Protons 78-82 Gy (RBE) No significant change Taira et al, 201215 221 Pd-103 BTx ± EBRT Not specified No significant change Kil et al, 20138 217 Protons 70-72.50 Gy (RBE) No significant change Current Series 447 2D-EBRT and 3D-CRT 66.6-68.4 Gy 9% decline at 3 months 2D, 2-dimensional; 3D, 3-dimensional; BTx, brachytherapy; CRT, conformal radiation therapy; EBRT, external beam radiation therapy; RBE, relative biological equivalence; SBRT, stereotactic body radiation therapy. Zagars and Pollack4 reported a 9% decline in ST 3 months after RT in 85 patients treated with RT for prostate cancer. Patients in the series received doses ranging from 66 to 78 Gy (median, 68 Gy) at 2 Gy per fraction to the prostate, only specified at the isocenter. No patient was treated with pelvic nodal RT. The decline in ST was statistically significant at the P = .0001 level. Daniell et al3 compared the ST levels of 33 men who had undergone RT with those of 55 men who had undergone radical prostatectomy 3 to 8 years after treatment. The irradiated patients had a 27.3% lower ST level compared with the surgically treated patients. The investigators suggested that these findings could be explained by radiation injury to the testicles. Pickles et al2 reported on 666 men who were treated with RT for localized prostate cancer between 1994 and 2001. No patient received hormonal therapy. According to the investigators, “few” of the patients received pelvic nodal RT. The vast majority was treated with fields that were limited to the prostate. Six months after completion of RT, the mean ST levels had declined to 83% of the baseline pre-RT levels. The authors attribute this decline to scatter radiation dose to the testicles. With further follow-up, the authors noted a normalization of ST in the vast majority of patients. Oermann et al1 reported on 26 men with low- or intermediate-risk prostate cancer treated with stereotactic body RT (SBRT) with robotic radiosurgery. Patients received 36.25 Gy in 5 fractions of 7.25 Gy to the prostate or prostate and proximal seminal vesicles over a 2-week period. The authors observed a 23.7% median decline in ST levels at 1 year, which was significant at the P = .013 level. One study of testosterone changes after brachytherapy failed to show declines in ST.15 This lack of testosterone suppression was seen in patients treated with brachytherapy alone as well as in patients treated with low-dose external beam RT (20-45 Gy) plus a brachytherapy boost. The authors suggest that the lack of testosterone suppression in this setting is explained by the lower testicular scatter dose associated with brachytherapy. Grigsby and Perez5 reported on 59 patients with prostate cancer who received 65 to 70 Gy to the prostate. In contrast to subsequently published studies, the authors did not identify significant changes is ST for 24 months after RT. However, significant declines in dihydroxytestosterone as well as increases in follicle-stimulating hormone and luteinizing hormone were seen. In contrast to the majority of studies on testosterone kinetics after external photon radiation, 2 recent studies on proton therapy did not demonstrate posttreatment testosterone suppression. Kil et al8 reported on 217 hormone therapy–naive patients with low- or intermediate-risk prostate cancer who received 70.00 (relative biological equivalence [RBE]) to 72.50 Gy (RBE) with hypofractionated passively scattered protons delivered at 2.5 Gy (RBE) per fraction in a prospective study. The median pretreatment ST level was 367.7 ng/dL. There were no significant changes in ST at treatment completion or 6 or 12 months after proton therapy. The authors imply that the lack of testosterone suppression after proton therapy, in contrast to the aforementioned studies of photon-based therapies, is consistent with studies that suggest that passively scattered proton therapy is associated with less out-of-field low-dose scatter radiation than photon-based therapy.16, 17, 18 Nichols et al9 reported on 150 similar patients from the same institution who were treated in an earlier prospective study and received 78.00 (RBE) to 82.00 Gy (RBE) with passively scattered protons delivered at 2.0 Gy (RBE) per fraction. The median pretreatment ST level was 357.9 ng/dL. There were no significant changes in ST at treatment completion or 6, 12, 18, or 24 months after proton therapy. A number of studies have attempted to estimate or measure the scatter radiation dose to the testicles in the setting of prostate RT. Oermann et al1 estimated a median dose of 2.1 Gy (range, 1.1-5.8 Gy) in the setting of robotic radiosurgery SBRT. The study by King et al19 estimated that testicular scatter doses from intensity modulated RT ranged from 0.84 Gy with PORT to 6.3 Gy in the setting of pelvic intensity modulated RT with a prostate boost. Previous studies used thermoluminescence dosimetry (TLD) measurements to estimate the testicular scatter dose to be in the range of 2 Gy, or approximately 3% of the prescribed dose to the prostate,2, 4, 20, 21 although an earlier TLD study by Grigsby5 estimated a testicular dose of 4.5 to 6.0 Gy. Leydig cell damage may be age dependent, with older patients experiencing a greater sensitivity to low-dose radiation in the 2 Gy range. Although the aforementioned studies address the effect of low-dose radiation, or lack thereof, in older men who are treated for prostate cancer, 2 earlier studies of higher-dose testicular irradiation in younger men showed no effect on ST. The study by Rowley et al22 involved the irradiation of the testes of 67 male prisoners with an age range of 25 to 52 years. The doses ranged from 0.08 to 6 Gy. The investigators reported that no significant effect was found on the ST levels of the irradiated patients. The study by Shapiro et al23 followed-up 27 men who had undergone RT for soft-tissue sarcomas. They failed to show changes in the ST levels within 30 months of follow-up. The patients in that series had received a wide range of testicular doses, from 0.01 to 25 Gy. BODY.CONCLUSIONS: External beam RT as delivered in the NRG Oncology RTOG 9408 study was associated with a median 9.3% decline in ST at 3 months after RT. There was no significant difference in this decline between patients who received WPRT and those who received PORT. These findings are consistent with most other series in the photon RT literature and suggest that low-dose scatter radiation outside of the beam path has a deleterious impact on testicular Leydig cell function.

TITLE: Effect of bupivacaine lozenges on oral mucositis pain: a randomized controlled multicenter phase II study A randomized controlled trial showing that a bupivacaine lozenge provided clinically significant pain relief in head and neck cancer patients with radiotherapy-related oral mucositis. ABSTRACT.ABSTRACT: ABSTRACT.INTRODUCTION:: A nonblinded parallel-group randomized controlled study investigated the efficacy and tolerability of repeated administration of a bupivacaine lozenge (25 mg) as pain management for oral mucositis pain in head and neck cancer patients as add-on to standard systemic pain management. ABSTRACT.OBJECTIVE:: The primary end point was the difference between the intervention group (Lozenge group) and the Control group in daily mean pain scores in the oral cavity or pharynx (whichever was higher). ABSTRACT.METHOD:: Fifty patients from 2 hospitals in Denmark were randomized 1:1 to 7 days of treatment with bupivacaine lozenges (taken up to every 2 hours) plus standard pain treatment minus topical lidocaine (Lozenge group) or standard pain treatment including topical lidocaine (Control group). The efficacy analysis included 38 patients, as 12 patients were excluded because of changes in study design and missing data. ABSTRACT.RESULTS:: Mean pain in the oral cavity or pharynx (whichever was higher) was significantly lower 60 minutes after taking lozenges (35 mm [n = 22]) than for the Control group (51 mm [n = 16]) (difference between groups −16 mm, 95% confidence interval: −26 to −6, P = 0.0032). Pain in the oral cavity was also significantly lower in the Lozenge group (18 mm) vs the Control group (36 mm, P = 0.0002). Pharyngeal mucositis pain did not differ significantly (37 mm [Lozenge group] vs 48 mm [Control group], P = 0.0630). No serious adverse events were reported. ABSTRACT.CONCLUSION:: These results show that the bupivacaine lozenge as an add-on to standard pain treatment had a clinically significant pain-relieving effect in patients with oral mucositis. ABSTRACT.CLINICALTRIALS.GOV:: NCT02252926. BODY.1. INTRODUCTION: Eighty to 100% of adult patients who undergo radiotherapy with or without concurrent chemotherapy for head and neck cancer will experience oral mucositis as a painful side effect of their cancer treatment.1,4,9,16 Oral mucositis is painful damage to the mucosa in the oral cavity and the pharynx and is associated with vital oral dysfunction such as swallowing problems.3 It has been identified by patients as the most severe side effect associated with cancer treatment in head and neck cancer patients as it induces severe problems with eating, drinking, and speaking.1,3 For patients with mild to moderate oral or pharyngeal mucositis pain, it would be advantageous to be able to manage pain locally rather than systemically, as local treatments have fewer side effects.15 Various lidocaine preparations, in the form of sprays or viscous solutions, are currently used for local anesthesia of the oral cavity and pharynx,15 but the effect is short lasting and the formulations are not very patient friendly because of unpleasant taste and texture. Such preparations are understood to be of limited, if any, effect in management of pain in patients with oral mucositis.16 Paracetamol, opioids, and gamma-aminobutyric acid analogs are currently the most frequently used systemic analgesic treatments, but a few data on the most beneficial analgesic therapy are available.1 At present, no effective pain treatment without substantial side effects exists for patients with severe oral mucositis pain, and in many patients treated with high-dose opioids there is a lack of sufficient pain relief.8 The lack of adequate pain management for oral mucositis–associated pain has led researchers to conduct numerous studies testing various active compounds in many different formulations in the search for new pain prevention and management options,15 with limited, if any, success. Two phase I studies, each conducted in 10 healthy subjects and 10 patients with head and neck cancer have investigated the safety of a lozenge containing 25 mg of the local anesthetic bupivacaine. They showed no signs of toxic plasma concentrations or risk of aspiration.10,11 Moreover, a pilot study in which a single dose of the lozenge was administered to patients with head and neck cancer showed a strong, long-lasting pain-relieving effect, with a mean duration of maximum pain relief in the oral cavity of 42 minutes and a significant pain relief even after 180 minutes.12 Lidocaine viscous solution has been reported to have a duration of 15 to 20 minutes.6 The aim of this nonblinded parallel-group randomized controlled study was to investigate the efficacy and tolerability of repeated administration of a bupivacaine lozenge (25 mg) as pain management for oral or pharyngeal mucositis pain in patients with head and neck cancer as an add-on to standard systemic pain management. The primary end point was pain in the oral cavity or pharynx (whichever was higher), scored on a 0 to 100 mm visual analog scale (VAS) every 2 hours (Control group) or 60 minutes after taking a lozenge (Lozenge group). BODY.2. METHODS: BODY.2.1. ETHICS: The study protocol and amendments were approved by the National Committee on Health Research Ethics (H-6-2014-034) and the Danish Health and Medicines Authority (EudraCT 2014-002346-42). The study was registered at the Danish Data Protection Agency (AHH-2014-034) and ClinicalTrials.gov (NCT02252926). The study was conducted at 2 sites in Denmark in accordance with the ethical principles that have their origins in the Declaration of Helsinki. The Good Clinical Practice (GCP) unit at Copenhagen University Hospitals monitored the study at both sites. A written declaration of informed consent was obtained from patients before their inclusion in the study. BODY.2.2. PARTICIPANTS: Head and neck cancer outpatients from the Departments of Oncology at Copenhagen University Hospital, Rigshospitalet and Copenhagen University Hospital, Herlev were included in this randomized controlled study. The inclusion criteria in the original protocol were diagnosis of head and neck cancer, age between 18 and 80 years, ability to speak and read Danish, ability to use electronic devices such as a tablet or smartphone, and ability to provide written informed consent. In an amendment to the original protocol (amendment 2.1) that was introduced after 7 patients had been randomized, oral- or pharyngeal mucositis pain of ≥40 mm on a 0 to 100 mm VAS was added as an inclusion criterion to ensure that patients had pain in either the oral cavity or pharynx when they were randomized. In a subsequent amendment (amendment 5), the ability to use electronic devices was removed and a paper diary was introduced instead, because the initial patients had problems registering pain and pain medication intake in the electronic diaries. Five patients (3 in the Lozenge group and 2 in the Control group) used the electronic diary and the remaining patients used the paper diary. The exclusion criteria in the original protocol were need for pain treatment with morphine before the start of radiotherapy, known allergy to bupivacaine or other local anesthetics of the amide type, pregnancy, and breastfeeding. The exclusion criterion related to morphine was removed in amendment 2.1. BODY.2.3. STUDY MEDICINE: The study medicine was manufactured by direct compression at the Capital Region Pharmacy, Denmark. Each lozenge contained 28.16 mg of bupivacaine hydrochloride, which corresponds to 25 mg of bupivacaine, and licorice powder and aspartame to mask the bitter taste of bupivacaine. The lozenge also contained mannitol, talc, and magnesium stearate as fillers. BODY.2.4. DESIGN: This was a nonblinded parallel-group phase II randomized controlled study. Patients with head and neck cancer who fulfilled the inclusion criteria and none of the exclusion criteria were included in the study by a study nurse or pharmacist and thereafter had daily prerandomization screening visits at the hospital (Fig. 1). At these visits, oral mucositis and pain in the oral cavity and pharynx were scored and pain medication use was registered. When their oral mucositis pain could not be relieved by paracetamol treatment (original protocol) or their VAS score for oral- or pharyngeal mucositis pain reached ≥40 mm (after amendment 2.1), patients were randomized to either standard pain management plus repeated administration of bupivacaine lozenges (Lozenge group) or standard pain treatment plus per need topical lidocaine and benzydamine mouthwash (Control group) for 7 days. Standard pain treatment was systemic analgesics (eg, morphine and paracetamol) as needed. The patients in the Lozenge group did not receive lidocaine or benzydamine during the study; otherwise their standard pain treatment was the same as for the Control group. During the 7-day treatment period, the patients had 6 daily study visits at the hospital in conjunction with their radiotherapy (there was no study visit on the 1 day in the week when they did not receive radiotherapy) (Fig. 1). On study day 8 or 9, there was an end of study visit. Figure 1.Overview of study visits after implementation of protocol amendment 2.1. *Day 8 or 9. VAS, visual analog scale. BODY.2.5. STUDY PROCEDURES: According to the original protocol, patients with head and neck cancer were assessed for pain in the oral cavity and pharynx on a daily basis from their first day of radiotherapy. After amendment 2.1 was introduced, patients were screened daily for oral- and pharyngeal mucositis pain from the first day of their third week of 6 scheduled weeks of radiotherapy. From their inclusion in the study to the end of study visit, patients scored their oral- and pharyngeal mucositis pain separately from 0 mm (no pain) to 100 mm (worst conceivable pain) on a VAS. During the prerandomization screening period, patients scored oral- and pharyngeal mucositis pain at each hospital visit. After randomization, the patients in the Lozenge group assessed pain in the oral cavity and the pharynx before and 60 minutes after administration of a lozenge and recorded their values in their patient diary (electronic or paper); patients in the Control group assessed pain in the oral cavity and the pharynx separately on the VAS every 2 hours when awake. Pain assessments were performed at home on each of the 7 days of the treatment period and at the hospital at the end of study visit. At each study visit, patients in the Lozenge group were given study medicine for the next day (on the day before their day without radiotherapy, study medicine for 2 days was supplied). Patients were informed to take lozenges every 2 hours during waking hours up to a maximum of 8 lozenges per day. Each time a patient entered a pain score in the electronic diary, the time of the scoring was recorded automatically. In the paper diary, the patient noted both the time of administration of lozenges as well as the time of pain scoring. Pain scores were also entered in the diaries by the patients in both groups each morning (before they took a bupivacaine lozenge, in the case of the Lozenge group). Pain scores were recorded separately for the oral cavity and for the pharynx in both groups. Along with pain scores and the time of scoring, the patients in both groups were asked to record their use of pain therapeutic drugs during the 7-day treatment period. For all pain therapeutic drugs, including the lozenges, the name and dose of the drug and time of administration were recorded. Pain medication use during the prerandomization screening period was registered by a study nurse or pharmacist. Oral mucositis was assessed using the World Health Organization scale for oral mucositis, which is based on subjective and objective symptoms,14 where 0 = no symptoms; 1 = erythema and/or soreness, no ulcers; 2 = erythema and ulcers, the patient can swallow solid food; 3 = ulcers and marked erythema, the patient cannot swallow solid food; and 4 = oral mucositis to a degree that makes normal nutrition impossible. Scoring was performed by a study nurse or pharmacist who was trained in assessing mucositis. The assessment was conducted during each visit from inclusion to the end of study visit. Adverse events (AEs) were registered at study visits on days 1 to 7 and at the end of study visit. Blood samples were drawn from 10 patients in the Lozenge group to assess whether the bupivacaine concentrations in plasma would accumulate after multiple administrations of the lozenges over 7 days. Method and results of these data on the 10 patients are reported in the study by Mogensen et al., 2017.10 BODY.2.6. RANDOMIZATION: Patients were randomized to either the Lozenge group or the Control group at a ratio of 1:1. The randomization was performed by an independent statistician at PCG Clinical Services AB, who created a computer-generated randomization schedule with randomly varying block sizes to avoid predictability in the assignment of therapy. The randomization schedule was uploaded to the web-based electronic database system Viedoc, supplied by the Contract Research Organization PCG Solutions AB, which assigned the patients to either the Lozenge group or the Control group. BODY.2.7. OUTCOMES: BODY.2.7.1. PRIMARY END POINT: The primary end point was the difference between the 2 groups in daily mean pain scores measured on a VAS. For the Lozenge group, the mean daily pain score for each patient was calculated as the mean of the pain scores recorded 60 minutes after administration of each bupivacaine lozenge. The window for pain registration 60 minutes after taking a lozenge was ±15 minutes. For the Control group, the mean daily pain score for each patient was calculated as the mean of the pain scores recorded every 2 hours. For each assessment (time point), the highest of the scores for the oral cavity and pharynx was used in the calculation of the mean daily pain score for each patient in both groups. BODY.2.7.2. SECONDARY END POINTS: The secondary end points included mean pain (VAS) for the oral cavity and the pharynx, scored separately 60 minutes after administration of a bupivacaine lozenge (Lozenge group) or every 2 hours (Control group); and mean pain (VAS) for the oral cavity and the pharynx, scored separately and as the highest of the values for these 2 anatomical sites immediately before administration of a bupivacaine lozenge (with the exception of the first lozenge of the day) (Lozenge group) or every 2 hours (Control group). For the Lozenge group, the difference between the first pain score (VAS) in the morning (before administration of the first bupivacaine lozenge of the day) and the pain score (VAS) 60 minutes after the first bupivacaine lozenge of the day was separately assessed for the oral cavity and pharynx. The patients registered their use of concomitant pain medication daily from randomization. BODY.2.8. STATISTICAL METHODS: BODY.2.8.1. DETERMINATION OF SAMPLE SIZE: The power calculation was based on pain scores (VAS) from 10 patients with head and neck cancer from a previous study12 and was based on a paired t test. With a minimum clinically relevant difference in pain between the 2 groups of 15 mm, a mean baseline pain of 57 mm, an SD of 15 mm, 80% power, and a significance level of 0.05; it resulted in a sample size of 32 patients completing the study. Approximately 40 patients were scheduled to be randomized to take account of an estimated dropout rate of 20%. BODY.2.8.2. EFFICACY EVALUATION: The objective of the statistical analysis of the primary end point was to determine whether there was a difference in mean pain scores between the 2 groups. This was performed by estimating a mixed model for repeated measures. Treatment day was regarded as the time variable. The response variable was mean daily pain (VAS) for each patient. For each time point with a pain recording, the higher of the recorded pain scores for the oral cavity and pharynx was used. Treatment group and oral mucositis scores were included as categorical independent variables. Baseline pain score (the last VAS score before randomization, recorded on day 1 of the treatment period) was included as a continuous independent variable. In the analysis of the primary end point, the higher of the baseline pain scores for the oral cavity and pharynx was used as the baseline for each patient. An interaction effect between treatment day and treatment group was also included in the model. Mixed model for repeated measures models were also calculated for the secondary end points. These models had the same correlation structure as that used in the analysis of the primary end point. Data for percentage of patients using opioids were analyzed by χ2 test. Results are given as estimate of parameters as a mean difference with a 95% confidence interval (CI). All statistical analyses, except for the analysis of percentage of patients using opioids, were performed using SAS (Version 9.4, SAS Institute Inc, Cary, NC). All tests were 2 sided and were performed at the 5% significance level. BODY.3. RESULTS: BODY.3.1. SUBJECTS: A total of 70 patients with head and neck cancer were screened from October 2014 to November 2015 at the Departments of Oncology at 2 Danish university hospitals. The last patient completed the study in December 2015. Seven patients were randomized under the original protocol, and 43 patients were randomized after approval of amendment 2.1. Twenty-six patients were randomized to the Lozenge group and 24 to the Control group. The 7 patients included under the original protocol were excluded from the efficacy analysis as their baseline pain scores were <40 mm. A total of 38 patients were included in the efficacy analysis (Fig. 2). Of these, 22 were from the Lozenge group and 16 were from the Control group. The median number of pain registrations (VAS scores) per day from baseline to day 7 was 7.5 (range: 0–14) in the Lozenge group and 6 (range: 0–8) in the Control group. Demographic data, cancer diagnosis, and baseline pain scores for the patients are shown in Table 1. There were no major differences in baseline data between the 2 groups, although there was a slight majority of ex-smokers, patients with oral cancer, and patients with oral mucositis score 0 at baseline in the lozenge group. Figure 2.Study flow chart. VAS, visual analog scale. Table 1Demographics and baseline characteristics. BODY.3.2. PRIMARY END POINT: Mean pain in the oral cavity or pharynx (whichever was higher), scored 60 minutes after administration of a bupivacaine lozenge in the Lozenge group and every 2 hours in the Control group, showed a significant difference between groups in favor of the lozenge. Mean pain was 35 mm in patients in the Lozenge group and 51 mm in patients in the Control group (difference between groups −16 mm, 95% CI: −26 to −6, P = 0.0032) (Table 2 and Fig. 3). Table 2Pain measurements. Figure 3.Mean pain in the oral cavity or pharynx (whichever was higher), scored on a visual analog scale (VAS) 60 minutes after administration of a lozenge in the Lozenge group (n=22) and every 2 hours in the Control group (n=16). The error bars show 95 confidence intervals. BODY.3.3. SECONDARY END POINTS: When pain scores for the oral cavity and pharynx were analyzed separately, the Lozenge group showed superior results compared with the Control group (Table 2, Fig. 4, and Fig. 5). When pain for the Lozenge group was measured as the mean pain score 60 minutes after lozenge administration, the intensity of oral cavity pain was significantly lower in the Lozenge group than in the Control group (mean difference: −18 mm, 95% CI: −27 to −10, P = 0.0002). Pharyngeal mucositis pain did not show a significant difference between groups (P = 0.0630) (Table 2, Fig. 4). Figure 4.Mean pain in (A) the oral cavity and (B) the pharynx, scored on a visual analog scale (VAS) 60 minutes after administration of a lozenge in the Lozenge group (n=22) and every 2 hours in the Control group (n=16). The error bars show 95% confidence intervals. Figure 5.Mean pain in (A) the oral cavity and (B) the pharynx, assessed on a visual analog scale (VAS) immediately before administration of the next lozenge in the Lozenge group (n=22) and every 2 hours in the Control group (n=16). The error bars show 95% confidence intervals. When pain for the Lozenge group was measured as the mean pain score immediately before the next lozenge, pain was significantly lower in the Lozenge group than in the Control group for both the oral cavity (mean difference: −15 mm, 95% CI: −23 to −7, P = 0.0004) and the pharynx (mean difference: −11 mm, 95% CI: −22 to 0, P = 0.0452) (Table 2, Fig. 5). BODY.3.4. PATIENT-REPORTED PAIN MEDICATION USE: Pain medications used by patients, in both groups, during the 7-day treatment period included paracetamol, ibuprofen, and opioids. Of the 23 patients assigned to the Lozenge group after amendment 2.1, 22 used the lozenges, and the median number of bupivacaine lozenges consumed per day was 4 (range: 0–7); 1 patient did not take any lozenges and was excluded from the efficacy analysis (Fig. 2). Eight patients in the Control group (50%) used topical lidocaine after randomization. BODY.3.5. ADVERSE EVENTS: In total, 5 AEs were reported in 3 of the 25 patients (12.0%) who consumed at least 1 bupivacaine lozenge. All AEs were assessed as mild and were categorized as follows: dysphagia (1), odynophagia (1), hyperalgesia (2), and salivary hypersecretion (1). The 2 cases of hyperalgesia were assessed as related to the bupivacaine lozenge; the other 3 AEs were assessed as possibly related to the bupivacaine lozenge. The 2 patients with hyperalgesia in the Lozenge group withdrew because of AEs. One AE (malaise) was reported in the control group. No serious AEs were reported in the study. BODY.4. DISCUSSION: Our aim of this nonblinded parallel-group randomized controlled study to test the efficacy and tolerability of repeated administration of a bupivacaine lozenge (25 mg), as an add-on to standard systemic pain management was fulfilled. The results showed that the pain in the Lozenge group 60 minutes after intake of a 25 mg bupivacaine lozenge was significantly lower than that experienced by the Control group. Moreover, the pain-relieving effect of the lozenge was sustained during a relevant period because there was a statistically and clinically significant reduction in pain in the Lozenge group compared with the Control group immediately before intake of the next lozenge. Pain scores for the oral cavity were significantly lower 60 minutes after lozenge administration in the Lozenge group than they were in the Control group. Many patients had pain in both the oral cavity and pharynx, but pain was often lower or absent in 1 of these 2 sites. The fact that all randomized patients included in the efficacy analysis had a minimum baseline pain score of 40 mm in the oral cavity and/or pharynx indicates inadequate standard pain management, as all patients were medicated with one or more systemic drugs at randomization. It was therefore surprising that patients in the Lozenge group only consumed a median of 4 of the 8 bupivacaine lozenges they were permitted to take each day. Possible explanations for the lower than anticipated consumption include that the lozenges may have had a prolonged pain-relieving effect, as shown previously.12 There is an immediate need for improved pain management in patients with head and neck cancer with oral mucositis. Ketamine has been tested in different studies as a topical analgesic administered as an oral mouthwash. In one study, 8 patients with oral mucositis were treated with ketamine mouthwash plus intravenously administered opioids. Five of the 8 patients experienced relief of their mucositis pain, but 4 patients experienced side effects which were probably related to the ketamine mouthwash. The side effects included mild confusion, hallucinations, nausea, and dizziness.17 The theory that inflammation causes increased expression of peripheral opioid receptors has led to studies of the effect of morphine-containing mouthwashes on oral mucositis pain, but the results are not consistent.13,18 A systematic review concluded that a 0.2% morphine mouthwash could be effective in the treatment of oral mucositis pain in patients with head and neck cancer.18 Tricyclic antidepressants, such as doxepin, have also been investigated as pain relief, and the above-mentioned systematic review concluded that a 0.5% doxepin mouthwash could be used to treat oral mucositis.18 In a study on 51 patients with oral mucositis due to radiotherapy, pain decreased after pain management with an oral rinse containing doxepin.5 However, both morphine and doxepin have systemic effects and some patients find the mouthwashes unpleasant, as they feel thick and sticky in the mouth and can cause nausea.7 The above-described studies on systemic drugs show various effects of different kinds of pain management. However, the tested systemic drugs have disadvantages in terms of efficacy, tolerability, and/or patient friendliness. By contrast, topical formulations, such as lozenges, used as local anesthetics in the oral cavity may possess the advantages that the concentration of the anesthetic agent will be high in the oral cavity.2 From a safety perspective, the AEs assessed as related to the bupivacaine lozenge in the present study were few and mild. Two patients withdrew because of hyperalgesia. Study personnel were instructed to be alert to any symptoms indicating systemic toxicity of bupivacaine, and no such symptoms were reported. The reporting of AEs focused on possible side effects of the treatment and did not focus on medical conditions that are common in the studied patient population, such as nausea and somnolence. Patients undergoing radiotherapy often experience dry mouth because of reduced saliva production. In the present study, this may give rise to a prolonged dissolution of the lozenge and reduce the effect of bupivacaine by delaying its transport to the oral cavity and pharynx. The fact that the patients were not hospitalized but instead used a patient diary at home to register pain scores as outpatients made it difficult to ensure that they registered all the required pain scores. However, compliance was judged to be satisfactory as there were enough pain registrations to analyze the data. The severity of oral mucositis was assessed using the World Health Organization scale for oral mucositis. Unfortunately, the study personnel who did the assessments focused solely on visible changes in the oral mucosa; they failed to appreciate that pain, by definition, renders a score of at least 1. This explains why some patients had a mucositis score of 0 at randomization despite a baseline pain score of ≥40 mm. The pain-relieving effect of bupivacaine lozenges has not been tested in a blinded placebo-controlled study. It is difficult to design a blinded placebo-controlled study for several reasons, not least that it would be difficult to blind patients because the local anesthetic effect of bupivacaine has been found to be instant and strong. Therefore, not only is there a risk of unintended bias in favor of the local anesthetic lozenge, but also patients will know whether or not they are receiving the active drug, which makes it very difficult to use a placebo-controlled design. For these reasons, a placebo group was not considered appropriate for this study and the most relevant control was considered to be standard pain treatment. In conclusion, the tested bupivacaine lozenge was found to be an effective and safe treatment to reduce pain due to oral mucositis in patients with head and neck cancer. Further clinical investigations are warranted to investigate whether the bupivacaine lozenge may improve pain relief in this difficult-to-treat condition. Indeed, a multicountry phase III trial is planned. Furthermore, the effect of the bupivacaine lozenge on other important outcomes should also be investigated. Such outcomes might include quality of life, patient nutrition, and the risk of an intermission in cancer treatment due to inadequate patient nutrition. Finally, because the bupivacaine lozenge is a noninvasive and patient-friendly way to alleviate oral mucositis pain, it should be investigated in patients with other cancer diagnoses who also have oral mucositis, such as patients with bone marrow transplant. BODY.DISCLOSURES: S. Mogensen, C. Treldal, and O. Andersen are inventors on a patent grant (EP2701681B1) published October 19, 2016. Proprietor Moberg Pharma AB, Bromma (Sweden). S. Mogensen, C. Treldal, and O. Andersen are stockholders in Oracain II Aps and have a European patent (Grant EP2701681B1) for the bupivacaine lozenge. The clinical development of the bupivacaine lozenge was supported by SEED capital in the period 2010 to 2014. In 2014, the IP rights for the lozenge were licensed to Moberg Pharma AB, Sweden, which is now in charge of further development of the bupivacaine lozenge. In 2014, Moberg Pharma awarded the Clinical Research Centre at Amager and Hvidovre Hospital, Denmark an unrestricted grant for pain research in the amount of 131.800 euros. The remaining authors have no conflicts of interest to declare.

TITLE: Effects of probiotic yogurt on performance, respiratory and digestive systems of young adult female endurance swimmers: a randomized controlled trial ABSTRACT.BACKGROUND: To determine the effects of probiotic yogurt on performance and health status of young adultfemale endurance swimmers. ABSTRACT.METHODS: In a randomized controlled trial, 46 endurance swimmers girls with mean age of 13.8 ±1.8 years,weight of 48.6±7.5kg and height of 159±5.6cm, were studied. Subjects were randomly assigned into two groups,receiving either 400 ml probiotic yogurt (intervention group) or ordinary yogurt (control group) daily for 8weeks. At the beginning and at the end of the study, the 400-m free swimming record was done and the HarvardStep test was employed to measure VO2max. Statistical analysis of the data was performed using SPSS software.This trial has been registered with IRCT ID of IRCT2012122311849N1. ABSTRACT.RESULTS: Average changes in the records of the intervention and control groups were 3.9 and 0.5 seconds, respectively(p= 0.22). The intervention group complained of dyspnea for 2.4 days and the value for the controlwas 4.4 days (p=0.024). Values for ear pain were 0.5 and 1.6 days (p=0.008) respectively. The average numberof episodes of respiratory infection in the intervention group was 0.9 day, which was statistically fewer than thatin the control group (1.4 days), P=0.009. ABSTRACT.CONCLUSIONS: A reduction in the number of episodes of respiratory infections and duration of some symptomssuch as dyspnea and ear pain was observed. Due to the reduction in upper respiratory tract infections of theathletes following intake of probiotic yogurt, improvement in VO2max is possible. BODY. INTRODUCTION : Elite athletes undertaking severe and intense exercises may suffer from an increased risk of upper respiratory tract infections and gastrointestinal symptoms (1-7). It is important for athletes to stay healthy during training period and competition but illnesses such as upper respiratory tract infections and gastrointestinal symptoms can seriously impair athlete’s performance to train (8). It is also known that intensive exercise and strenuous physical training causes a rise in chronic exhaustion and a decrease in athlete’s efficiency (9,10). Impaired athletic performance and fatigue in well trained athletes and its relation to infections and decreased mucosal immunity has been reported (11). Based on reports, 89% of presentations of elite athletes were associated with the viral or bacterial upper respiratory tract infection (12). Illness during training and competition may negatively affect athletic performance (13). Therefore, identifying strategies to improve host resistance and minimizing the risk of illnesses that may compromise athletic performance, is necessary. Probiotic bacteria being defined as live food ingredients occur naturally in fermented food products such as yogurt (14). Probiotics may improve athlete’s efficiency by maintaining healthy gastrointestinal tract function, reducing susceptibility to illnesses such as acute infectious diarrhea and its associated symptoms, enhancing host resistance to upper respiratory tract infections and improved immune function (1,14–17). Many studies have been made on the beneficial effects of the probiotics for human health including effects on gastrointestinal tract function and diseases, immune response, hyperlipidemia, hypertension, and allergic conditions within such groups as children, infants, adults and the elderly (8,12,18,19). Although, there is an increasing interest in the effect of probiotics on the incidence of respiratory tract infections (18), few studies have been carried out on the role of probiotics in the improvement of athletes’ capability during exercise period and competitions (8,11). The initial investigations in athletes have thus far not been convincing (8). This study was performed on elite athletes, who are more susceptible to infection. This study was carried out to determine the effect of receiving probiotic yogurt on the status of respiratory and digestive system of young adult female swimmers in summer training period. BODY. METHODS : Subjects: The studied subjects were 46 female endurance swimmers aged between 11 to 17 years who had taken part in the national 400 and 800 meter crawl swimming competitions of 2009. Subjects had exercise for three times a week. The swimming distance was 3800 meter in 2 hour and 30 minutes for each session. Inclusion criteria were being healthy and having the 400 meter crawl swimming time less than 6 min. and 20 Sec (20). Exclusion criteria included acute gastrointestinal symptoms such as: diarrhea, vomiting and stomachache and intake of antibiotics within the past two months before the study. This study was approved by the University Human Research Review Committee and the National Nutrition and Food Technology Research Institute Ethics Committee. This study has been registered in the IRCT‏ website with the ID of‏ IRCT20121223 11849N1. Procedures: In this randomized controlled trial the subjects were randomly assigned into two groups as follows: group 1) Receiving 400 ml of probiotic yogurt containing 4 × 1010 cfu/ml (Colony forming unit per millimeter) comprising of Lactobacillus Acidophilus SPP, Lactobacillus Delbrueckii Bulgaricus, Bifidobacterium Bifidum, and Streptococcus Salivarus Thermnophilus, (n=23) and group 2) Receiving similar dose of ordinary yogurt, as a control (n=23). The 400 meter free swimming record was conducted, and Harvard step test was also employed to measure VO2max‏. The athlete stepped up and down off a 41cm high bench for 3 minutes at a rate of 22 steps/minute.‏ On finishing the test the number of heart beats was counted for 15 seconds. The number of beats was multiplied in 15 seconds by 4 to give the "step test pulse rate". This final value was used to assess the athlete's VO2max in ml/kg/min (21). This test was performed at the beginning and at end of week eight. All subjects were asked to record the digestive symptoms (such as stomachache, vomiting and diarrhea), respiratory infections (such as rhinitis, fever, sore throat, cough, chest ailing breath by sound, ear pain), the extent of exercise per meter, the intake of medicine, and quantity of the consumed foodstuff product which had been recommended, on the provided questionnaires, daily. The subjects were advised to refrain from other probiotic products which were outside the project plan. Statistical analysis: Statistical analysis of the data was performed using SPSS version 17 (Chicago, IL, USA). Data was presented as mean with standard deviation (SD). Data were checked for normality. The differences between the groups were tested using the t test as a parametric and Mann Whitney U test as a nonparametric test. Comparisons between the 2 groups was performed using 2-sided statistical tests. Differences were considered significant at p< 0.05. BODY. RESULTS : The mean age, weight and height of subjects were 13.8 ±1.8 years, 48.6±7.5 kg and 159 ±5.6 cm, respectively. Mean of anthropometry and the extent of exercise per meter, has been shown in Table 1.‏ Table 2 shows the average change in the performance indices including 400 m crawl swimming record and VO2max in subjects during the study. The average changes in the records of the intervention and control group were 3.9±0.09 and 0.5±0.02 seconds, respectively (p= 0.22). On the other hand, the change in VO2max in the intervened group was 0.56±0.096, and in the control group was 0.01±0.55, ml/kg-1.min-1; that was statistically significant in both groups (p=0.02). Table 3 shows the average duration of respiratory infections in subjects. On the average, the intervention group complained of dyspnea, as a symptom of respiratory infection, for 2.4±2.6 days; the value in the controls was 4.4±2.8 days (p=0.024). For ear pain, another symptom of respiratory infection, the value were 0.5±0.9 and 1.6±1.7 days (p=0.008). As shown in Tables 3 and 4, after 8 weeks of intervention, no significant difference was noted between the 2 groups with regard to the average duration of symptoms of respiratory infections including rhinitis, fever, sore throat, cough, and also digestive disorders such as diarrhea, vomiting and stomachache. Table 1BODY. MEAN OF ANTHROPOMETRY DATA AND EXERCISE DISTANCE IN SUBJECTS, AT THE BEGINNING OF THE STUDY. : Study group Index Probiotic Control p n=23‏(Mean±SD‏) >n=23‏(Mean±SD‏) Age (year) 13.7±1.9 14.0±1.8 0.636 Weight (kg) 46.7±6.3 50.4±8.3 0.097 Height (cm) 158.3±5.1 159.9±6.2 0.328 Exercise distance (m) 11358.7±152.0 11436.9±139.2 0.075 Table 2BODY. MEAN OF THE CHANGE RATE OF PERFORMANCE INDICES IN SUBJECTS. : Study group Index’s change rate Probiotic Control p n=23‏(Mean±SD‏) n=23‏(Mean±SD‏) 400 m crawl swimming record (min-sec) -0.039±0.09 -0.005±0.02 0.222 VO2max (ml/kg-1.min-1) 0.56±096 0.01±055 0.022 Table 3BODY. AVERAGE DURATION OF RESPIRATORY INFECTIONS BASED ON STUDY GROUPS IN SUBJECTS. : Study group Duration of exposure respiratory infections(day) Probiotic Control p n=23‏(Mean±SD‏) n=23‏(Mean±SD‏) Rhinitis (day) 3.2±2.5 4.3±3.0 0.268 Fever (day) 0.5±0.7 1.0±0.9 0.152 Sore throat (day) 0.8±0.9 1.8±1.7 0.08 Cough (day) 2.0±2.4 2.9±3.3 0.348 Dyspnea (day) 2.4±2.6 4.4±2.8 0.024 Ear pain (day) 0.5±0.9 1.6±1.7 0.008 Table 4BODY. AVERAGE DURATION OF DIGESTIVE DISORDERS BASED ON STUDY GROUPS IN SUBJECTS. : Study group Duration of exposure digestive disorders Probiotic Control p n=23‏(Mean±SD‏) n=23‏(Mean±SD‏) Diarrhea(day) 1.0±1.5 1.2±1.3 0.394 Vomiting(day) 0.8±0.9 1.0±1.5 0.981 Stomachache(day) 1.4±1.3 2.0±1.1 0.102 Table 5 presents average number of episodes of respiratory infection and digestive disorders in subjects. The average number of episodes of respiratory infection in the intervention group was 0.9±0.8 day, which was statistically fewer than that in the control group (1.4±0.6 days); the difference was statistically significant (p=0.009). In addition, the average number of episodes of digestive disorders was 0.9±0.8 in the intervention group and 1.6±0.8 in the controls that did not reach statistical significance (P=0.57). Average duration of symptoms of respiratory infections in the intervention group was 4.0±2.7 days which was not statistically fewer than that in the control group (5.4±3.3 days). Average duration of symptoms of digestive problems in the intervention and control group was 1.8±1.7 and 2.5±1.6 days, respectively. After 8 weeks of intervention, no significant difference was found between the 2 groups with regard to the average duration of symptoms of respiratory infection or digestive disorders. Table 5BODY. AVERAGE NUMBER OF EPISODES OF RESPIRATORY INFECTIONS AND DIGESTIVE PROBLEMS IN SUBJECTS. : Study group Average number of episodes of problems Probiotic Control p n=23‏(Mean±SD‏) n=23‏(Mean±SD‏) Digestive disorders (number) 0.9±08 1.6±0.8 0.057 Respiratory infections (number) 0.9±0.8 1.4±0.6 0.009 BODY. DISCUSSION : This study investigated the effect of probiotic yogurt on performance, status of respiratory and digestive system in young adult women endurance swimmers. An important finding in this study was the reduction in the number of episodes of respiratory infections and in duration of some of its symptoms such as dyspnea and ear pain, following the consumption of probiotic yogurt. However, by consumption of such type of yogurt, the decrease in digestive disorders of the athletes did not appear meaningful. According to the results of the present study a non-significant decline in the records of 400 m crawl swimming was observed. In a review of studies on probiotics, while probiotics have not been reported to directly affect athletic performance, probiotics help athletes avoid becoming ill from severe and intense exercises and increase the chances that athletes will stay healthy (1,14). In the present study, mean rate change of 400 m crawl swimming record before and after taking the probiotics yogurt was similar to results of Cox in 2007. In his study on twenty healthy elite male distance runners, he found a reduction in the number of days and severity of respiratory illness in a cohort of highly trained distance runners. He suggested that the effect of amount of yogurt consumed and the level of weekly training were more important among all factors (2). According to the results of our study a significant increase in VO2max was observed which is similar to previous studies (22-24). Previous research have indicated a decrease in severity and duration of colds in both children (25,26) and adults (27). The present study confirms the previous findings that probiotics have similar effects in athletes in training. In our study, the number of subjects with upper respiratory tract infections was lower than that observed in the previous studies on marathon runners (8,28). This is possibly due to differences in the season of the study (summer vs. winter), age groups (14 vs. 40 years), type of sport (swimming vs. marathon) and intervention period. Our findings on the average number of episodes of respiratory infection are in agreement with those of Heath et al (29). In our study the number of healthy days in the intervention group was higher than the control group; it was similar to previous studies (8). One possible explanation is that the present study has been carried out in summer, with a lower chance of colds and flu. Previous studies were carried out in wintertime (18,19,25,30). However the Cox study on 20 healthy male long-distance runners to examine the effect of probiotic capsules containing Lactobacillus fermentum showed a significant reduction in the actual number of respiratory infections, the number of episodes in the probiotics group was less than half the corresponding number in the control group (2). A statistically non-significant reduction in the number of days of digestive disorders included diarrhea, vomiting and stomachache was noted in the probiotic group. Improvement in health status of athletes following intake of probiotics have been reported by Cox and Nicols (2,14). BODY. CONCLUSION : It is concluded that consumption of probiotic yogurt results in a reduction in the number of episodes of respiratory infections and in duration of some of its symptoms such as dyspnea and ear pain; the reduction in digestive disorders did not reach statistical significance. Intake of probiotic yogurt also resulted in a significant improved in VO2max and a non-significant decline in the records of 400 m crawl swimming. Possibly, improvement in VO2max is due to the reduction of upper respiratory tract infections (reduction in the number of episodes of respiratory infections and in duration of some of its symptoms) of athletes following intake of probiotic yogurt. The results also showed that the athletes who take probiotics stay healthier overall. It is necessary to plan the athlete’s regimen carefully and to make sure they eat properly. We recommend further studies using a larger sample size, longer period time, different sexes and age groups, and different seasons to find the effects of probiotic on the health and performance of athletes.

TITLE: Tranexamic acid for hyperacute primary IntraCerebral Haemorrhage (TICH-2): an international randomised, placebo-controlled, phase 3 superiority trial ABSTRACT.SUMMARY: ABSTRACT.BACKGROUND: Tranexamic acid can prevent death due to bleeding after trauma and post-partum haemorrhage. We aimed to assess whether tranexamic acid reduces haematoma expansion and improves outcome in adults with stroke due to intracerebral haemorrhage. ABSTRACT.METHODS: We did an international, randomised placebo-controlled trial in adults with intracerebral haemorrhage from acute stroke units at 124 hospital sites in 12 countries. Participants were randomly assigned (1:1) to receive 1 g intravenous tranexamic acid bolus followed by an 8 h infusion of 1 g tranexamic acid or a matching placebo, within 8 h of symptom onset. Randomisation was done centrally in real time via a secure website, with stratification by country and minimisation on key prognostic factors. Treatment allocation was concealed from patients, outcome assessors, and all other health-care workers involved in the trial. The primary outcome was functional status at day 90, measured by shift in the modified Rankin Scale, using ordinal logistic regression with adjustment for stratification and minimisation criteria. All analyses were done on an intention-to-treat basis. This trial is registered with the ISRCTN registry, number ISRCTN93732214. ABSTRACT.FINDINGS: We recruited 2325 participants between March 1, 2013, and Sept 30, 2017. 1161 patients received tranexamic acid and 1164 received placebo; the treatment groups were well balanced at baseline. The primary outcome was assessed for 2307 (99%) participants. The primary outcome, functional status at day 90, did not differ significantly between the groups (adjusted odds ratio [aOR] 0·88, 95% CI 0·76–1·03, p=0·11). Although there were fewer deaths by day 7 in the tranexamic acid group (101 [9%] deaths in the tranexamic acid group vs 123 [11%] deaths in the placebo group; aOR 0·73, 0·53–0·99, p=0·0406), there was no difference in case fatality at 90 days (250 [22%] vs 249 [21%]; adjusted hazard ratio 0·92, 95% CI 0·77–1·10, p=0·37). Fewer patients had serious adverse events after tranexamic acid than after placebo by days 2 (379 [33%] patients vs 417 [36%] patients), 7 (456 [39%] vs 497 [43%]), and 90 (521 [45%] vs 556 [48%]). ABSTRACT.INTERPRETATION: Functional status 90 days after intracerebral haemorrhage did not differ significantly between patients who received tranexamic acid and those who received placebo, despite a reduction in early deaths and serious adverse events. Larger randomised trials are needed to confirm or refute a clinically significant treatment effect. ABSTRACT.FUNDING: National Institute of Health Research Health Technology Assessment Programme and Swiss Heart Foundation. BODY.INTRODUCTION: Spontaneous (non-traumatic) intracerebral haemorrhage is the cause of up to 20% of all strokes, yet accounts for nearly half of all stroke deaths worldwide. Survival after intracerebral haemorrhage has not changed for several decades,1 and the only intervention that improves functional outcome is early intensive blood pressure lowering.2 Around a quarter of intracerebral haemorrhages are complicated by haematoma expansion, which most often occurs within the first few hours, but can occur at up to 24 h, and is associated with poor outcomes.3, 4, 5 Radiological markers, including the CT angiography (CTA) spot sign, have been used to try to predict which patients are at greater risk of haematoma expansion.6 Drug therapies aimed at limiting haematoma expansion include recombinant factor VII, but a meta-analysis of this and other haemostatic therapies found no benefit on functional outcome.7 In patients with traumatic haemorrhage (including from head injuries), tranexamic acid, an antifibrinolytic drug, significantly reduces death due to bleeding and all-cause mortality, with no increase in vascular occlusive events.8 A post-hoc analysis of the CRASH-2 trial showed that because death due to bleeding occurred early after trauma, timely administration of tranexamic acid was necessary for patients to receive any benefit.9 A meta-analysis of tranexamic acid in traumatic intracranial haemorrhage showed that it was associated with a significant reduction in subsequent intracranial bleeding,10 and a larger trial is ongoing.11 Tranexamic acid also reduced the number of deaths due to bleeding in women with post-partum haemorrhage.12 Use of tranexamic acid after acute intracerebral haemorrhage has been tested in two small randomised studies,13 including TICH-1,14 which assessed the feasibility of a larger trial. The administration of tranexamic acid was feasible and well tolerated. BODY.RESEARCH IN CONTEXT: Evidence before this study We searched the Cochrane Stroke Trials register, the Cochrane Central Register of Controlled Trials, MEDLINE Ovid, and Embase Ovid for randomised controlled trials of antifibrinolytics and tranexamic acid up to Nov 27, 2017, using the terms “tranexamic acid” and “exp basal ganglia hemorrhage”or “intracranial hemorrhages” or “cerebral hemorrhage” or “intracranial hemorrhage, hypertensive”. To identify further published, ongoing, and unpublished randomised controlled trials we scanned bibliographies of relevant articles and searched international registers of clinical trials in Nov 27, 2017. We searched for trials in all languages. The quality of evidence was assessed with the GRADE approach. We found two small randomised controlled trials of tranexamic acid with a total of 54 participants, with no clear evidence of benefit or harm associated with tranexamic acid. Five further randomised controlled trials are ongoing. Added value of this study TICH-2 is, to our knowledge, the first large multicentre, international, randomised controlled trial of tranexamic acid in acute spontaneous intracerebral haemorrhage. It included an older population than in previous tranexamic acid trials after trauma and post-partum haemorrhage. Tranexamic acid was not associated with any significant improvement in functional outcome at 90 days, despite a significant reduction in the number of participants with haematoma expansion and fewer deaths by days 2 and 7 among those allocated tranexamic acid. Tranexamic acid was safe, with fewer serious adverse events and no increase in thromboembolic events compared with placebo. Implications of all the available evidence Although there is insufficient evidence to support the routine use of tranexamic acid in clinical practice for spontaneous intracerebral haemorrhage, the results do not exclude a possible small effect. The reductions in haematoma expansion and early deaths are promising, but larger randomised trials are needed to confirm or refute a clinically significant treatment effect. Future research should also investigate which subgroups of patients are most likely to benefit. Therefore, the Tranexamic acid for hyperacute primary IntraCerebral Haemorrhage (TICH-2) trial tested the hypothesis that intravenous tranexamic acid reduces death and dependence when given within 8 h of spontaneous intracerebral haemorrhage. BODY.METHODS: BODY.STUDY DESIGN AND PARTICIPANTS: TICH-2 was an international double-blind, randomised, placebo-controlled, parallel group, phase 3 trial. Participants were enrolled by investigators from acute stroke units at 124 hospital sites in 12 countries: Denmark, Georgia, Hungary, Ireland, Italy, Malaysia, Poland, Spain, Sweden, Switzerland, Turkey, and the UK. Ethics approval was obtained at each site and country before the start of the study. The trial was adopted in the UK by the National Institute Health Research (NIHR) Clinical Research Network and registered as ISRCTN93732214. The full TICH-2 trial protocol and statistical plan have been published.15 Adults with acute intracerebral haemorrhage were eligible for inclusion if they were admitted to a participating hospital within 8 h of stroke symptom onset (or time last seen well). We chose 8 h as the treatment window for consistency with both of the previous trials of tranexamic acid in traumatic intracerebral haemorrhage.9 We sought to include participants as quickly as possible because haematoma expansion usually occurs in the first few hours after intracerebral haemorrhage,3, 5 but allowed randomisation up to 8 h because some patients present later and haematoma expansion can occur up to 24 h.16, 17 Key exclusion criteria were intracerebral haemorrhage secondary to anticoagulation, thrombolysis, trauma, or a known underlying structural abnormality; patients for whom tranexamic acid was thought to be contraindicated; prestroke dependence (modified Rankin Scale [mRS] score >4); life expectancy less than 3 months; and Glasgow Coma Scale score less than 5. The complete list of exclusion criteria has been published previously.15 Investigators obtained written informed consent from each participant if they had the capacity to provide it. If participants could not give consent, a relative or representative gave proxy consent. When consent was deferred or given by a proxy, we informed the participant about the trial as soon as possible and sought their consent. BODY.RANDOMISATION AND MASKING: Randomisation was done centrally in real time. A secure website was used to randomly assign all participants eligible for inclusion to receive tranexamic acid or matching placebo, with 1:1 allocation. The random allocation sequence was generated by the trial programmer. Randomisation was stratified by country, with minimisation for key prognostic factors: age, sex, time since onset, systolic blood pressure, stroke severity on the National Institutes of Health Stroke Scale (NIHSS), presence of intraventricular haemorrhage, and known history of antiplatelet treatment use immediately before stroke onset. Sharp Clinical Services (Crickhowell, UK) prepared individual masked treatment packs containing four 5 mL glass ampoules of tranexamic acid 500 mg or sodium chloride 0·9%, which were made identical in appearance by the addition of a heat shrink sleeve. Ampoules and the treatment pack were labelled with a unique pack number. Sharp Clinical Services stored the treatment packs and distributed them to pharmacies within trial sites using a web-based system of control. The pharmacy at each participating site received numbered supplies from Sharp Clinical Services. The packs were stored at room temperature and protected from excessive heat and freezing in a restricted access area (more detail is provided in the protocol). The randomisation system allocated each participant a unique number corresponding to a treatment pack containing either tranexamic acid or placebo. Treatment allocation was concealed from all staff and patients involved in the trial. BODY.PROCEDURES: The intervention, tranexamic acid, was given intravenously as a 1 g loading dose in 100 mL normal saline 0·9% infused over 10 min, followed by another 1 g in 250 mL normal saline 0·9%, which was infused over 8 h. The comparator was a matching placebo (normal saline 0·9%), administered with an identical regimen. At randomisation, investigators recorded the participants' age, sex, and medical history, as well as their assessment of intracerebral haemorrhage location, intraventricular haemorrhage, and spot sign. Investigators assessed prestroke dependence with the mRS, and stroke severity using the NIHSS and Glasgow Coma Scale. Participants were reviewed at day 2, day 7, and on the day of death or hospital discharge, whichever came first, to gather information on clinical assessment (NIHSS), the process of care measures (eg, blood pressure lowering treatment, neurosurgical intervention), and discharge date and destination (eg, home or institution). Adherence was assessed by examining the participant's drug chart and recording the trial treatment administered at day 2 (ie, whether all treatment was given, the time and date of the two doses, and any other comments). Adherence was verified by both central review of the drug chart and pharmacies recording returns of residual or unused trial medications. Central assessors, who were trained and certified in administration of the mRS and masked to treatment allocation, did the final follow-up at 90 days by telephone from the coordinating centre in each country. If the participant or carer could not be contacted, they received a questionnaire covering the same outcome measures by post. Brain imaging by CT was done as part of routine care before enrolment; a second research CT scan was done after 24 h of treatment to assess haematoma expansion. When multiple scans were done, the scan closest to 24 h after randomisation was used. Central independent expert assessors, who were masked to treatment assignment, assessed CT scans for the location of the intracerebral haemorrhage using a web-based adjudication system. Semi-automated segmentation of the intracerebral haemorrhage was done on Digital Imaging and Communications in Medicine-compliant images to give intracerebral haemorrhage volumes. The user-guided three-dimensional active contour tool18 in the ITK-SNAP software (version 3.6) was used for segmentation and one of three assessors did manual editing as required. All assessments were masked to treatment assignment. Haematoma expansion was defined as an absolute increase of more than 6 mL or a relative growth of greater than 33%. As part of standard routine care, participants received blood pressure lowering treatment, neurosurgery, and venous thromboembolism prophylaxis as appropriate, in accordance with clinical guidelines. BODY.OUTCOMES: The primary outcome was functional status at day 90, as assessed with the mRS, which was administered by telephone or postal questionnaire and masked to treatment allocation. This scale is a 7-level ordered outcome ranked as 0 (no symptoms), 1 (no disability despite symptoms), 2 (slight disability but able to look after own affairs), 3 (moderate disability but able to walk without assistance), 4 (moderately severe disability; unable to walk or attend to own bodily needs), 5 (severely disabled; bedridden and requiring constant nursing care), or 6 (death). It is the most commonly used primary outcome in the context of acute stroke trials, and is recommended in international guidelines. Prespecified secondary outcomes included neurological impairment at day 7 or discharge (whichever came first) assessed with the NIHSS, health-related quality of life measured with EuroQoL-5 dimensions (EQ-5D) health utility status and visual analogue scale, activities of daily living according to the Barthel index, cognition assessed via a modified Telephone Interview for Cognitive Status-modified.(TICS-M) and verbal fluency, mood assessed with the Zung depression scale (ZDS), costs (length of hospital stay and discharge destination), and radiological efficacy (change in haematoma volume from baseline to 24 h and haematoma location). Data on the day 365 outcomes specified in the protocol are still being collected and will be reported separately. We prespecified subgroups in the statistical analysis plan:19 age (<70 years vs ≥70 years), sex (female vs male), time from onset to randomisation (<3 h vs ≥3 h), mean systolic blood pressure (<170 mm Hg vs ≥170 mm Hg), stroke severity (NIHSS score <15 vs ≥15), presence of intraventricular haemorrhage (no vs yes), known history of antiplatelet treatment before stroke onset (no vs yes), spot sign on CTA (yes vs no), intracerebral haemorrhage location (supratentorial deep vs supratentorial lobar), and ethnicity (other vs white). Prespecified safety outcomes were death, venous thromboembolism, ischaemic events (stroke, transient ischaemic attack, myocardial infarction, acute coronary syndrome, peripheral artery disease), and seizures. These were reported up to day 90, along with all serious adverse events in the first 7 days. Safety outcomes and serious adverse events were independently adjudicated masked to treatment assignment. Serious adverse events were categorised in accordance with the medical dictionary for regulatory authorities (MeDRA). BODY.STATISTICAL ANALYSES: The total sample size based on an ordinal primary analysis was estimated at 2000 participants, assuming significance of 5%, power of 90%, an ordinal odds ratio (OR) of 0·79, a distribution of the mRS based on data from participants with primary intracerebral haemorrhage in the ENOS trial20 (4% had mRS 0, 17% had 1, 16% had 2, 19% had 3, 24% had 4, 7% had 5, and 13% had 6), increases due to loss of follow-up at 5%, and a 20% reduction for baseline covariate adjustment. The OR of 0·79 was chosen because it lay in the range seen in related trials.10, 21 The effect of tranexamic acid on an unfavourable functional status (ie, death, vegetative state, or fully dependent requiring attention day and night or dependent but not requiring constant attention) had a relative risk of 0·77 (95% CI 0·59–1·02) when given within 8 h of traumatic intracerebral haemorrhage.11 We followed a prespecified statistical plan19 and analysed the primary outcome as a shift in the mRS at 90 days, using ordinal logistic regression with adjustment for the stratification and minimisation criteria. We tested the assumption of proportional odds with the likelihood ratio test. We also did sensitivity analyses of the day 90 mRS without adjustment and as a binary outcome (dichotomised at mRS 0–3 vs 4–6). Analyses were done in accordance with the intention-to-treat (ITT) principle, with participants kept in the groups to which they were allocated by the minimisation algorithm. We defined the ITT group as all participants who underwent randomisation. Safety outcomes were also analysed in the ITT population. 19 As detailed in the statistical analysis plan, any missing minimisation criteria were given the highest risk value in order for that participant to be included in the analyses. For one participant for whom we did not know if they were taking previous antiplatelet therapy before their intracerebral haemorrhage, we assigned the highest risk value, which in this case was yes. No other missing values were imputed. In case treatment was associated with asymmetric effects on death and other outcome measures, an extreme value was used in participants who had died before day 90 for outcomes that do not include death as part of their scale (0 for EQ-5D health utility status, −1 for EQ visual analogue scale, −1 for TICS-M, and 102·5 for ZDS). The trial was overseen by a trial steering committee, and an international advisory committee consisting of each national coordinator. A trial management committee based at the Stroke Trials Unit in Nottingham, UK, was responsible for day-to-day conduct of the trial. An independent data monitoring committee reviewed the unmasked data every 6 months. Study data were collected, monitored, and analysed in Nottingham. The trial was done in accordance with the principles of good clinical practice and the Declaration of Helsinki. BODY.ROLE OF THE FUNDING SOURCE: The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had overall responsibility for the decision to submit for publication. BODY.RESULTS: Recruitment started on March 1, 2013, and ended on Sept 30, 2017, after a 12-month extension was sought and approved to enable the trial to reach its target sample size. This slower than planned recruitment was due to delays in opening trial sites outside the UK. Subsequently, recruitment increased, and the Trial Steering Committee agreed that the study should exceed the target of 2000 and continue until the end of the extension. Therefore, a total of 2325 participants were recruited from 124 sites in 12 countries over 55 months. 1161 participants were randomly assigned to receive tranexamic acid and 1164 to receive placebo. Most participants were recruited in the UK (1910 [82%] of 2325; appendix). The mean age was 68·9 years (SD 13·8) and 1301 (56%) participants were male (table 1). The median time from stroke onset to randomisation was 3·6 h (IQR 2·6–5·0) and 833 (36%) participants were recruited within 3 h. Mean baseline systolic blood pressure was 173 mm Hg (SD 27·5) and diastolic blood pressure was 93 mm Hg (18·4). 1371 (59%) participants had a haematoma that was deep and supratentorial, whereas 738 (32%) had one that was lobar and supratentorial; 745 (32%) participants had intraventricular haemorrhage. Mean haematoma volume was 24·0 mL (SD 27·2) and median haematoma volume was 14·1 mL (IQR 5·9–32·4). Contrast-enhanced imaging in the form of CTA was done in 249 (11%) participants. Of these individuals, 24 (20%) of 121 in the tranexamic acid group and 32 (25%) of 128 in the placebo group were spot positive. Treatment groups were well balanced at baseline (table 1).Table 1Baseline characteristics Tranexamic acid (n=1161)Placebo (n=1164)Age*, years 69·1 (13·7) [29–97] 68·7 (13·9) [20–101] >70 584 (50%) 580 (50%) Sex*, male 642 (55%) 659 (57%) Ethnic origin White 986 (85%) 992 (85%) Other 174 (15%) 172 (15%) Onset to randomisation*, h 3·6 (2·6–5·1) [1·0–20·8] 3·7 (2·6–5·0) [0·8–8·0] ≤3 421 (36%) 412 (35%) ≤4·5 779 (67%) 796 (68%) History Previous antiplatelet therapy* 316 (27%) 295 (25%) Statin use prior to admission 319 (28%) 303 (26%) Previous stroke or transient ischaemic attack 173 (15%) 156 (14%) Ischaemic heart disease 110 (10%) 92 (8%) Prestroke mRS 0 (0–1) [0–4] 0 (0–1) [0–4] Glasgow Coma Scale 13 (2·2) [5·0–150] 14 (2·1) [5·0–15·0] NIHSS score* 13 (7·5) [0·0–41·0] 13 (7·5) [0·0–42·0] Systolic blood pressure*, mm Hg 172 (27·5) [98·0–265] 174 (26·8) [99·0–265] Diastolic blood pressure, mm Hg 93 (18·4) [46·0–179] 94 (17·8) [35·5–162] Haematoma location Supratentorial lobar 379 (33%) 359 (31%) Supratentorial deep 675 (58%) 696 (60%) Infratentorial 73 (6%) 76 (7%) Combination 34 (3%) 33 (3%) Intracerebral haematoma volume (mL) 14·1 (5·9–32·4) [0·0–207] 12·5 (5·1–31·9) [0·0–163] Intraventricular haemorrhage* 382 (33%) 363 (31%) CT angiography done 121 (11%) 128 (11%) Spot positive 24 (20%) 32 (25%) Spot negative 97 (80%) 96 (75%) Data are n (%), mean (SD), or median (IQR). Values in square brackets are ranges. Baseline information was missing for one participant for ethnic origin, one for history of previous antiplatelet therapy, 18 for history of statin use, 24 for history of previous stroke or transient ischaemic attack, 27 for history of ischaemic heart disease, 29 for history of thromboembolism, 48 for CT angiography, and 52 for haematoma volume. mRS=modified Rankin Scale. NIHSS=National Institutes of Health Stroke Scale. *Minimisation criteria. Adherence to per protocol treatment for the allocated treatment was high: 2207 (95%) of 2325 participants received all of their randomised treatment, whereas only 15 (1%) received no treatment (appendix). Adherence did not differ between the groups. The median time from randomisation to treatment was 21 min (IQR 13–33). The primary outcome of mRS at day 90 was assessed in 2307 (99%) of 2325 participants; nine (<1%) were lost to follow-up and nine (<1%) withdrew from their day 90 follow-up (figure 1). There was no difference in the distribution (shift) in the mRS at day 90 after adjustment for stratification and minimisation criteria, with an adjusted odds ratio (aOR) of 0·88 (95% CI 0·76–1·03, p=0·11; table 2 and figure 2). A formal goodness-of-fit test showed no evidence that the proportional odds assumption was violated (p=0·97). In a sensitivity analysis, we detected no difference between the groups in the proportion of participants who were dead or dependent at day 90 (mRS >3) and the aOR was 0·82 (95% CI 0·65–1·03, p=0·08; table 2).Figure 1Trial profile Figure 2Shift plot of day 90 mRS An mRS score of 0 represents no symptoms, 1 represents no disability despite symptoms, 2 represents slight disability but able to look after own affairs, 3 represents moderate disability but able to walk without assistance, 4 represents moderately severe disability (unable to walk or attend to own bodily needs), 5 represents severely disabled (bedridden and requiring constant nursing care), and 6 represents death. mRS=modified Rankin Scale. Table 2Primary and secondary outcomes Tranexamic acid (n=1161)Placebo (n=1164)AdjustedEffect estimate (95% CI)p valuePrimary outcome, day 90 Participants with mRS outcome 1152 1155 Ordinal OR 0·88 (0·76 to 1·03) 0·11 mRS 0 26 (2%) 24 (2%) .. .. mRS 1 115 (10%) 124 (11%) .. .. mRS 2 197 (17%) 181 (16%) .. .. mRS 3 187 (16%) 194 (17%) .. .. mRS 4 213 (18%) 221 (19%) .. .. mRS 5 164 (14%) 162 (14%) .. .. mRS 6, death 250 (22%) 249 (22%) .. .. Sensitivity analysis, day 90 mRS, unadjusted .. .. Ordinal OR 1·00 (0·86 to 1·15) 0·97 mRS >3 814 (71%) 826 (72%) Binary OR 0·82 (0·65 to 1·03) 0·08 Haematoma Change in volume from baseline to 24 h*, mL 3·72 (15·9) 4·90 (16·0) MD −1·37 (−2·71 to −0·04) 0·0432 Participants with haematoma expansion† 265 (25%) 304 (29%) Binary OR 0·80 (0·66 to 0·98) 0·0300 Day 7 Death by day 7 101 (9%) 123 (11%) Binary OR 0·73 (0·53 to 0·99) 0·0406 NIHSS day 7 10·13 (8·3) 10·29 (8·3) MD −0·43 (−0·94 to 0·09) 0·10 Day 90 Death by day 90 250 (22%) 249 (21%) HR 0·92 (0·77 to 1·10) 0·37 EQ-5D HUS, out of 1 0·34 (0·4) 0·34 (0·4) MD 0·01 (−0·01 to 0·04) 0·30 EQ-VAS, out of 100 48·81 (33·8) 48·34 (33·1) MD 1·75 (−0·52 to 4·02) 0·13 Barthel index, out of 100 52·92 (44·0) 53·21 (43·7) MD 1·70 (−0·90 to 4·31) 0·20 TICS-M, out of 39 13·57 (12·5) 13·94 (12·8) MD −0·19 (−1·12 to 0·74) 0·69 ZDS, out of 100 67·28 (29·5) 67·29 (29·9) MD −0·35 (−2·60 to 1·90) 0·76 Global analysis (Wei-Lachin test) .. .. MWD 0·00 (−0·05 to 0·04) 0·85 Discharge information Length of stay in hospital, days 63·12 (47·1) 63·73 (48·1) MD 1·09 (0·97 to 1·24) 0·16 If discharged, days well at home 69·94 (28·6) 72·15 (29·1) MD −0·72 (−3·73 to 2·28) 0·64 Disposition at discharge Home 465 (40%) 453 (39%) Binary OR 1·14 (0·93 to 1·40) 0·20 Institution 505 (43) 506 (43%) Binary OR 0·99 (0·83 to 1·18) 0·90 Died by discharge 190 (16%) 205 (18%) Binary OR 0·83 (0·65 to 1·07) 0·15 Data are n (%) or mean (SD), unless noted otherwise. OR=odds ratio. MD=mean difference. HR=hazard ratio. MWD=Mann-Whitney difference. mRS=modified Rankin Scale. NIHSS=National Institutes of Health Stroke Scale. EQ-5D HUS=EuroQol-5 dimensions health utility status. EQ-VAS=EuroQol visual analogue scale. TICS-m=Telephone Interview for Cognitive Status-modified. ZDS=Zung Depression Scale. *Adjusted for baseline haematoma volume. †Haematoma expansion defined as an increase of >6 mL or a growth of >33%. When the primary outcome was assessed in prespecified subgroups (figure 3), the only significant interaction was between mRS and baseline systolic blood pressure (interaction p=0·0188), such that participants with a baseline systolic blood pressure less than or equal to 170 mm Hg had a favourable shift in mRS with tranexamic acid compared with those with a systolic blood pressure greater than 170 mm Hg. There was no heterogeneity of treatment effect by time of administration (figure 3), whether dichotomised as less than 3 h versus 3 h or longer (interaction p=0·75) or as less than 4·5 h versus 4·5 h or longer (interaction p=0·28); similarly, there was no interaction between treatment effect and time when analysed as a continuous variable (aOR 0·98, 95% CI 0·90–1·07, p=0·69).Figure 3Primary outcome by subgroups All subgroups were predefined except for intracerebral haemorrhage volume, which was added as a post-hoc analysis. OR=odds ratio. NIHSS=National Institutes of Health Stroke Scale. Fewer participants had haematoma expansion at day 2 in the tranexamic acid group (265 [25%] of 1054 participants) than in the placebo group (304 [29%] of 1058 participants; aOR 0·80, 95% CI 0·66 to 0·98, p=0·0300). The mean increase in haematoma volume from baseline to 24 h was also less in the tranexamic acid group (3·72 mL, SD 15·9) than in the placebo group (4·90 mL, 16·0; adjusted mean difference −1·37, 95% CI −2·71 to −0·04, p=0·0432). Neurological impairment (mean NIHSS score at day 7) did not differ between the tranexamic acid group and placebo group (adjusted mean difference −0·43, 95% CI −0·94 to 0·09; p=0·10). There were no significant differences in any of the day 90 functional outcomes between treatment groups—ie, activities of daily living, mood, cognition, or quality of life (table 2). Length of hospital stay and discharge disposition did not differ between treatment groups (table 2). By day 7, fewer patients had died in the tranexamic acid group (101 [9%] of 1161) than in the placebo group (123 [11%] of 1164; table 2). However, the numbers of deaths by day 90 did not differ between the tranexamic acid group (250 [22%] patients) and the placebo group (249 [21%] patients; table 2). Survival did not differ between the treatment groups over 90 days (adjusted hazard ratio 0·92, 95% CI 0·77–1·10, p=0·37; appendix). Participants in the tranexamic acid group had fewer predefined safety outcomes and serious adverse events than those in the placebo group at day 2 (379 [33%] patients vs 417 [36%] patients, p=0·0272), day 7 (456 [39%] vs 497 [43%], p=0·0200), and 90 days (521 [45%] vs 556 [48%], p=0·0393; appendix). There was no increase in venous thromboembolic events (39 [3%] patients in the tranexamic acid group vs 37 [3%] in the placebo group; p=0·98) or arterial occlusions (myocardial infarction, acute coronary syndrome, or peripheral arterial occlusion) in the tranexamic acid group compared with the placebo group (appendix). Seizure was the most common safety outcome (77 [7%] patients in the tranexamic acid group vs 85 [7%] in the placebo group) and nervous system disorders were the most common serious adverse events (149 [13%] vs 163 [14%]), followed by infections (98 [8%] vs 116 [10%]). BODY.DISCUSSION: In this trial of tranexamic acid versus placebo after acute intracerebral haemorrhage, there was no significant difference between the groups in the primary outcome of functional status at day 90. However, in the tranexamic acid group, we detected significant reductions in the prespecified secondary outcomes of early death, haematoma expansion, and serious adverse events, consistent with tranexamic acid having an antifibrinolytic effect after intracerebral haemorrhage. Tranexamic acid was associated with a small but significant reduction in haematoma expansion and smaller haematoma volumes, key factors that are known to affect outcomes after intracerebral haemorrhage.4, 6 However, the small reduction in haematoma volume (1·37 mL smaller in the tranexamic acid group than in the placebo group) might have been insufficient to translate into improved functional status in this population. One explanation for our findings could be that the anticipated treatment effect (OR 0·79) was too large, which the trial was unable to detect. Indeed, previous randomised controlled trials of tranexamic acid in other settings have enrolled more than ten times the number of participants to identify smaller effects on bleeding-related deaths after trauma (OR 0·85)8 and post-partum haemorrhage (OR 0·81).12 Furthermore, the findings of an individual patient data meta-analysis of 40 138 participants have subsequently shown that it is necessary to start tranexamic acid within 3 h of the start of bleeding to receive any benefit in other conditions.14 We found no evidence of an increase in serious adverse effects with tranexamic acid; notably, there was no increase in venous thromboembolism in this significantly older population with more comorbidities than participants in previous studies of tranexamic acid.8, 12 It is therefore unlikely that any potential benefit of tranexamic acid was offset by harm, as has been suggested with recombinant factor VIIa.22 In a phase 3 trial, there was no evidence of clinical benefit from recombinant factor VIIa, which was associated with a reduction in haematoma expansion but an increased risk of arterial occlusive events.22 Although tranexamic acid and recombinant factor VIIa are both haemostatic agents, tranexamic acid acts through antifibrinolytic mechanisms and recombinant factor VIIa is a procoagulant, so they have different risk-benefit profiles. To date, the only intervention to improve functional outcome after intracerebral haemorrhage is early intensive blood pressure lowering.2 Although no significant effects on haematoma growth were detected in INTERACT-2,2 secondary analysis suggested that blood pressure lowering did attenuate bleeding in a dose-dependent manner.23 The interaction between baseline systolic blood pressure and treatment in our study suggests that participants with lower blood pressure were more likely to benefit from tranexamic acid. This finding could have been confounded by stroke severity, given that larger haematomas have increased blood pressure and worse outcomes.21, 24 Baseline haematoma volume is the strongest predictor of outcome after spontaneous intracerebral haemorrhage, and in an exploratory post-hoc analysis, participants with a baseline haematoma volume of between 30 mL and 60 mL who received tranexamic acid seemed to have better outcomes (figure 3). Although this finding could be due to chance, it is also compatible with the notion that patients with moderately sized haematomas might be more likely to benefit from tranexamic acid, and hence could be targeted for future studies, as has been postulated with recombinant factor VIIa.21, 22 The strengths of this study include its double-blinding, allocation concealment, low risk of bias, high adherence to treatment, and very few missing data on primary outcomes. Treatment groups were well balanced for baseline factors. The use of approved brief and proxy consent processes allowed the rapid enrolment of patients without the capacity to consent, which is important to avoid bias in acute stroke studies. Our inclusion criteria were deliberately broad to reflect the clinical population and to facilitate recruitment from multiple international sites. Nevertheless, most participants were recruited from the UK. The study had several other limitations. We did not collect screening logs so are unable to present data on eligibility. Wide inclusion criteria led to a heterogeneous population with more severe strokes, larger haematoma volumes, and a greater proportion of lobar haematomas and intraventricular haemorrhage than populations in other intracerebral haemorrhage trials,2, 21, 22, 25 which could have diluted any potential treatment effect. Finally, despite efforts to ensure rapid treatment, most participants were enrolled more than 3 h after the onset of intracerebral haemorrhage, which could explain the absence of a significant interaction with time in the subgroup analysis. Several smaller randomised controlled trials of tranexamic acid in intracerebral haemorrhage are ongoing (STOP-MSU [NCT03385928], STOP-AUST [NCT01702636], TRAIGE [NCT02625948], TRANSACT [NCT03044184], and TICH-NOAC [NCT02866838]; total enrolment is fewer than 1000 participants across the trials), and a meta-analysis of individual patient data is planned.26 Future trials would need to enrol far more patients to allow the detection of a small treatment effect, or enrich their population with patients at greatest risk of haematoma expansion, primarily by treating patients earlier.27 Although patients presenting early are at greater risk of haematoma expansion, they also have more severe strokes and larger haematoma volumes.24 Identification of patients most likely to benefit from haemostatic therapy on the basis of factors other than time of onset has been suggested,28 but enrichment with the CTA spot sign has yet to be successful.29 In summary, tranexamic acid did not affect functional status at day 90, although potential benefits were seen with reductions in haematoma expansion, early death, and serious adverse events. The observed effect size was smaller than anticipated and is compatible with a lack of efficacy or the presence of a smaller treatment effect than expected. Future research should investigate which subgroups of patients might benefit. Tranexamic acid is inexpensive, easy to administer, seems to be safe, and is widely available, so even a modest treatment effect could have an important impact on the global scale. Larger randomised trials are warranted.

TITLE: Open- and Closed-Skill Exercise Interventions Produce Different Neurocognitive Effects on Executive Functions in the Elderly: A 6-Month Randomized, Controlled Trial This study aimed to explore the effects of open- and closed-skill exercise interventions on the neurocognitive performance of executive functions in the elderly. Sixty-four healthy elderly males were randomly assigned to either a closed-skill (bike riding or brisk walking/jogging, n = 22), open-skill (table tennis, n = 21), or control (n = 21) group. Various neuropsychological [e.g., accuracy rates (AR) and reaction time (RT)] and electrophysiological [e.g., event-related potential (ERP) P3 component] measures were assessed during a variant of the task-switching paradigm, as well as an N-back task at baseline and after either a 6-month exercise intervention or control period. The results showed that, when performing the task-switching paradigm, the two exercise groups relative to control group showed significantly faster RTs in the switch trials after the exercise intervention. However, the RT facilitation in the non-switch and switch trials post-exercise relative to pre-exercise only emerged in the open-skill group. In terms of the N-back task, the two exercise groups significantly increased ARs in the 1-back condition after the exercise intervention, and the beneficial AR effect on the 2-back condition only emerged in the closed-skill group. In addition, the two exercise groups exhibited significantly larger P3 amplitudes on the frontal-to-parietal cortex areas after the exercise intervention relative to the baseline when performing the two cognitive tasks. These neurocognitive results still remained unchanged even when the confounding factors (e.g., cardiorespiratory fitness, social participation, and BMI) were controlled for. The present study concluded that, although 6-month open- and closed-skill exercise interventions facilitate overall electrophysiological effects (i.e., increased ERP P3 amplitudes) on the frontal-to-parietal cortices in the elderly, the two exercise modes produced different levels of neuropsychologically beneficial effects on RTs of the task-switching paradigm (i.e., lessened RTs) and ARs of the N-back task (i.e., enhanced ARs). The distinctive neurocognitive changes induced by open- and closed-skill exercise have implications for task switching and working memory in elderly individuals, especially with such cognitive functioning impairments. BODY.INTRODUCTION: Life expectancy has been increasing in developed countries, resulting in a rapid growth in the elderly population. Since aging is the main risk factor for neurodegenerative diseases (e.g., Alzheimer's disease), which affect a substantial and growing part of the global population (Rodríguez-Arellano et al., 2016), one important issue is how to counteract such neurocognitive declines in order to reduce the medical costs associated with geriatric care. Although a decrease in certain cognitive functions is an unavoidable part of normal aging, the degree to which this occurs varies within the healthy older population. Among an array of cognitive functions, the executive-control processes and brain areas that support them have been shown to undergo large age-related performance declines (West, 1996; Colcombe and Kramer, 2003; Anderson and McConnell, 2007), as seen in capacities such as working memory (Wingfield et al., 1988), visuospatial attention (Greenwood et al., 1993), and task switching (Friedman et al., 2008). The vast majority of related studies have proposed that physical fitness and exercise are factors that strongly promote healthy cognitive aging (Lee et al., 2012; Kimura et al., 2013; Tsai et al., 2015). Improvements in physical fitness via exercise training are thus reflected in enhancements of a number of cognitive functions, such as processing speed, visuospatial function, and control processes (e.g., inhibition, planning, scheduling, and working memory) in older adults, with the largest effect sizes being on tests thought to depend more on the executive function (e.g., task switching, inhibitory control, and working memory; Diamond, 2013) (Hall et al., 2001; Voelcker-Rehage et al., 2010; Guiney and Machado, 2013; Tsai et al., 2015). However, a broad range of physical exercise types is possible, and different kinds of exercise seem to have specific effects on neurocognitive performance, due to the differences in the secretion of some biomarkers (e.g., brain-derived neurotrophic factor, insulin-like growth factor-1, and homocysteine) in the neurochemical system (Neeper et al., 1995; Liu-Ambrose et al., 2010; Cassilhas et al., 2012; Tsai et al., 2014b,c), and the differences in brain tissue volumes and activation patterns induced by different types of exercise (Luft et al., 2008; Park et al., 2008; Liu-Ambrose et al., 2010; Erickson et al., 2011; Tsai and Wang, 2015; Tsai et al., 2016). These earlier works seem to support the view that different types of physical exercise could affect the brain in different ways. Different forms of physical exercise with different cognitive executive process loads and different motor-coordination skills have been reported to be strongly associated with improved neurocognitive performances (Voelcker-Rehage et al., 2011; Tsai et al., 2016). Therefore, the present study divided exercise into two main modes, open- and closed-skill (Di Russo et al., 2010; Dai et al., 2013; Tsai and Wang, 2015), since the former (e.g., table tennis and badminton) requires rich cognitive and executive loadings and different sets of motor-coordination skills to adapt to a unpredictable/changing environment and various opponents (van Praag et al., 2000; Artola et al., 2006; Di Russo et al., 2010), while the later (e.g., running and biking) is performed according to the individual's own pace in a stable and predictable environment (Di Russo et al., 2010). From the perspective of motor-coordination skill, Voelcker-Rehage et al. (2011) found that older adults experienced beneficial effects on executive control (assessed using the Flanker task) and perceptual speed (assessed using the Visual Search task) due to cardiorespiratory and coordination training. However, the two exercise modes produced different effects on speed and accuracy, with coordination training leading to improved accuracy rates (ARs) on executive control and perceptual speed, but cardiorespiratory training only leading to better ARs on executive control. In terms of reaction time (RT), only the perceptual speed task was significantly improved by coordination training. In addition, cardiorespiratory training increased activation of the sensorimotor network in the elderly, while coordination training elevated activation of the visual–spatial network (Voelcker-Rehage et al., 2011). In contrast, Hötting et al. (2012) found that, although significant increases in episodic memory learning scores were found for both the cycling and stretching/coordination groups as compared with the sedentary control group, cycling training had greater effects on the episodic memory recognition scores than the stretching/coordination training. They also found that the latter was particularly effective in improving selective attention as compared with the cycling training. This suggests a specific relation between particular types of exercise and cognitive functions, with the increase in memory functions being linked to an increase in cardiovascular fitness, whereas the increase in attention is more pronounced after stretching/coordination training. The two exercise modes (i.e., open- and closed-skills) thus seem to be capable of producing different effects on the various cognitive domains (Voss et al., 2010) and neural processes (Tsai and Wang, 2015; Tsai et al., 2016). Although recent studies have explored the effects of open- and closed-skill exercise on neuropsychological performance in disabled athletes (Di Russo et al., 2010) and the young adults (Wang et al., 2013a,b), and neurocognitive (neuropsychological and electrophysiological) performances in the elderly (Dai et al., 2013; Tsai and Wang, 2015; Tsai et al., 2016), the findings in the rather limited research literature remain somewhat ambiguous. More importantly, even though previous cross-sectional studies have demonstrated that regular participation in open- and closed-skill exercise has distinct benefits for neurocognitive performances (e.g., specific cost, RT, P3 amplitudes, and strength of inhibitory control) in the elderly when performing the task switching paradigm (Dai et al., 2013; Tsai and Wang, 2015) and visuospatial attention task (Tsai et al., 2016), the elderly subjects who showed more of the benefits of open-skill exercise on neurocognitive performance might have had some inherently better aspects of their executive control functions (e.g., visuospatial attention and task switching) as compared to their counterparts participating in the closed-skill exercise mode, and this may have induced them to adopt this kind of exercise (Snowden et al., 2011). Therefore, these cross-sectional studies cannot establish causality between exercise and cognitive aging, which is nonetheless required for more accurate and effective public health recommendations (Snowden et al., 2011; Miller et al., 2012), as well as to better explain the beneficial effects of the two exercise modes. Two cognitive tasks, the task-switch paradigm and N-back task, were adopted in the current study to investigate the impacts of the various exercise-mode mechanisms responsible for specific kinds of executive-control functioning, since earlier works found that open- and closed-skill exercise could have different neurocognitive effects on different cognitive tasks executive functions (Di Russo et al., 2010; Dai et al., 2013; Tsai and Wang, 2015; Tsai et al., 2016), and, crucially, open-skill exercise (e.g., table tennis) affects more of the prefrontal cortex areas responsible for attention, task-switching and inhibition, while closed-skill exercise (e.g., jogging) works more on the hippocampus, which is important for memory (e.g., long-term memory and working memory; Axmacher et al., 2010; Burrel, 2015). Moreover, there are previous reports of age- and physical-activity-related impacts on the neuropsychological (e.g., AR and RT) and electrophysiological [e.g., event-related potential (ERP) P3 component] outcomes of the task-switching paradigm (Hillman et al., 2006; Friedman et al., 2008; Adrover-Roig and Barceló, 2010; Guiney and Machado, 2013) and N-back task (Voelcker-Rehage et al., 2010; Guiney and Machado, 2013; Saliasi et al., 2013) among elderly subjects. ERP recordings made during the cognitive task performance permitted on-line measures of cognitive processes on the order of milliseconds, which cannot be obtained by neuropsychological performance alone (Tsai et al., 2014b). Given that the P3 activity has nonspecific qualities that are often associated with indexing stimulus evaluations and the intensity of the concomitant executive function processes (e.g., task-set updating processes and reconfiguration, updating working memory, integrating information into existing networks) (Kok, 2001; Kieffaber and Hetrick, 2005; Nicholson et al., 2005; Polich, 2007), the ERP component was used to illustrate the effects of the different exercise-mode interventions on executive cognitive functions in the elderly in the present study. With regard to the electrophysiological index, the P3 activity induced by the task-switch paradigm represents the set of processes subsumed under the construct of the task-set reconfiguration (Kieffaber and Hetrick, 2005; Nicholson et al., 2005). ERP P3 represents the memory-related neural processing that is involved in categorizing incoming information and updating the context of the working memory (e.g., encoding, rehearsal, recognition, and retrieval) (Duncan-Johnson and Donchin, 1977; Donchin and Coles, 1988; Rugg, 1995). To date there is a lack of intervention research on the impact of open- and closed-skill exercise modes on various forms of executive function (e.g., task switching and working memory) involved in cognitive aging in older adults. The main goal of this study was thus to clarify the distinctive effects of a 6-month open- and closed-skill exercise intervention on the neuropsychological (e.g., AR and RT) and electrophysiological (e.g., ERP P3 latency and amplitude) performances in older adults with a sedentary life-style when performing the task-switching paradigm and N-back task, with rigorous controls on the confounding factors in neurocognitive performance [e.g., cardiorespiratory fitness, social participation, and body mass index (BMI), since these parameters could be changed to different extents after exercise] (Messier and Gagnon, 2009; Miller et al., 2012). The elderly subjects with regular open-skill exercise participation in the literature general show better switch-related neurocognitive performances than those with only closed-skill experience (Dai et al., 2013; Tsai and Wang, 2015), and short-term closed-skill exercise intervention (e.g., resistance exercise) cannot improve task-switching performance (Kimura et al., 2010), while closed-skill exercise training with the goal of enhancing cardiorespiratory fitness can facilitate white matter integrity, increase the size of the hippocampus, and improve memory performance in the elderly (Erickson et al., 2011; Ruscheweyh et al., 2011; Voss et al., 2013; Maass et al., 2016) and have more benefits on memory functions than stretching/coordination training (Hötting et al., 2012). As such, we hypothesized the following: (1) that a 6-month open-skill exercise intervention (e.g., table tennis) in contrast to a closed-exercise one (e.g., bike riding or brisk walking/jogging) would have more benefits for neurocognitive performance with regard to task-switching in the elderly; and (2) that closed-skill exercise would have more beneficial effects on the cognitive functioning involving the memory domains. BODY.MATERIALS AND METHODS: BODY.PARTICIPANTS: Sixty-four community-dwelling men were recruited with the use of an informative flyer and underwent screening by a standardized telephone interview, with subjects being eligible for inclusion in this study if they (1) were aged 60–80 years old and a non-smoker; (2) were living independently in their own home with a sedentary life-style; and (3) did not have a current medical condition for which exercise is contraindicated. The participants consisted solely of men because neurocognitive and endocrinological responses to exercise could be gender-dependent (Baker et al., 2010). They then underwent a routine laboratory testing and medical examination, including blood pressure and heart rate measurements, electrocardiography, a standardized neurological and psychiatric examination, and a structured interview on previous medical history to ascertain whether they were free of a history of neurological disorders, brain injury, depressive symptoms [scores above 13 on the Beck Depression Inventory, 2nd edition (BDI-II)], and cognitive impairment [scores below 26 on the Mini Mental State Examination (MMSE)] (Ruscheweyh et al., 2011). The Edinburgh Handedness Inventory assessed all participants as right-handed (Oldfield, 1971). Written informed consent, as approved by the Institutional Ethics Committees in the organization within which the study was performed, was obtained from all the participants. BODY.PROCEDURES: The Consolidated Standards of Reporting Trials (CONSORT) flowchart outlining the number of participants from first contact to study completion is shown in Figure 1. The original cohort consisted of 79 participants. After the assessment of a physician specializing in geriatric care, two subjects were excluded due to a history of heart disease, five due to neurological disorders, musculoskeletal problems, or psychiatric illness (e.g., scores above 13 on the BDI-II or below 26 on the MMSE), and three due to regular participation in open- and/or closed-skill exercise in the previous 3 months. The remaining 69 participants were randomly assigned to either an open-skill, closed-skill, or active control group by drawing an envelope with the treatment assignment enclosed after matching for age. Figure 1The CONSORT (Consolidated Standards of Reporting Trials) flowchart. Before the exercise intervention, the participants visited the cognitive neurophysiology laboratory on two separate occasions. During the first session, each participant completed an informed consent form, the basic information form (e.g., a medical history and demographic questionnaire), and a handedness inventory. Two certified fitness instructors then completed all assessments of senior functional physical fitness for each participant. On a separate day within 1 week of the completion of the baseline evaluation, the participants performed two cognitive tasks (i.e., a task-switching paradigm and an N-back task) in a counter balanced order with concomitant electrophysiological recording (i.e., event-related potentials, ERPs). Before the final exercise interventions were completed, one participant in the open-skill group, two in the closed-skill group, and two in the active control group, terminated their participation, leaving 64 participants in the final sample (open-skill, n = 22; closed-skill, n = 21; active control, n = 21). The three groups did not significantly differ at baseline in any of the demographic characteristics, including years of formal education, body mass index, systolic and diastolic pressure, social participation, MMSE, BDI-II, and senior functional physical fitness (see Table 1). Within 1 week after completing a 24-week exercise intervention, the participants completed the same questionnaires and senior functional physical fitness assessment, and received the same neurocognitive measurements, as in the pre-exercise procedure. Compliance with the prescribed training protocol remained high throughout the study period (90 ± 2%). Table 1Demographic characteristics of the open-skill exercise, closed-skill and control groups before and after the exercise intervention. Open-skill (n = 22)Closed-skill (n = 21)Control (n = 21)Pre-exercisePost-exercisepPre-exercisePost-exercisepPre-exercisePost-exercisepAge (years) 66.88 ± 4.74 – 66.15 ± 4.90 – 65.70 ± 3.54 – Education (years) 12.50 ± 4.09 – 12.62 ± 2.97 – 10.62 ± 3.20 – Height (cm) 162.90 ± 6.71 162.88 ± 6.74 0.329 159.70 ± 7.24 159.57 ± 7.12 0.186 161.20 ± 7.94 160.68 ± 8.54 0.329 Weight (kg) 62.65 ± 9.11 62.71 ± 8.99 0.329 60.98 ± 9.11 58.86 ± 9.55 0.005 62.04 ± 10.57 61.95 ± 10.47 0.329 BMI (kg/m2) 23.65 ± 3.64 23.68 ± 3.61 0.329 23.79 ± 3.65 23.04 ± 2.99 0.007 23.83 ± 3.29 23.94 ± 3.20 0.458 Systolic pressure (mmHg) 128.68 ± 22.95 125.82 ± 18.02 0.076 129.29 ± 21.79 124.57 ± 20.99 0.125 132.05 ± 25.12 129.33 ± 24.59 0.313 Diastolic pressure (mmHg) 75.86 ± 11.62 74.14 ± 9.13 0.296 78.62 ± 11.65 74.76 ± 10.76 0.151 79.48 ± 10.58 77.67 ± 12.07 0.347 Social participation 10.23 ± 2.07 10.73 ± 1.91 0.031* 9.57 ± 2.40 9.90 ± 2.28 0.090 9.95 ± 2.04 9.62 ± 1.99 0.090 Memory depth 18.50 ± 5.57 19.45 ± 5.41 0.404 17.10 ± 7.15 20.14 ± 4.60 0.059 18.24 ± 3.89 18.67 ± 4.46 0.410 MMSE 28.73 ± 1.28 28.05 ± 2.73 0.252 27.48 ± 3.03 28.95 ± 1.40 0.082 27.67 ± 1.80 28.19 ± 1.43 0.299 BDI-II 3.68 ± 3.72 3.09 ± 3.05 0.221 5.04 ± 4.14 4.71 ± 8.16 0.877 3.38 ± 3.38 2.47 ± 3.03 0.092 FITNESS Grip (kg) 28.90 ± 6.75 29.13 ± 6.74 0.834 26.98 ± 10.03 24.65 ± 9.50 0.368 27.81 ± 7.62 26.84 ± 6.82 0.338 Arm Curl (number) 18.09 ± 4.60 21.82 ± 3.40 <0.001* 17.24 ± 3.22 17.29 ± 2.57 0.942 17.47 ± 4.32 16.95 ± 3.69 0.451 Chair Stand (sec) 15.18 ± 3.80 19.41 ± 5.90 0.001* 18.08 ± 6.35 17.95 ± 4.04 0.932 15.80 ± 4.98 16.10 ± 2.96 0.757 8-Foot Up-and-Go (sec) 6.03 ± 1.38 5.53 ± 1.03 0.012* 6.97 ± 3.73 5.54 ± 1.04 0.086 6.32 ± 1.09 6.35 ± 1.05 0.859 Back Scratch (cm) −3.23 ± 11.98 −2.11 ± 9.66 0.315 −1.43 ± 11.14 −1.55 ± 9.37 0.924 −0.64 ± 7.31 0.43 ± 9.58 0.383 Chair Sit-and-Reach (cm) 19.85 ± 5.27 20.40 ± 3.06 0.532 17.09 ± 2.02 17.04 ± 2.16 0.782 18.58 ± 2.38 19.33 ± 2.84 0.181 VO2max (mL/kg/min) 25.63 ± 9.66 30.32 ± 12.03 0.071 26.83 ± 5.44 31.01 ± 4.25 0.001* 24.11 ± 6.61 23.79 ± 6.71 0.735 BMI, body mass index; MMSE, Mini Mental State Examination; BDI-II, Beck Depression Inventory, 2nd edition; VO2max, maximal oxygen uptake. *p < 0.05. BODY.TRAINING PROTOCOL: BODY.OPEN-SKILL EXERCISE CONDITION: The participants in the open-skill group were trained individually and regularly in a series of 40-min sessions that took place three times per week for 24 weeks, with the following structure: a warm-up, the main part of table tennis training, playing games of table tennis with the coach, and cooling down at the end. The training intervention was carried out in a sequence of increasing complexity. The table tennis training program was intended to improve the participants' general skills, and had seven main components over the whole training session: (a) footwork (e.g., ready position, one-, two-, and cross-step), (b) serving (including how to give sidespin, backspin, topspin, no-spin, and so on), (c) forehand and backhand driving, (d) forehand bouncing, backhand bouncing, and alternate bouncing, (e) smashing, (f) continuously hitting back a ball that was randomly delivered by the ball-projection machine from fixed or random directions, and (g) comprehensive practice. Training for each skill began with a simple movement and then progressed to more complex variations. The assessor for the neurocognitive tests and the exercise leader/coach were blinded to group membership. A more detailed training manual is available on request from the authors. BODY.CLOSED-SKILL EXERCISE CONDITION: Participants in the closed-skill group attended three supervised exercise sessions per week for 24 consecutive weeks on a bicycle ergometer or a motor-driven treadmill (Medtrack ST55, Quinton Instrument Company, United States), with exercise intensity corresponding to 50–60% of the individual target heart rate reserve (HRR) during the first 2 weeks and 70–75% of the HRR for the remainder of the program. Each aerobic exercise session involved a 5-min warm-up period, followed by 30 min of continuous bike riding or brisk walking/jogging at an intensity that would maintain the heart rate within the assigned training range, and 5 min of cool-down. A Polar HR monitor (RX800CX, Finland) was used to monitor each participant's heart rate during the exercise. BODY.CONTROL CONDITION: Participants in the active control group attended a balance and stretching program led by a trained exercise leader three times a week for 24 weeks. Every class included a 5-min warm-up period, static stretching and balance training, and a 5-min cool-down period. Different stretching and balancing techniques used various equipment, such as balance boards and fitness balls, to maintain the participants' interest. BODY.PHYSICAL FITNESS ASSESSMENT: The Senior Functional Physical Fitness (SFPF) test (Rikli and Jones, 2012) is a battery of tests that was used to assess the participants' physical fitness in the current study. The participants first undertook 10 min' warm-up before the test and then completed the component tests in the designated order. Five items in the SFPF test were measured, as follows: (1) the Arm Curl test, which assesses arm muscle (specifically of the biceps) strength endurance, with the score being the number of repetitions in 30 s using the elbow of the dominant hand to flex and extend with a weight (men: 8 lb; women: 5 lb) through the complete range of motion; (2) the Chair Stand test, which measures lower body strength, based on the number of repetitions in 30 s using a full standing position from a chair and then returning to a fully seated position; (3) the 8-Foot Up-and-Go test, which evaluates agility and dynamic balance, using the number of seconds needed to get up from a seated position from a chair, walk eight feet, and return to a fully seated position on the chair; (4) the Back Scratch test, which assess upper body (shoulder) flexibility based on the number of centimeters being short of touching (minus score) or overlapping (plus score) between the third fingertip of each hand; (5) the Chair Sit-and-Reach test, which assesses the flexibility of the lower extremities, with the score being the distance in centimeters between the fingertips and toes; the number of centimeters short of reaching the toes (minus score) or reached beyond the toes (plus score). With regard to cardiorespiratory fitness, the Rockport Fitness Walking Test was used to estimate VO2max (Kline et al., 1987), in which the participants were required to walk one mile as quickly as possible, during which their heart rate was continuously recorded using a Polar HR monitor. BODY.COGNITIVE TASKS: BODY.TASK-SWITCHING PARADIGM: The task switching paradigm employed in the present study has been shown to effectively assess the variations among elderly subjects who regularly participate in open- and closed-skill types of exercise (Tsai and Wang, 2015). The stimulus used in this test was a white digit (1–9, excluding 5) shown focally in the center of the screen against a black background. The same digit was not repeated in successive trials, and the digits were put into eight task blocks (blocks 1–2 and 7–8: homogeneous tasks; blocks 3–6: heterogeneous tasks), with a short rest period in the middle of each. The homogenous (i.e., non-switch) blocks each included 56 trials. Within each homogenous block, the participants only responded if the focal digit was greater or less than 5 (e.g., blocks 1 and 7), or if it was odd or even (e.g., blocks 2 and 8). The heterogeneous (i.e., task-switching) blocks contained 224 trials, each with 20 switches. With the heterogeneous blocks, the participants started with one task (e.g., even/odd) and then switched to the other (e.g., more/less than 5), as signaled by a simultaneously presented rectangle drawn around the digit, after at least seven or no more than 13 intervening trials. The participants were asked to press one of two buttons on a small response box that they held in the right hand as quickly and accurately as possible. The digit was shown on the screen until the participants pressed the response button, and the following trial began 500ms after the RT response. The prompts “more less” or “even odd” appeared simultaneously with and below the digit during all trials, dependent on which was appropriate to the task. The homogenous and heterogeneous blocks were counterbalanced across participants. All the participants were given instructions about the tasks, and both single-task and task-switch trials were practiced before the formal test until they understood the whole procedure. BODY.N-BACK TASK: A continuous stream of white letters (stimuli) with pseudo-random sequences of vowels and consonants was presented with 10% gray noise, embedded in a 50% random noise gray rectangular background patch, on a computer screen. Targets were defined according to the N-back design. Participants pressed a button with the index finger of their right hands as soon as a target appeared, and no motor response was needed for non-target trials. Stimuli were presented for a duration of 500 ms, followed by a 3 s inter-trial-interval during which a dot helped participants maintain fixation. The cognitive task consisted of three different working-memory-load conditions: (1) a simple detection (control) condition with sequential letters or background patches without any letters being presented, during which the participants responded when the background patches without letters appeared (memory-free condition); (2) the 1-back condition, with the target being any letter identical to the one immediately preceding it (moderately demanding); (3) the 2-back condition, with the target being any letter that was identical to the one presented two trials back (highly demanding). Before the formal test, the participants were given the task instructions and initially practiced a brief version of the task, consisting of two blocks of 45 trials each (one block of moderately demanding and one of highly demanding trials). Following this practice, the participants completed nine blocks of trials with 120 trials in each (three blocks per condition). Nine blocks were tested following the sequence: blocks 1, and 8–9: the control condition, blocks 2, and 6–7: the 1-back condition, and blocks 3–5: the 2-back condition. No feedback was provided during this period. BODY.ERPS RECORDING AND ANALYSIS: Continuous electroencephalographic (EEG) signals were acquired from 18 electrodes (F7, F3, Fz, F4, F8, C3, Cz, C4, T5, T3, T4, T6, P3, Pz, P4, O1, Oz, and O2) placed using a 10/20 extended Quik-Cap system (Compumedics Neuroscan, Inc., El Paso, TX). Horizontal and vertical electrooculogram (EOG) activity for eye movements was monitored bipolarly with ocular electrodes placed on the supero-lateral right canthus and infero-lateral to the left eye. A ground electrode was placed on the mid-forehead on the Quik-Cap. References were placed at vertex by default, but were subsequently re-referenced off-line to averaged mastoids. The values of interelectrode impedance were kept at 5 KΩ for all electrodes. The raw EEG signal was recorded with an A/D rate of 500 Hz/channel, a band-pass filter of 0.1–50 Hz, and a 60-Hz notch filter using an on-line amplifier. These data were written continuously to hard disk for off-line analysis using Neuroscan Scan 4.3 analysis software (Compumedics Neuroscan, Inc., El Paso, USA). Trials with a response error or EEG artifacts (e.g., VEOG, HEOG, and electromyogram) exceeding peak-to-peak deflections over 100 μV were discarded before averaging. ERPs were extracted off-line and averaged in epochs starting 200 ms prior to stimulus activity onset, and lasting for 1,000 ms of post-stimulus activity. Since the ERP P3 component is widely examined among studies that independently and simultaneously investigate the externalizing spectrum and executive functioning (Baskin-Sommers et al., 2014), it was the focus of the current work. The effects of the two cognitive tasks on the P3 component in the elderly were clearly visible in the frontal-parietal regions of the scalp in the current study (see also Kieffaber and Hetrick, 2005; Friedman et al., 2008; Baskin-Sommers et al., 2014). Three electrodes (Fz, Cz, Pz) were thus analyzed in the present work (Themanson et al., 2006; Tusch et al., 2016). P3 mean amplitudes were calculated for the time-window between 300 and 600 ms post stimulus. BODY.DATA PROCESSING AND STATISTICAL ANALYSES: One-way analysis of variance (ANOVA) was used to examine the homogeneity of the demographic backgrounds of the participants at the baseline in the open-skill, closed-skill, and control groups. A two-tailed paired t-test was used to analyze the differences within the three groups at baseline and at 24 weeks. Different trials/conditions in the two cognitive tasks were subjected to neuropsychological (i.e., AR and RT) and electrophysiological (i.e., P3 amplitude) statistical analyses. Only the RT and ERP data corresponding to correct answers were averaged according to the task trial/condition. With regard to the task-switching paradigm, two switch costs were determined by the RTs performance: (1) general-switch cost, which was obtained by subtracting the mean RT between homogeneous trials during homogeneous blocks and non-switch trials during heterogeneous blocks; (2) specific-switch cost, which was calculated by subtracting the mean RT between non-switch trials and switch trials during heterogeneous blocks. BODY.TASK SWITCHING PARADIGM: With regard to the neuropsychological performance, the AR and RT were separately submitted to a 3 (Group: open-skill vs. closed-skill vs. control) × 2 (Time: pre-exercise vs. post-exercise) × 3 [Trial: homogeneous (during homogeneous blocks) vs. non-switch vs. switch (during heterogeneous blocks)] mixed repeated measures analysis of variance (RM–ANOVA). In terms of the electrophysiological performance, the P3 latency and amplitude from ERP recordings were submitted to a 3 (Group: open-skill vs. closed-skill vs. control) × 2 (Time: pre-exercise vs. post-exercise) × 3 (Trial: homogeneous vs. non-switch vs. switch) × 3 (Electrode: Fz vs. Cz vs. Pz) RM–ANOVA. BODY.N-BACK TASK: With regard to the neuropsychological performance, the AR and RT were separately submitted to a 3 (Group: open-skill vs. closed-skill vs. control) × 2 (Time: pre-exercise vs. post-exercise) × 3 (Condition: 0-back vs. 1-back vs. 2-back) RM–ANOVA. In terms of the electrophysiological performance, the P3 latency and amplitude from ERP recordings were submitted to a 3 (Group: open-skill vs. closed-skill vs. control) × 2 (Time: pre-exercise vs. post-exercise) × 3 (Condition: 0-back vs. 1-back vs. 2-back) × 3 (Electrode: Fz vs. Cz vs. Pz) RM–ANOVA. Bonferroni post-hoc analyses were performed when there was a significant difference. Simple main effects were determined following observation of any significant interaction effects. Because cardiorespiratory fitness, social participation, and BMI are confounding factors on cognitive performance (Messier and Gagnon, 2009; Miller et al., 2012), the neuropsychological and electrophysiological performances of the three groups after the exercise intervention were assessed separately using an analysis of covariance (ANCOVA) to examine the effects on the neurocognitive performance in relation to different types of intervention, with the three post-exercise factors as a covariate. The Greenhouse–Geisser (G–G) correction adjusted the significance levels of the F ratios whenever RM–ANOVA detected a major violation of the sphericity assumption. Partial Eta squared (ηp2) was used to calculate effect sizes for significant main effects and interactions, with the following criteria used to determine the magnitude of mean effect size: 0.01–0.059 indicated a small effect size; 0.06–0.139, a medium effect size; and >0.14, a large effect size. A p-value less than 0.05 for the differences between the mean values is considered statistically significant. BODY.RESULTS: BODY.DEMOGRAPHIC CHARACTERISTICS: Table 1 presents an overview of the pre- and post-exercise characteristics of the participants. At baseline there were no significant differences (all ps > 0.05) at the group level with regard to age, height, weight, BMI, systolic and diastolic pressure in the three groups (i.e., open-skill, closed-skill, and control). Other confounding factors (e.g., the years of education, social participation, memory depth, global cognitive function, depression, and cardiorespiratory fitness) in relation to cognition and the tests of functional fitness also did not achieve significant difference in the three groups (all ps > 0.05) across the three groups at baseline. Paired t-tests showed that after the exercise intervention the open-skill group had significantly improved scores for the social participation, arm curl, chair stand, and 8-foot up-and-go items, and approached significance for the level of cardiorespiratory fitness (VO2max); and the results for the closed-skill group showed that the values of weight and BMI decreased significantly, the level of cardiorespiratory fitness was significantly enhanced, and the performance of memory depth approached significance. BODY.ACCURACY RATE (AR): BODY.TASK-SWITCHING PARADIGM: As shown in Figure 2, RM–ANOVA performed on the ARs only highlighted the main effects of Time [F(1, 61) = 10.36, p = 0.002, ηp2 = 0.15] and Trial [F(2, 122) = 35.57, p < 0.001, ηp2 = 0.37], with a higher AR post- (90.96%) than pre-exercise (88.53%) and with the following trial gradient across the three groups: homogeneous (93.52%) > non-switch (88.96%) > switch (86.75%). Figure 2Grand mean accuracy rate (the left side) and reaction time (the right side) performance (mean ± SE) of the open-skill, closed-skill, and control groups during the task-switching (upper) and N-back (lower) tasks before and after the exercise intervention. *Significantly different (p < 0.05). BODY.N-BACK TASK: RM–ANOVA performed on the ARs for the N-back task revealed the main effects of Time [F(1, 61) = 20.45, p < 0.001, ηp2 = 0.25] and Condition [F(2, 122) = 129.89, p < 0.001, ηp2 = 0.68], with a higher AR for post- (90.61%) than pre-exercise (88.43%), and with the following condition gradient: 0-back (98.01%) > 1-back (90.17%) > 2-back (80.37%). The interactions between Time × Group [F(2, 61) = 4.74, p = 0.012, ηp2 = 0.14], Time × Condition [F(2, 122) = 10.51, p < 0.001, ηp2 = 0.15], and Time × Group × Condition [F(4, 122) = 2.61, p = 0.012, ηp2 = 0.39] were also significant. Post-hoc analysis for the Time × Group × Condition interaction showed that (1) there were no significant differences at any condition among the three groups at baseline and after the exercise intervention (all ps > 0.05); and (2) when post- compared to pre-exercise, the open- [F(1, 21) = 4.44, p = 0.047] and closed-skill [F(1, 20) = 21.45, p < 0.001] groups exhibited significantly higher ARs in the 1-back condition, and only the closed-skill [F(1, 20) = 36.59, p < 0.001] group exhibited a significantly higher AR in the 2-back condition. In addition, the significant post- and pre-exercise differences in ARs among the three groups only emerged in the 2-back condition, with a greater improvement after the exercise intervention for the closed-skill group than the open-skill and control groups (closed-skill vs. open-skill: p = 0.002; closed-skill vs. control: p = 0.001). Even when the post-exercise cardiorespiratory fitness, social participation, and BMI were controlled for, the results of ANCOVA on the ARs in the 2-back condition still indicated a significant difference in the three groups [F(2, 58) = 9.90, p < 0.001], with post-hoc analysis indicating that closed-skill group performed significantly better than the open-skill (closed-skill vs. open-skill: p < 0.001) and control (closed-skill vs. control: p < 0.001) groups after the exercise intervention. BODY.REACTION TIME (RT): BODY.TASK-SWITCHING PARADIGM: As illustrated in Figure 2, RM–ANOVA conducted on mean RTs revealed the main effects of Time [F(1, 61) = 10.22, p = 0.002, ηp2 = 0.14] and Trial [F(2, 122) = 490.16, p < 0.001, ηp2 = 0.89], suggesting that RTs were faster post- (999.2 ms) rather than pre-exercise (1,056.1 ms), and that RTs followed the trial gradient: homogeneous (587.4 ms) < non-switch (1,136.6 ms) < switch (1,358.9 ms). The interactions of Time × Group [F(2, 61) = 7.34, p = 0.001, ηp2 = 0.19], Time × Condition [F(2, 122) = 5.55, p = 0.005, ηp2 = 0.08], and Time × Condition × Group [F(4, 122) = 3.69, p = 0.007, ηp2Post-hoc analysis for the Time × Condition × Group interaction showed that (1) the open- and closed-skill groups showed significantly faster responses than the control group in the switch trials after the exercise intervention [F(2, 61) = 10.31, p < 0.001; open-skill vs. control: p < 0.001; closed-skill vs. control: p = 0.027], and (2) when post- compared to pre-exercise, the open-skill group responded faster in the non-switch [F(1, 21) = 24.80, p < 0.001] and switch [F(1, 21) = 27.15, p < 0.001] trials. Even when the post-exercise cardiorespiratory fitness, social participation, and BMI were controlled for, the results of ANCOVA on the RTs in switch trials still indicated a significant difference in the three groups [F(2, 58) = 7.70, p = 0.001], with post-hoc analysis indicating that the two exercise groups showed significantly faster responses than the control group (open-skill vs. control: p < 0.001; closed-skill vs. control: p = 0.027) after the exercise intervention. In terms of RT switch costs, RM-ANOVA for the general-switch cost revealed the main effect of Time [F(1, 61) = 15.01, p < 0.001, ηp2 = 0.20], indicating that the general-switch cost was smaller post- (501.9 ms) rather than pre-exercise (596.5 ms) across the three groups. RM-ANOVA for the specific-switch cost revealed that the interaction of Time × Group [F(2, 61) = 3.35, p = 0.042, ηp2 = 0.10] was significant. Post-hoc analysis showed that only the value of the specific-switch cost approached significance [F(1, 21) = 4.33, p = 0.050] post-exercise (149.2 ± 163.0 ms) relative to pre-exercise (227.5 ± 208.1 ms) in the open-skill group. BODY.N-BACK TASK: RM–ANOVA conducted on mean RTs for the N-back task only revealed a main effect of Condition [F(2, 92) = 210.94, p < 0.001, ηp2 = 0.82], with the following gradient: 0-back (586.8 ms) < 1-back (693.5 ms) < 2-back (812.4 ms). BODY.P3 LATENCY: BODY.TASK-SWITCHING PARADIGM: As shown in Figures 3, 5, no significant difference was observed with regard to any main effect or interaction in the P3 latency. These results indicate that the P3 latencies did not show obvious changes between three groups when performing the task-switching paradigm throughout the exercise intervention stage. Figure 3Grand average event-related potential waveforms of three electrodes (Fz, Cz, and Pz) for three conditions (homogeneous, non-switch, and switch) for the P3 component in the task-switching paradigm at baseline (dashed line) and after exercise intervention (solid line) in the open-skill (red), closed-skill (green), and control groups (blue). BODY.N-BACK TASK: No significant difference was observed with regard to any main effect or interaction in the P3 latency. These results indicate that the P3 latencies did not show obvious changes when performing the N-back task between three groups throughout the exercise intervention stage. BODY.P3 AMPLITUDE: BODY.TASK-SWITCHING PARADIGM: As shown in Figures 3, 5, RM–ANOVA performed on the P3 amplitudes of the task-switching paradigm showed main effects of Time [F(1, 61) = 30.30, p < 0.001, ηp2 = 0.33], Condition [F(2, 122) = 39.35, p < 0.001, ηp2 = 0.39] and Electrode [F(2, 122) = 15.99, p < 0.001, ηp2=0.21], with the post-hoc analyses indicating that the P3 amplitudes were larger post- (4.89 μV) rather than pre-exercise (3.82 μV); the P3 amplitudes in the three conditions had the following gradient: homogeneous (5.85 μV) > non-switching (3.99 μV) > switching (3.22 μV); and significantly greater amplitudes at the Fz (4.73 μV) and Cz (4.57 μV) electrodes were found as compared to the Pz (3.77 μV) electrode. The interactions of Time × Group [F(2, 61) = 11.47, p < 0.001, ηp2 = 0.27] and Condition × Electrode [F(4, 244) = 13.96, p < 0.001, ηp2 = 0.19] were also significant. Post-hoc analysis for the Time × Group interaction showed that (1) there were no significant differences among the three groups at baseline [F(2, 61) = 0.04; p = 0.961]; (2) there were significant differences among the three groups after the exercise intervention [F(2, 61) = 6.37; p = 0.003], with post-hoc analysis showing that both open- (5.79 μV) and closed-skill (5.38 μV) groups showed significantly larger P3 amplitudes as compared to the control group (3.50 μV) after the intervention (open-skill vs. control: p = 0.004; closed-skill vs. control: p = 0.025); and (3) both the open- and closed-skill groups exhibited significantly larger P3 amplitudes [open-skill: F(1, 21) = 32.99, p < 0.001; closed-skill: F(1, 20) = 23.34, p < 0.001] across all three conditions and electrodes after the intervention relative to baseline. Even when the post-exercise cardiorespiratory fitness, social participation, and BMI were controlled for, the results of ANCOVA on the averaged P3 amplitudes in the three conditions and three electrodes still indicated significant post-exercise group differences [F(2, 58) = 8.07, p = 0.001], with post-hoc analysis indicating that the two exercise groups showed significantly larger P3 amplitudes than the control group (open-skill vs. control: p < 0.001; closed-skill vs. control: p = 0.003) after the intervention. BODY.N-BACK TASK: As illustrated in Figures 4, 6, RM–ANOVA performed on the P3 amplitudes of the N-back task showed the main effects of Time [F(1, 61) = 13.42, p = 0.001, ηp2 = 0.18] and Condition [F(2, 122) = 9.85, p < 0.001, ηp2 = 0.14], with the post-hoc analyses indicating the P3 amplitudes were larger post- (3.57 μV) rather than pre-exercise (2.27 μV); and significantly greater amplitudes at the 0-back (3.31 μV) and 1-back (3.27 μV) conditions were found as compared to the 2-back (2.19 μV) condition. The interaction of Time × Group [F(2, 61) = 3.89, p = 0.026, ηp2 = 0.11] was also significant, with post-hoc analysis indicating that (1) there were no significant differences among the three groups at baseline [F(2, 61) = 0.02; p = 0.985]; (2) there were significant differences among the three groups after the exercise intervention [F(2, 61) = 6.09; p = 0.004], with post-hoc analysis showing that both open- (4.02 μV) and closed-skill (4.52 μV) groups showed significantly larger P3 amplitudes as compared to the control group (2.17 μV) after the intervention (open-skill vs. control: p = 0.032; closed-skill vs. control: p = 0.005); and (3) both the open- and closed-skill groups exhibited significantly larger P3 amplitudes [open-skill: F(1, 21) = 7.59, p = 0.012; closed-skill: F(1, 20) = 16.83, p = 0.001] across all three conditions and electrodes after the intervention relative to baseline. Even when the post-exercise cardiorespiratory fitness, social participation, and BMI were controlled for, the results of ANCOVA on the averaged P3 amplitudes in the three conditions and three electrodes still indicated significant post-exercise group differences [F(2, 58) = 5.56, p = 0.006], with post-hoc analysis indicating that the two exercise group showed significantly larger P3 amplitudes than the control group (open-skill vs. control: p = 0.016; closed-skill vs. control: p = 0.002). Figure 4Grand average event-related potential waveforms of three electrodes (Fz, Cz, and Pz) for three conditions (0-, 1-, and 2-back) for the P3 component in the N-back task at baseline (dashed line) and after exercise intervention (solid line) in the open-skill (red), closed-skill (green), and control groups (blue). Figure 5P3 amplitudes (mean ± SE) of three electrodes (Fz, Cz, and Pz) for three conditions (homogeneous, non-switch, and switch) during the task-switching paradigm in the open-skill, closed-skill, and control groups before and after the exercise intervention. Figure 6P3 amplitudes (mean ± SE) of three electrodes (Fz, Cz, and Pz) for three conditions (0-, 1-, and 2-back) during the N-back task in the open-skill, closed-skill, and control groups before and after the exercise intervention. BODY.DISCUSSION: BODY.MAIN FINDINGS: The present study aimed to explore the effects of 6-month open- (e.g., table tennis) and closed-skill (e.g., bike riding or brisk walking/jogging) exercise interventions on neurocognitive performance in the elderly when performing the task-switching paradigm and N-back task. The findings showed that, with regard to the neuropsychological performance, after participation in the two types of exercise the elderly participants did not increase their ARs in the task-switching paradigm, but showed significantly faster responses than the control group in the switch trials of the heterogeneous condition after the exercise intervention. In addition, compared to the baseline, regular participation in open-skill exercise for 6 months could effectively enhance RTs in the elderly when performing the non-switch and switch trials of the heterogeneous condition, and the specific-switch cost approached significance post- relative to pre-exercise in the open-skill group. In terms of the N-back task, although performing the open- and closed-skill exercise for 6 months did not improve RTs in the elderly when performing such a cognitive task, the ARs in the 1-back condition were significantly enhanced after the exercise intervention in both exercise groups, and the beneficial effects on the 2-back condition only emerged in the closed-skill group. In terms of electrophysiological performance, both open- and closed-skill groups exhibited significantly larger P3 amplitudes across conditions and electrodes after the exercise intervention relative to baseline when performing not only the task-switching paradigm, but also the N-back task. These post-exercise neurocognitive benefits still remained even when the confounding factors (e.g., cardiorespiratory fitness, social participation, and BMI) were controlled for. BODY.NEUROPSYCHOLOGICAL PERFORMANCES: In the present study, the older adults participating in the open-skill types of exercise (e.g., table tennis) not only had to follow the rules of the game, but also switch strategies and select relevant sensory information when encountering the various skill levels of the other players within a constantly changing environment (Di Russo et al., 2010). In addition, they needed to continually adapt or switch to more suitable movements/responses to initiate appropriate actions or inhibit inappropriate ones based on the opponent's actions. The capabilities of motoric and cognitive switching are thus facilitated during this type of exercise. Indeed, with regard to neuropsychological performance in the task-switching paradigm, although no differences in ARs were found among the three groups after the exercise intervention in the present study, only the open-skill group showed faster responses in the non-switch and switch trials of the heterogeneous condition and a lower specific-switch cost after participation in 6-month table tennis exercise training. This result is in line with the previous findings of cross-sectional research (Tsai and Wang, 2015). Since task switching involves stimulus perception and identification, attentional reallocation, task-set updating, response conflict detection, and monitoring processing (Friedman et al., 2008), the elderly subjects participating in the open-skill exercise modes could show greater cognitive flexibility at switching from one task to another. However, both the open and closed-skill groups showed significantly faster responses when compared to the control group in the switch trials of the heterogeneous condition after the exercise intervention, partly supporting the findings of earlier studies which showed that, relative to the control group, the older adults who regularly participated in physical exercise or regular participation in open- or closed-skill exercise displayed a generalized reduction in the time efficiency of the central processing of cognitive functions when performing a task switching paradigm (Hillman et al., 2006; Themanson et al., 2006; Dai et al., 2013; Tsai and Wang, 2015). In addition, it is worth pointing out that, after engaging in 6-month open- or closed-skill exercise, no improvement in the general-switch cost was found in the older adults in the present study, suggesting that the two exercise modes could not facilitate the process of selecting between and coordinating the two competing tasks (Friedman et al., 2008). Although the older adults participating in the closed-skill types of exercise (e.g., bike riding and jogging) in the present study stayed in a predictable and stable environment to perform the related exercise at their own pace (Di Russo et al., 2010), and thus they had a lower cognitive load than seen with the open-skill exercise, their cardiorespiratory fitness was significantly enhanced. This was because, compared to the open-skill exercise, repeatedly performing similar movements coupled with continuously higher HR maintenance could much more effectively improve cardiorespiratory fitness. Previous studies have demonstrated that physical exercise interventions aimed at increasing cardiorespiratory fitness are associated with improvements in the neuropsychological (e.g., response accuracy) and electrophysiological (e.g., P3 and CNV components) performances of working memory in preadolescent children (Pesce et al., 2009; Kamijo et al., 2011; Tsai et al., 2014a) and the elderly (Voss et al., 2013). The potential mechanisms could be that such an exercise mode can increase cerebral blood flow (Seifert and Secher, 2011), cerebral structure (Colcombe et al., 2003), and brain-derived neutrophic factors (Seifert et al., 2010), and regulate hippocampal neurogenesis and synaptic plasticity (Erickson et al., 2009). Given that previous studies have linked cardiorespiratory fitness to enhanced memory in the elderly (Kramer et al., 2001; Voss et al., 2013), and aerobic exercise is associated with increased hippocampal size and function (Erickson et al., 2011), it is not surprising that the closed-skill group in the present study participating in the exercise type that had the greatest impact on cardiorespiratory fitness showed improved ARs on the N-back task involving moderate and high working-memory demands, which require continual processing and updating (Bopp and Verhaeghen, 2005), although there were still no significantly different ARs among the three groups post-exercise. Nevertheless, it should be noted that the open-skill group also exhibited significantly higher ARs on the N-back task involving a moderate working-memory demand (i.e., the 1-back condition) after the exercise intervention, suggesting that the potentially neuropsychological benefit derived from the open-skill exercise on the working memory should not be negated in the elderly. One possible explanation for this is that the effect of improved cardiorespiratory fitness approached significance in the elderly participating in the table tennis intervention in the present study. The results on the relation between the N-back task and exercise modes in the present study were partly in line with Hötting et al.'s (2012) findings that cycling training improved learning and recognition scores in an episodic memory test in middle-aged adults, while stretching/coordination training only improved the learning score, and such beneficial effects could be attributed in an increase in cardiovascular fitness. However, it is worth noting that, when the cofounding factors (also including cardiorespiratory fitness) were controlled for in the present study, the beneficial effect on the AR in the 2-back condition still remained for the closed-skill group relative to the open-skill and control groups. Therefore, the potential mechanisms of the effects (e.g., the increases in the hippocampal size/function and the cerebral blood flow) of the closed-skill exercise on the working memory in the elderly are worth exploring in the future (Erickson et al., 2011). BODY.ELECTROPHYSIOLOGICAL INDICES: P3 latencies did not show obvious changes after 6-month exercise intervention in the two exercise groups, suggesting that the perceptual/central processing could not be facilitated by the open- and closed-skill exercise in the elderly when performing the two cognitive tasks. In line with a previous study reporting a larger P3 amplitude for the active rather than for the sedentary elderly when performing the task switching paradigm (Hillman et al., 2006), the older adults participating in the 6-month open- and closed-skill exercises programs in the present study could effectively increase their P3 amplitudes and so have larger P3 amplitudes across all conditions relative to the control group, suggesting that the two physical exercise modes could facilitate the attentional set that makes it possible to better evaluate the stimulus in either of the two tasks. However, the open- and closed-skill groups exhibited similar benefits on the neural processes at work in processing the current task-switching paradigm. These results are somewhat inconsistent with those of a previous cross-sectional study (Tsai and Wang, 2015), in which the older adults regularly participating in open-skill exercise (e.g., table tennis and badminton) showed a significantly larger P3 amplitude in the switch condition when performing the task-switching paradigm compared to their counterparts participating in closed-skill exercise. The lack of consistency in these results may be attributable to the inherently better task-switching capacity that may encourage some older adults to choose an open-skill exercise mode (Snowden et al., 2011; Tsai and Wang, 2015). The current 6-month exercise intervention study seems to clarify that both open- and closed-skill exercise modes could produce similar electrophysiological benefits across all conditions when older adults perform the task-switching paradigm. However, even though lower P3-and-RT correlation was found in the older individuals (Pfefferbaum et al., 1980), the distinctive effects of open-skill exercise on neuropsychological performance (i.e., better exercise-training-induced effects on specific-switch cost and RTs in the heterogeneous conditions) in the elderly, as mentioned above, cannot be ignored. Similarly, relative to the open-skill exercise, although a closed-skilled exercise intervention could have more neuropsychological benefits (i.e., significantly increasing ARs under the high load condition) on the elderly when performing the N-back task, the effects of greater P3 amplitudes were comparable in the two exercise groups in the present study. This suggests that not only closed- but also open-skill exercise could facilitate the memory-related neural processing which is involved in categorizing incoming information and updating the context of the working memory (e.g., encoding, rehearsal, recognition, and retrieval), due to the greater efficiency by which cognitive resources are allocated (Duncan-Johnson and Donchin, 1977; Donchin and Coles, 1988; Rugg, 1995). Although there were no significant between-group AR differences post-exercise among the three groups, the increase in exercise-induced P3 amplitudes observed in the two exercise groups after the 6-month interventions, as compared with the figures seen before training, could reveal that they allocated more resources for target classification and evaluation, which might result in higher ARs in the working memory task since larger overall P3 amplitudes during the N-back could be associated with better task performance in older adults (Tusch et al., 2016). However, the P3 amplitude could be influenced by a greater latency jitter of P3 in the high than in the low memory condition (Kok, 2001), and regular exercise could change P3 latency in the older adults (see review, Hillman et al., 2003), the potential response jitter thus needs to be clarified in further investigations, since different conditions in the two cognitive tasks are involved in different cognitive loads in the present study. It is worth noting that there was no significant interaction of Time × Group× Electrode in the present study, suggesting that 6-month open- and closed-skill exercise interventions induced similar electrophysiological effects (i.e., increased P3 amplitudes) from the frontal to parietal cortices. However, previous studies have suggested that different physical exercises could affect the brain in different ways (Erickson et al., 2011; Voelcker-Rehage et al., 2011; Burrel, 2015). For example, Erickson et al. (2011) found that 1-year of aerobic exercise training could effectively increase the size of the anterior hippocampus in the elderly, but not the caudate nucleus and thalamus volumes, accompanied by improved memory function. Moreover, such effects were not shown in the individuals performing a stretching and toning program. Voelcker-Rehage et al.'s (2011) longitudinal research reported that although the older adults participating in cardiorespiratory training aimed at enhancing cardiorespiratory fitness or coordination training to increase fine- and gross-motor body coordination could improve their executive functioning and perceptual speed, although with different effects on speed and accuracy, the two types of exercise had different impacts on neural activity, with an increased activation of the sensorimotor network and less prefrontal activation in the cardiovascular-training group, and increased activation in the visual-spatial network (e.g., right inferior frontal gyrus, superior parietal cortex, thalamus, and caudate body) in the coordination-training group. In addition, animal studies showed that regular cardiovascular training in rats did not increase the number of synapses, but could increase the density of capillaries (Black et al., 1990) and shorten the diffusion distance from the blood vessels in the molecular layer of the paramedian lobule (Isaacs et al., 1992). In contrast, regular complex motor-skill training in the rats did not increase the density of capillaries, but could substantially increase the number of synapses per Purkinje neuron and blood vessels, thus maintaining the diffusion distance (Isaacs et al., 1992). These findings from both human and animal studies suggest that different types of exercise intervention (e.g., open- vs. closed-skill) could produce distinct training effects on the brain tissues and neural activations. However, in the present study the effects of exercise interventions on neural activity in the frontal-to-parietal cortices seem to be comprehensively covered. In fact, in terms of aging, cognition, and brain function, the phenomenon of dedifferentiation, which characterizes a simple marker of cognitive decline, is often found in the elderly. That is, the regions of the brain that are recruited to perform a variety of cognitive tasks are less specific among older rather than younger adults (Cabeza, 2001), possibly due to additional cortical areas being recruited to compensate for losses in neural efficiency in the former. Therefore, while increased prefrontal activation to compensate for processing impairment, particularly in posterior areas, is a consequence of age-related structural and functional declines in various brain regions (Greenwood, 2007), the findings of the present study suggest that both open- and closed-skill exercise modes could not only facilitate anterior cortical processing efficiency, but also compensate for neural processing impairment in the posterior cortical areas due to cognitive aging. In addition, whether the improvement in cognitive function that results from the open- and closed-skill exercise would produce more distinctive effects in different age groups is one area for future works to address. It is likely that longitudinal assessments of the effects of open- and closed exercise interventions on electrophysiological performance in young adults would help clarify the benefits of different types of exercise with regard to cognitive functions. Colcombe and Kramer's (2003) meta-analytic study suggested that aerobic fitness training increases cognitive performance by 0.5 SD on average, regardless of the exercise training method and types of cognitive task, especially in the executive-control processes. Therefore, although the distinct benefits of open- and closed-skill exercises on both working memory and task switching performance were found in the present research, cardiorespiratory fitness could play an important role and have a beneficial influence on these two types of cognitive functions in older adults (Netz et al., 2011; Voelcker-Rehage et al., 2011; Wang et al., 2016). Additionally, in light of evidence that depression, education, social stimulation, and BMI could also mediate the exercise-cognition association in the elderly (Miller et al., 2012; Ronan et al., 2016; Tomioka et al., 2016), adequate controls to take into account the confounding factors that participants in the intervention groups are impacted by, in addition to physical exercise, need to be considered. Indeed, in the present study the two groups showed different levels of improvements in cardiorespiratory fitness, social participation, and BMI after participation in either open- or closed-skilled exercise. However, when including these improved confounding factors as covariates in the analysis of the improved neurocognitive performance after the exercise intervention, the difference among the three groups remained significant, showing that both types of exercise modes do indeed lead to improved neurocognitive performance. BODY.STRENGTHS AND WEAKNESSES: Although a number of confounding factors which could mediate the exercise-cognition association were rigorously controlled for in the current study, some potential limitations of this work need to be addressed. First, since changes in the brain and neurocognitive performance are not always proportional to each other, a decrease in brain size and plasticity that results in cognitive changes is associated with normal aging (Peters, 2006). Further MRI/fMRI studies to explore the changes in the sizes/densities of the brain tissues and in patterns of brain activation would be helpful to understand the complex relationship between different exercise modes and neurocognitive performance, and to determine the exact mechanism of cognitive enhancement in the elderly. Second, some exercise items that do not aim to increase cardiorespiratory fitness are also included in t2001). However, the adopted reference did not change the topography map of the P300 components (Yang et al., 2017), and the P3 amplitudes decreased significantly with increasing cognitive loads (Kok, 2001) in the present study. These findings suggest that the electrophysiological findings from such a reference site are still reliable. However, further EEG-fMRI studies aiming to explore the effects of exercise interventions might consider applying the Reference Electrode Standardization Technique (REST) (Yao, 2001; Yao et al., 2005) as the reference method for ERP data (Yang et al., 2017). BODY.CONCLUSIONS: Extending earlier cross-sectional studies on aging that relied on volunteer participants, which could inevitably include some selection bias with an overrepresentation of individuals with inherently higher executive functioning, the present study of a 6-month exercise intervention confirmed recent cross-sectional results showing that open- and closed-skill exercise modes relate differently to various forms of executive functioning (e.g., task switching and working memory) in relation to cognitive aging in older adults. However, the current findings more clearly revealed that both open- and closed-skill exercise could effectively enhance overall brain cortical activity. These beneficial effects of the two exercise interventions on the neuropsychological and electrophysiological performances in the elderly remained unchanged after statistical adjustment for improved cardiorespiratory fitness, social participation, and BMI. Although exercise is a simple and healthy lifestyle factor that has been proposed to be protective against neurocognitive declines during aging, possibly even retarding or reversing age-associated degeneration in the brain, different exercise modes seem to have different effects on various forms of executive function in the elderly. BODY.AUTHOR CONTRIBUTIONS: CT designed the study, wrote the protocol, and the first draft of the manuscript. CP analyzed the data. FC and YT helped collect data. 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: Lost in translation? A multilingual Query Builder improves the quality of PubMed queries: a randomised controlled trial ABSTRACT.BACKGROUND: MEDLINE is the most widely used medical bibliographic database in the world. Most of its citations are in English and this can be an obstacle for some researchers to access the information the database contains. We created a multilingual query builder to facilitate access to the PubMed subset using a language other than English. The aim of our study was to assess the impact of this multilingual query builder on the quality of PubMed queries for non-native English speaking physicians and medical researchers. ABSTRACT.METHODS: A randomised controlled study was conducted among French speaking general practice residents. We designed a multi-lingual query builder to facilitate information retrieval, based on available MeSH translations and providing users with both an interface and a controlled vocabulary in their own language. Participating residents were randomly allocated either the French or the English version of the query builder. They were asked to translate 12 short medical questions into MeSH queries. The main outcome was the quality of the query. Two librarians blind to the arm independently evaluated each query, using a modified published classification that differentiated eight types of errors. ABSTRACT.RESULTS: Twenty residents used the French version of the query builder and 22 used the English version. 492 queries were analysed. There were significantly more perfect queries in the French group vs. the English group (respectively 37.9% vs. 17.9%; p < 0.01). It took significantly more time for the members of the English group than the members of the French group to build each query, respectively 194 sec vs. 128 sec; p < 0.01. ABSTRACT.CONCLUSIONS: This multi-lingual query builder is an effective tool to improve the quality of PubMed queries in particular for researchers whose first language is not English. BODY.BACKGROUND: Evidence based medicine is increasingly encouraged in medical practice and decision-making, which requires evidence based on valid research. MEDLINE, created by the US National Library of Medicine (NLM), is the most widely used medical bibliographic database in the world. It is the largest component of PubMed, which is the largest free online database of biomedical journal citations and abstracts. PubMed currently contains 26,415,890 citations from 5,650 indexed journals from 81 countries and in 60 languages. Each PubMed record is indexed with the NLM’s controlled vocabulary, the Medical Subject Headings (MeSH) [1]. More than 82% of PubMed citations are in English and this can be an obstacle for some researchers to access the information the database contains [2]. Nevertheless, some tools are, in fact, available to help non–native-English speakers to access PubMed references written in their native language: i.e. BabelMeSH [3, 4], Patient, Intervention, Comparison, Outcome (PICO) Linguist [4] and LiSSa [5]. Although some of these tools have demonstrated a high level of precision and coverage [5], they can only permit limited access to available evidence. Recent research has also confirmed the lack of skills to perform a literature search among physicians: they not only are unable to master the specific querying process of medical databases but also feel uneasy in performing research [6]. The English used in the PubMed querying process might possibly explain some of the difficulties. Therefore, we were prompted to create a multilingual query builder to facilitate access to the PubMed subset using a language other than English (e.g. French, German, Spanish, or Norwegian), with an advanced multifunctional system. This practical tool relies on the MeSH translation in multiple languages to boost information retrieval. The aim of this study was to assess the impact of a multilingual query builder on the quality of PubMed queries for physicians and medical researchers, in particular those whose first language is not English. BODY.METHODS: BODY.THE MULTI-LINGUAL QUERY BUILDER: Many users, including the French, have difficulties writing queries when performing research on PubMed. Therefore, we designed a multi-lingual query builder to facilitate information retrieval. Our objective was to develop an “easy-to-use” tool to select relevant MeSH terms. This tool is based on available MeSH translations, providing users with both an interface and a controlled vocabulary in their own language. Currently, the interface has been translated into French, English and Spanish while the MeSH thesaurus is available in more than fifteen languages (e.g. Dutch, English, Finnish, French, German, Italian, Portuguese and Spanish). Users can fill the search box with natural language. The Autocomplete search function helps to find the relevant MeSH terms. Full MeSH information is available, including definitions, relations and a hierarchical qualifiers list. The selected MeSH term is added to the query. Other MeSH terms can be added, using usual Boolean operators. When a satisfactory query is obtained, the user can submit it to PubMed. The tool allows to go back and forth: the user can observe the number of results given by the query - or the results itself - and reformulate the query if needed. An example for a given query appears in Fig. 1 (English interface). The query is then completed by using a wide range of synonyms such as natural language terms, in order to maximize recall and precision. As shown by Thirion et al., adding terms to the query significantly increases the precision when compared to the default query used by PubMed (54.5% vs 27%) [7].Fig. 1Query Builder web application detailed screenshot The query builder is a web application (written in Java thanks to the Vaadin framework) connected to four services. Each service is dedicated to a specific task: i) the autocomplete function provides the MeSH terms related to the query; ii) the terminology server retrieval collects data of the selected MeSH term; iii) the InfoRoute service is the main application service, it builds the PubMed URL using the advanced PubMed search syntax (search tags [8], MeSH terms, Boolean operators) and iiii) a function to get the results number in PubMed for the generated query. BODY.RECRUITMENT AND STUDY SET-UP: A randomised controlled study was conducted at the Department of General Practice of Rouen University during the month of January 2015. Native French speaking general practice residents were recruited by email by one researcher (MS) and randomly allocated either the French or the English version of the query builder web tool, using a computer-generated randomisation sequence. The residents were asked to translate 12 short medical questions (the same for each participant) into MeSH queries. The questions were written in French for both groups. They received a 15 minutes training about the query builder. During the training session, the participants used the query builder in the language for which they were randomized. This training was performed in French for all participants. It focused on the different stages of bibliographic search, the description and the use of MeSH thesaurus, Boolean operators and subheadings. The evaluation took place in two adjacent rooms of the Rouen University Medical School, at the same time. Residents allocated to the first room had access to the French version of the query builder, residents allocated to the second one had access to the English version. Twelve short medical questions were designed with three levels of complexity. The clinical questions were drafted by an experienced medical librarian (GK). They were chosen arbitrarily in the logs of a document database developed in the Rouen University hospital (http://www.chu-rouen.fr/cismef/). They were then validated by two physicians (MS and NG) for medical relevance. The level of complexity was decided in regard to the number of MeSH terms (descriptors or subheadings) required for a correct query. Low difficulty questions required two terms. Medium difficulty questions required three terms. High difficulty questions required four terms. The difficulty to express an idea with MeSH terms was also taken into account for one question (salty taste in the mouth). The list of short medical questions is shown in Table 1.Table 1Short medical questions (EN/FR) and queries considered as correct Low difficulty questions  Fibroid uterus spontaneous rupture/Rupture spontané d’un fibrome utérin Leiomyoma AND rupture, spontaneous  Alopecia areata prevention/Prévention des pelades Alopecia areata/prevention and control  Vitamin D determination in blood/Dosage de la vitamine D dans le sang Vitamin D/blood; vitamin D/analysis OR vitamin D/blood; vitamin D/blood OR (vitamin D AND blood chemical analysis)  Sarcopenia for over 65 years old patients/Sarcopénie chez les patients de plus de 65 ans Sarcopenia AND aged Medium difficulty questions  Vaccination induced pain in infant/Douleur au cours de la vaccination des nourrissons Infant AND Pain AND vaccination; infant AND (Pain OR pain measurement OR pain management) AND vaccination  Guidelines for breast cancer treatment/Recommandations sur le traitement du cancer du sein Breast neoplasms/therapy AND practice guidelines as topic; breast neoplasms/therapy AND practice guideline  Asthma epidemiology in USA/Epidémiologie de l’asthme aux Etats-Unis Asthma/epidemiology AND united states; (asthma/epidemiology OR (asthma AND epidemiology)) AND united states  Screening for uterine cervical neoplasm/Dépistage du cancer du col de l’utérus Mass screening AND uterine cervical neoplasms/prevention and control; mass screening AND uterine cervical neoplasms/diagnosis; High difficulty questions  Salty taste in the mouth/Goût sale dans la bouche Sodium chloride AND dysgeusia; sodium chloride AND taste disorders; sodium chloride AND taste perception  Allopurinol cutaneous side effect/Effets secondaires cutanés de l’allopurinol Allopurinol/adverse effect AND skin diseases/chemically induced; allopurinol/adverse effect AND (skin diseases/chemically induced OR skin manifestations/chemically induced)  Glucocorticoids effects on asthmatic patient’s growth/Impact des glucocorticoïdes sur la croissance du patient asthmatique ((Glucocorticoids/adverse effects) AND (growth/drug effects OR growth disorders/chemically induced) AND asthma/drug therapy; ((glucocorticoids/adverse effects) AND (growth/drug effects OR growth disorders/etiology) AND asthma/drug therapy  Antibiotics dosage for overweight or obese patient/Posologie des antibiotiques chez le patient en surpoids ou obèse Anti-bacterial agents/administration and dosage AND (Obesity OR overweight); (anti-bacterial agents/administration and dosage OR (drug dosage calculations AND anti-bacterial agents)) AND (Obesity OR overweight) Questions were presented in French to all the participants. Queries only contain MeSH terms and/or MeSH terms/subheadings, except “practice guideline” which is a publication type For evaluation purposes, the query builder was embedded in a light web application. It allowed investigators to lock the interface language (English vs. French), to propose one different page for each short medical question, record the submitted query and the overall response time. Participants were free to navigate between each medical question and change their queries. BODY.OUTCOMES AND STATISTICAL ANALYSIS: The main outcome was the quality of the query. The expected queries were built by a team of medical librarians and are available in Table 1. Both groups delivered queries in English and these queries were then submitted to the librarians for evaluation. The query quality was assessed according to a modified published classification [9] that differentiated eight types of errors: irrelevant MeSH term, over-specification (descriptors or subheadings), under-specification (descriptors or subheadings), incorrect operator and syntax error. The description of these types of errors is shown in Table 2. Queries may present more than one mistake of each kind. If a query had two irrelevant MeSH terms and one under-specified qualifier, three errors were counted. Queries without any errors were considered as perfect. Two librarians blinded to the arm independently evaluated each query and resolved discrepancies by consensus. Proportions were compared using Fisher’s test. Analyses were stratified according to the difficulty level.Table 2Summary of main results Error type and its description (from Vanopstal et al)French versionn (%)English versionn (%) p Perfect queries 89 (37.9) 46 (17.9) <0.01 Irrelevant MeSH term 52 (22.1) 83 (32.3) <0.01 Query contains at least one incorrect MeSH term Over-specification (descriptors) 28 (11.9) 39 (15.2) 0.3 Query contains at least one MeSH term that is too narrow Over-specification (qualifiers) 17 (7.2) 17 (6.6) 0.8 Under-specification (descriptors) 41 (17.4) 87 (33.9) <0.01 Query contains at least one MeSH term that is too broad Under-specification (qualifiers) 48 (20.4) 72 (28) 0.05 Incorrect operator 1 (0.4) 1 (0.4) 1 Misuse of “AND” or “OR” Syntax error 2 (0.9) 4 (1.6) 0.7 Query contains unmatched brackets or quotes, or truncated words The time spent in building queries was measured as a secondary outcome. It was measured by the web form from the reading of the clinical question to the submission of the final MeSH query by the end-user. Times were compared using the Mann-Whitney test. For an expected improvement of 15% in the group ‘native language’ vs. ‘English language’, from 25 to 40%, 17 end-users per group were required (alpha = 0.05, beta = 0.1, var = pq/n). All statistical tests were performed with R 3.0.2 software. BODY.RESULTS: Forty-two residents participated in this study, each of them had at least one year of experience. Twenty residents used the French version of the query builder and 22 used the English version. The mean age was 26.9 years old in the French group and 26.7 years old in the English group (p = 0.58). The proportion of male was 35% in the French group and 45.5% in the English group (p = 0.54). After the exclusion of 11 non-responses and one obvious mistake (query related to a previous question), 492 queries were analysed. Please see flow chart in Fig. 2.Fig. 2Application screenshot Queries based on low difficulty questions contained 0.83 error on average [0.71–0.96], queries based on medium difficulty questions 0.92 error [0.80–1.03] and queries based on high difficulty questions 1.69 errors [1.53-1.85] (p < 0.01). One hundred and thirty-five queries (27.4%) were considered as perfect. There were significantly more perfect queries in the French group vs. the English group (respectively 37.9% vs. 17.9%; p < 0.01). The most frequent errors were the use of an underspecified MeSH term (44.7%) or an irrelevant MeSH term (27.4%). Members of the French group performed significantly better for these two kinds of mistake, respectively 35.7% vs 52.9% (p < 0.01) and 22.1% vs. 32.3% (p = 0.01). No differences were found between the two groups concerning over-specification errors, the use of incorrect operator or syntax errors. See Table 2 for detailed results. There were significantly more perfect queries in the French group vs. the English group for low difficulty questions (48.1% vs 31.8%; p = 0.04), medium difficulty questions (45.6% vs 18.4%; p < 0.01) and high difficulty questions (20.3% vs 3.5%; p < 0.01). The fastest evaluator spent 85 seconds on average for each query, the slowest 260 seconds. It took significantly more time for the English participants than the French participants to build each query, respectively 194 sec vs. 128 sec; p < 0.01 (Fig. 3).Fig. 3Distribution of average time taken by participants to write one query, according to language BODY.DISCUSSION: BODY.SUMMARY OF MAIN RESULTS: Our research findings show that a multi-lingual query builder to access PubMed could be a useful tool in research and clinical practice for non-native English speakers. Participants querying in their first language built twice more perfect queries than participants querying in English. These results were found for low, medium and high difficulty questions. The impact of querying in first language increased with the level of difficulty. Participants querying in their first language took less time to build each query than participants querying in English. BODY.DISCUSSION OF THE MAIN RESULTS: Many barriers to query building and information retrieval among healthcare professionals have previously been identified in the literature. Currently, the most reported obstacles are: (i) the amount of time required to find information, (ii) difficulties in reformulating the original question and finding an optimal search strategy, (iii) lack of a good source of information, uncertainty as to whether all relevant information has been found and (iiii) inadequate synthesis of any pieces of evidence into a clinically useful approach [10, 11]. The literature also shows that physicians, and especially primary care doctors, express a need for database training, regardless of their first language [6, 12]. Physicians’ difficulties in building search queries are well known. In 2007, a web log analysis was undertaken in a meta-search engine covering 150 health resources and a variety of guidelines. It showed that most queries were built using a single search term and no Boolean operator [13]. A similar study was conducted on PubMed queries. If PubMed queries had a median of three terms, only 11% of them contained Boolean operators [14]. Many factors can influence the physicians’ ability to build relevant search queries, including the level of English skills [9, 15]. These findings suggest that our query builder may be of significant value for non-native English speaking healthcare professionals. As previously mentioned, our data suggest that the impact of using the first language increases with the complexity of clinical questions. Complexity appears to play a key role in physicians’ difficulties in information retrieval. They fail to master the use of Boolean operators and, when dealing with complex clinical questions, GP trainees tend to refer to their colleagues more than electronic sources [13, 16]. Participants querying in their first language took less time to build each query than participants querying in English. This appears to be an important finding, as time constraints are always cited as a major obstacle when seeking information and may improve the PubMed’s use as searches with PubMed are not as frequent as searches with Google or UpToDate [10, 17]. Nevertheless, only one-third of the queries were considered perfect, even among participants querying in their first language. Irrelevant MeSH terms and the lack of specification in descriptors and subheadings lead to poor precision and recall [9]. Querying in first language will not solve all the problems faced by researchers and physicians, especially as the overwhelming majority of PubMed references remain in English. Some research tools already provide an automatic translation of a biomedical text, including titles and abstracts and using the MEDLINE database [18]. Other tools increase the information retrieval task performance by allowing non–native-English speakers to access PubMed references written in their native language: BabelMeSH [3, 4], Patient, Intervention, Comparison, Outcome (PICO) Linguist [4] and LiSSa [5]. According to Gagnon et al., educational meetings currently seem to be the only type of interventions showing a significant positive effect on clinical information retrieval technologies adoption by healthcare professionals [19]. BODY.STRENGTHS AND LIMITATIONS: This study has several limitations. First, only the quality of the query was assessed and not the quality of the results. However, the quality of the query is strongly associated to the quality of the results. Vanopstal et al. demonstrated that under-specified queries led to an increase of noise and our data show that under-specification is the main error compensated by querying in users’ first language [9]. An evaluation of the queries is planned as a second step. A sample of discrepant results (using queries built by English group versus French group) will be rated by a group of physicians. This will allow us to assess the impact of the multilingual query builder on the quality of the results. Second, this study only involves French residents in general medicine and this could affect the external validity of the results. Nevertheless, we do know that PubMed querying issues are encountered among physicians and medical researchers worldwide [14]. In order to enhance external validity, a similar trial will soon be conducted among Spanish speaking residents and physicians at the Buenos Aires Italian Hospital (Argentina). This study is, to our knowledge, the first published evaluation of a multi-lingual query builder to access the PubMed subset. In order to avoid any before and after studies bias, a randomised controlled trial was carried-out. The clinical questions were drafted by an experienced medical librarian (GK) and validated by two physicians (MS and NG). The theoretical difficulty levels of the clinical questions were proven due to the significant association between the average number of errors and the dif9]. BODY.PERSPECTIVES: As might be expected, this study clearly demonstrates that querying in first language is easier than querying in English. This study will soon be repeated among Argentinean healthcare professionals, comparing the use of Spanish and English. The multi-lingual query builder permits to overcome the obstacle of English when building queries, and could be of major interest for students, clinicians and researchers worldwide. The query builder is already available in more than fifteen languages including Dutch, English, Finnish, French, German, Italian, Portuguese and Spanish. Some translations of MeSH terms or of the web site interface may still be lacking and we actively encourage all the teams working on MeSH translation, i.e. INSERM and Inist-CNRS in France. Our tool permits physicians and medical researchers to perform a request in the most relevant PubMed database fields. Full MeSH information is available, including definitions, relations and a hierarchical qualifiers list. A wide range of synonyms is also used automatically as natural language terms to complete the query, in order to maximize recall. Some features are currently lacking, like combining previous requests, however our priority was to first build an effective easy-to-use tool. BODY.CONCLUSION: Physicians often feel incompetent when seeking medical information, especially when using bibliographic databases. This sentiment is sometimes associated with a feeling of illegitimacy, as these databases were not created to meet their needs. This shows that there is a gap between an idealized academic search model and the practical requirements of everyday life [20]. This multi-lingual query builder is an effective tool to improve the quality of PubMed queries and should narrow the gap, particularly for physicians and researchers whose first language is not English.

TITLE: Administration of rocuronium based on real body weight versus fat-free mass in patients with lymphedema ABSTRACT.OBJECTIVE: To compare the clinical pharmacokinetics of rocuronium when applied according to fat-free mass versus real body weight during anesthetic induction of patients with lymphedema. ABSTRACT.METHODS: Sixty patients with lymphedema (age, 18–60 years; American Society of Anesthesiologists physical status, I–II) undergoing elective surgery with general anesthesia were randomly divided into two groups. Rocuronium was administered based on the fat-free mass in 30 patients and real body weight in 30 patients. General anesthesia was induced with propofol and remifentanil by target-controlled infusion. Intubation was attempted when the onset time (T1) (time from end of bolus injection to 100% twitch depression) reached maximal inhibition, and respiratory support with mechanical ventilation was then applied. The T1, clinical duration (time from end of bolus injection to recovery of twitch tension to 25% of control), recovery index (time from 25% to 75% of recovery of T1), and dosage were recorded. ABSTRACT.RESULTS: Complete data were recorded for 59 patients, and there were no significant differences in the general condition, intubation condition, or median duration of action of rocuronium between the two groups. However, the median T1, recovery index, and dosage of rocuronium were significantly different. ABSTRACT.CONCLUSION: Good intubation conditions and a shortened clinical duration can be obtained for patients with lymphedema when induction with rocuronium is based on the fat-free mass. BODY.INTRODUCTION: Lymphedema is a lymphatic circulation disorder with complex pathological causes. With the recent advancements in surgical procedures, increasing numbers of lymphedema patients now undergo surgical treatment.1 Previous pharmacodynamics studies on rocuronium mainly focused on obese patients. The results showed that administration of rocuronium based on the fat-free mass (FFM) in obese patients achieved a neuromuscular block similar to that based on the real body weight (RBW) in patients of normal body weight, resulting in reduced interindividual variations in drug responses among obese patients.2 Most patients with lymphedema are overweight. However, they differ from obese patients in terms of body mass composition and pathophysiological functions.3 In the present study, we investigated the pharmacodynamics of rocuronium administered based on different body weight scalars to provide a reference for safe drug administration in patients with lymphedema. BODY.MATERIALS AND METHODS: BODY.GENERATION INFORMATION: The study was approved by the Medical Ethics Committee of Beijing Shijitan Hospital. Written informed consent was obtained from the patients before the surgical procedure. Fifty-nine patients with lymphedema who underwent elective lymphatic venous anastomosis (LVA) (upper and lower extremities) from October 2015 to May 2016 were included in the study. The patients were aged 18 to 60 years and had an American Society of Anesthesiologists physical status of I to II and body mass index (BMI) of 30 to 40 kg/m2. All patients underwent nasogastric intubation before the surgery, and their Mallampati score ranged from I to III. The exclusion criteria were renal and hepatic dysfunction, allergic constitution, anticipated airway difficulties, and intraoperative blood loss of >200 ml. Patients were randomly divided into two groups: Group A (based on FFM) and Group B (based on RBW). BODY.GENERAL ANESTHESIA: Before anesthesia, all patients were routinely monitored via electrocardiography, peripheral oxygen saturation, heart rate, and blood pressure using multi-parameter anesthesia monitors. The bispectral index (BIS) was recorded with a BIS monitor. Ringer’s sodium acetate was administered via peripheral intravenous infusion. Anesthesia was induced with propofol (plasma concentration of 3–4 µg/ml) and remifentanil (effect site concentration of 3–6 ng/ml) by target-controlled infusion (TCI). When the BIS reached 60, all patients in each group was given a bolus intravenous injection of rocuronium (0.6 mg/kg, twice the effective dose in 95% of the population [ED95] of rocuronium).4 Drug administration in Group A was based on the FFM:FFMMale = 9.27 × l03 × total body weight/(6.68 × l03 + 216 × BMI) FFMFemale = 9.27 × l03 × total body weight/(8.78 × l03 + 244 × BMI) BMI = body weight (kg)/height (cm)2 5 Drug administration in Group B was based on the RBW. Calibration of neuromuscular blockade was initiated before rocuronium injection. Acceleromyography (TOF-Watch SX Organon Limited, Co. Dublin Ireland Monitor) was used to evaluate the degree of neuromuscular blockade at the adductor pollicis muscle using train-of-four stimulations. Tracheal intubation was attempted when the first twitch of train-of-four (onset time, T1) reached maximal depression, and mechanical ventilation was used to maintain the end-tidal carbon dioxide pressure at 35 to 45 mmHg. When T1 recovered to 25% of the baseline value, patients in Group A were administered a supplementary dose of rocuronium (0.3 mg/kg, ED95) based on the FFM, and those in Group B were administered 0.3 mg/kg of rocuronium based on the RBW. General anesthesia was maintained with propofol and remifentanil by TCI, and the BIS was maintained at 45 to 55. TCI was discontinued 20 min before the end of surgery. After regaining spontaneous respiration and consciousness, the patients were extubated and sent to the recovery room. BODY.INDICATORS FOR EVALUATION: The rocuronium dosage, T1 (time from end of bolus injection to 100% twitch depression), maximum depression of T1, clinical duration (time from end of bolus injection to recovery of twitch tension to 25% of baseline), and recovery index (time lapse of recovery from 25% to 75% of T1) were recorded, and the coefficient of variation of each parameter was calculated. Intubation conditions were assessed using the Cooper scoring system: a total score of 8 to 9 was considered excellent, 6 to 7 good, 3 to 5 fair, and 0 to 2 poor.6 BODY.STATISTICAL ANALYSIS: The aim of this study was to compare the effects of rocuronium between two types of administration in patients with lymphedema undergoing surgery. With statistical significance set at 0.05, power at 80%, and loss-to-follow-up at about 6%, the estimated sample size of each group was 68. In total, 140 patients were finally enrolled in this study. A biostatistician who did not participate in the data management or analysis used SAS 9.2 software (SAS Institute, Cary, NC) to generate random numbers in a 1:1 ratio. The results of randomization were sealed in sequentially numbered envelopes. Patients with lymphedema were then consecutively recruited and randomly assigned to one of the two above-described groups. The biostatistician, patients, and doctors were blinded to the treatment allocation. Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., Chicago, IL). The total dosage, T1, clinical duration, and recovery index are presented as mean ± standard deviation. The differences in these indicators between the two groups were analyzed with an independent-samples t test. Other indexes, including sex and type of surgery, were compared with the χ2 test. A P value of < 0.05 was considered statistically significant. BODY.RESULTS: BODY.COMPARISON OF GENERAL CONDITIONS BETWEEN GROUPS A AND B: All patients had complete statistical data. There were no significant differences between the two groups of patients in terms of sex, age, BMI, FFM, or type of surgery. Therefore, the two sets of data were comparable (Table 1). Table 1.Comparison of general conditions between the two groups. GroupPatients (n)SexAge (years)BMI (kg/m2)FFM (kg)Type of surgery (n)Male (n)Female (n)Upper extremity anastomosisLower extremity anastomosisA 30 9 21 41 ± 6 36 ± 2 52 ± 1 22 8 B 29 7 22 43 ± 5 35 ± 1 49 ± 2 21 8 P >0.05 >0.05 >0.05 Data are presented as mean ± standard deviation. BMI: body mass index, FFM: fat-free mass. Patients in Group B had a longer clinical duration of rocuronium than those in Group A; however, this difference was not statistically significant. Statistically significant differences were observed in the T1, recovery index, and dosage between the two groups (P < 0.05). No significant difference was observed between the two groups in the intubation condition as assessed by the Cooper score or in the first-pass intubation success rate (Table 2). Table 2.Comparisons of rocuronium onset time, rocuronium dosage, and Cooper scores between the two groups. GroupPatients (n)Onset time (s)Clinical duration (min)Recovery index (min)Dosage (mg/h)Cooper scoreA 30 75.1 ± 6.9 44.8 ± 3.9 2.2 ± 0.5 41.5 ± 4.5 8.6 ± 0.7 B 29 58.1 ± 5.2 51.6 ± 5.9 5.3 ± 0.8 58.2 ± 5.6 8.3 ± 0.9 P <0.05 >0.05 <0.05 <0.05 >0.05 Data are presented as mean ± standard deviation. BODY.DISCUSSION: Lymphedema can be classified as primary or secondary. Primary lymphedema is hereditary, whereas secondary lymphedema is mainly caused by surgical treatment of tumors, radiotherapy and chemotherapy, lymphatic obstruction, and chronic venous insufficiency.7 For this select group of patients, the lack of neuromuscular monitoring will lead to difficulties for anesthesiologists in terms of deciding whether to administer a supplementary dose of the neuromuscular blocking agent and determining the timing of administration. When the supplementary dose is administered based solely on past experience, it often results in prolonged action of the neuromuscular blocking agents, leading to side effects such as postoperative residual neuromuscular blockade. Clinically, dosage calculation for neuromuscular blocking agents is based on the RBW and ED95. However, there is substantial interindividual variation in the response to neuromuscular blocking agents among patients with special body mass compositions. Therefore, evidence-based medicine has been increasingly proving the rationality of drug administration based on the corrected body weight in this special population. Although patients with lymphedema are not equivalent to obese patients, most are overweight, which will also affect the volume of distribution, plasma concentration, and elimination half-life of neuromuscular blocking agents. Previous studies on the clinical application of neuromuscular blocking agents in obese patients have shown that changes in the pathophysiological functions and body mass composition of obese patients could affect the pharmacodynamics of these drugs. Meyhoff et al.2 showed that calculation of the rocuronium dosage should be based on the ideal body weight of obese patients to avoid a prolonged onset time of neuromuscular blocking agents. Wang et al.8 found that while administration of rocuronium based on the body surface area did not reduce interindividual variations in the onset of neuromuscular blockade, the total drug dosage required was reduced. In contrast, Meyhoff et al.2 reported that administration of rocuronium based on the FFM achieved a clinical duration in obese patients similar to that in patients of normal body weight, while also reducing interindividual variation in pharmacodynamics among obese patients. The target sites of neuromuscular blocking agents are located at the neuromuscular junctions, and the lean body mass determines the number of neuromuscular junctions.8 Furthermore, the pharmacological effect of a neuromuscular blocking agent is primarily dependent on its plasma concentration, which is closely associated with the blood volume. Nondepolarizing neuromuscular blocking agents are mainly distributed in the lean muscles because of their high water solubility and low fat solubility. Therefore, calculation of the administration dosage of these agents is more accurate and reasonable when based on the FFM than on the RBW.2 Our study also showed that for patients with lymphedema, the FFM is a better body weight scalar than the RBW for the administration of rocuronium. The widely accepted Janmahasatian formula was applied to calculate the lean body weight in the present study. This formula allows for a more accurate calculation of the lean body weight of patients with lymphedema.5 Substantial variations in lymphatic drainage occur before and after LVA and may affect the plasma protein volume and concentration. This is especially true for drugs with high fat solubility and may result in fluctuations in the drug plasma concentration. Research related to the lymphatic circulatory system is less advanced than that related to the blood circulatory system. We hope that further investigations will be conducted on the changes in the circulation steady state during LVA and lymphatic liposuction and their effects on drug concentrations and pharmacodynamics. BODY.CONCLUSION: Good intubation conditions and a shortened clinical duration can be obtained for patients with lymphedema when induction with rocuronium is based on the FFM.

TITLE: Sufentanil Sublingual Tablet System for the Management of Postoperative Pain Following Open Abdominal SurgeryA Randomized, Placebo-Controlled Study ABSTRACT.BACKGROUND AND OBJECTIVES: This study evaluates the efficacy and safety of a sufentanil sublingual tablet system (SSTS) for the management of postoperative pain following open abdominal surgery. ABSTRACT.METHODS: At 13 hospital sites in the United States, patients following surgery with pain intensity of greater than 4 on an 11-point numerical rating scale were randomized to receive SSTS dispensing a 15-μg sufentanil tablet sublingually with a 20-minute lockout or an identical system dispensing a placebo tablet sublingually. Pain intensity scores were recorded at baseline and for up to 72 hours after starting study drug. The primary end point was time-weighted summed pain intensity difference (SPID) over 48 hours. Secondary end points included SPID and total pain relief (TOTPAR) for up to 72 hours and patient and health care provider global assessments of the method of pain control. ABSTRACT.RESULTS: Summed pain intensity difference over 48 hours was significantly higher in the SSTS group than in the placebo group (least squares mean [SEM], 105.60 [10.14] vs 55.58 [13.11]; P = 0.001). Mean SPID and TOTPAR scores were significantly higher in the SSTS group at all time points from 1 hour (SPID) or 2 hours (TOTPAR) until 72 hours (P < 0.05). In the SSTS group, patient global assessment and health care provider global assessment ratings of good or excellent were greater than placebo at all time points (P < 0.01). Safety parameters, including adverse events and vital signs, were similar for SSTS and placebo. ABSTRACT.CONCLUSIONS: These results suggest that SSTS is effective and safe for the management of postoperative pain in patients following open abdominal surgery. Administration of opioids to postoperative patients using intravenous patient-controlled analgesia (IV PCA) results in lower pain scores and higher patient satisfaction compared with nurse-administered modalities.1,2 However, the requirement of a patent IV line and tethering of the patient to an IV PCA pump mounted on an IV pole results in risk of infection, reduced mobility, and analgesic gaps due to IV catheter infiltration or IV tubing obstruction.3,4 The programming of the pump, which is required to be completed by the nurse to set up the device, can result in dosing errors.5,6 The sufentanil sublingual tablet system (SSTS; Zalviso; AcelRx Pharmaceuticals, Redwood City, California), currently under review by the FDA, is a preprogrammed, noninvasive patient-activated bedside system to allow patients to manage moderate to severe pain in a hospital setting. The systemic uptake of sublingual sufentanil is rapid because of its high lipophilicity, and the resultant pharmacokinetics demonstrates a blunted peak plasma level and longer plasma half-time than IV administered sufentanil.7 The device has a preprogrammed 20-minute lockout interval and uses a radiofrequency identification (RFID) thumb tag to allow only the patient to operate the device (Fig. 1). Upon setup of the system, completed without a need for programming decisions, the nurse inserts a small cartridge containing 40 sufentanil tablets (approximately a 2-day supply) into the dispenser tip, which is then locked into the controller base, and the system is tethered to the bedside or other secure location. The controller base has a graphic user interface screen that facilitates patient training by the nurse and displays setup instructions and system data for authorized health care professionals. Phase 2 dose-finding studies in patients following major surgery demonstrated that sufentanil 15 μg per tablet was the optimal dosage strength, resulting in high patient satisfaction and a similar adverse event profile to lower dosage strengths.7 Sufentanil 15 μg dosed sublingually is equivalent to 3 to 4 mg IV morphine based on 300 to 400 potency factor and 60% bioavailability of sublingual sufentanil. Therefore, this sufentanil dose, available every 20 minutes, reflects an approximately equianalgesic dose to the standard 1 mg morphine on-demand every 6 minutes with typical IV PCA settings. The objectives of the present study were to evaluate the efficacy and safety of SSTS for the management of postoperative pain in adult patients who had undergone open abdominal surgery. The study hypothesis was that sublingually administered sufentanil using the SSTS device would allow patients to titrate rapidly to acceptable levels of analgesia and would result in a good safety profile and ease of use for both patients and nurses. FIGURE 1Sufentanil sublingual tablet system with RFID patient thumb tag and security tether attached to the bottom of the controller. BODY.METHODS: This phase 3, randomized, placebo-controlled, double-blind study was conducted at 13 hospitals in the United States between March 2012 and January 2013 (clinicaltrials.gov; NCT01539642). The study protocol and statement of informed consent were approved by a centralized institutional review board (Copernicus Group, Research Triangle Park, North Carolina) or the local institutional review board at each study site. All patients provided written informed consent before undergoing any study procedures. Male and nonpregnant female patients were eligible for inclusion if they were at least 18 years old, American Society of Anesthesiologists physical status I to III, and scheduled to undergo open abdominal surgery (including open abdominal surgeries that were laparoscopic assisted, such as partial colectomies) under general anesthesia or spinal anesthesia that did not include intrathecal opioids. Fully laparoscopic surgeries were not included. Patients were excluded if they were opioid tolerant (use of >15 mg oral morphine equivalent per day within the past 3 months); had documented sleep apnea, alcohol or drug abuse, or a need for outpatient oxygen therapy; or had any medical condition that would interfere with pain assessments. The use of any drug that may affect postoperative pain levels, such as gabapentanoids, steroids, or anti-inflammatory drugs were not allowed intraoperatively or postoperatively. Therefore, patients with a chronic pain condition necessitating treatment with these agents were also excluded from the study. During surgery, IV opioids were allowed as needed for analgesia, but the use of any regional anesthetic technique to provide postoperative analgesia, such as epidural, peripheral nerve block, or local anesthetic wound infiltration was prohibited. Following surgery, IV morphine, hydromorphone, or fentanyl could be administered as needed to keep the patient comfortable in the postanesthesia care unit (PACU). Antiemetic prophylaxis and treatment was allowed per standard hospital protocol at each site. Patients were randomized 2:1 to receive SSTS 15 μg or an identical system containing placebo tablets (“placebo system”) using an interactive web-response system. The sponsor, investigator, other study center personnel, and patients were blinded to treatment group assignment. Patients were randomized in the PACU provided they continued to meet entrance criteria and had a respiratory rate of 8 to 24 breaths/min, oxygen saturation greater than 95%, were able to answer questions and follow commands, and had no vomiting that was unresponsive to standard treatment. Following randomization, but before receiving study drug, patients were required to have a pain intensity of less than 5 at some point while in the PACU to demonstrate their pain was able to be managed, have been discharged or were ready for discharge from the PACU, and lastly have a pain intensity that escalated back greater than 4 just before the first dose of study drug. Pain intensity was based on an 11-point numerical rating scale (NRS), where 0 = no pain and 10 = worst possible pain. When these conditions were met and the patient requested medication for pain, baseline vital signs, oxygen saturation, and pain intensity were assessed before the patient self-administered the first dose of study drug. The SSTS or placebo system was used for 48 hours because that is the typical duration of use for IV PCA following surgery. If patients continued to require strong opioid analgesia following 48 hours, then sites had the option to extend the study up to 72 hours; however, a completer was considered any patient finishing 48 hours in the study. Patients were educated about proper use of the SSTS device by study personnel using patient-training screens displayed on the system both during the initial patient screening process and before the first dose of study drug. In order to maintain patients in both arms of the study as long as possible to minimize missing data, inadequate analgesia was treated using 2 mg IV morphine. Morphine dosing was allowed after only 10 minutes had passed following study drug dosing and not more than once per hour throughout the study. Patients who required additional analgesia beyond this were discontinued from the study because of inadequate analgesia and could receive any standard analgesic available at the clinical site. Patients who had oxygen saturation levels that could not be maintained at 95% or greater with or without the use of supplemental oxygen, respiratory rate less than 8 breaths/min, or excessive sedation were not allowed to have access to study drug or morphine until their vital signs had improved. BODY.EFFICACY AND SAFETY ASSESSMENTS: Efficacy was assessed by patient reports of pain intensity (based on the 11-point NRS) and pain relief (based on a 5-point scale where 0 = no relief, 1 = a little relief, 2 = moderate relief, 3 = a lot of relief, 4 = complete relief). Patients recorded pain intensity and pain relief scores at 15, 30, 45, and 60 minutes; every hour until 12 hours; every 2 hours until 48 hours; and every 4 hours from 52 to 72 hours after the first dose of study drug. Pain intensity and pain relief scores were also obtained just prior to IV morphine dosing for inadequate analgesia. The primary efficacy end point was the time-weighted summed pain intensity difference (SPID) over 48 hours (SPID48). This cumulative pain intensity measure is recommended by regulatory agencies to demonstrate the efficacy of acute pain products. Secondary efficacy end points included SPID at each evaluation time point; total pain relief (TOTPAR), pain intensity difference (PID), and pain relief at each evaluation time point; the proportion of patients discontinuing the study or requiring additional opioid medication due to inadequate analgesia; and the patient global assessment (PGA) and health care professional global assessment (HPGA) of method of pain control at 24, 48, and 72 hours. The PGA and HPGA were assessed using a 4-point categorical scale, where 1 = poor, 2 = fair, 3 = good, and 4 = excellent. Validated patient and nurse Ease-of-Care (EOC) questionnaires8,9 were completed to assess patient and nurse impressions of the SSTS. The patient EOC questionnaire has 23 questions, 21 of which are scored on a scale of 0 to 5 (where 0 = not at all and 5 = a very great deal) and summarized into 6 subscale scores (confidence with device, comfort with device, movement, dosing confidence, pain control, and knowledge/understanding) and a total EOC score. The other 2 questions (satisfaction with level of pain control and satisfaction with method of administration of pain medication) are scored on a 6-point scale (extremely dissatisfied to extremely satisfied) and combined into an overall satisfaction score. The nurse EOC questionnaire has 22 questions, 20 of which are scored on a scale of 0 to 5 (where 0 = not at all and 5 = a very great deal) and summarized into 2 subscale scores (time-consuming and bothersome) and a total EOC score. Two other questions (satisfaction with level of pain control and satisfaction with device) were scored on a 6-point scale (extremely dissatisfied to extremely satisfied) and combined into a total satisfaction score. Safety assessments included spontaneously reported adverse events based on the Medical Dictionary for Regulatory Activities (medDRA version 11.0); clinical laboratory evaluations including alanine aminotransferase, aspartate aminotransferase, total bilirubin, creatinine, and blood urea nitrogen; vital signs; and continuous oxygen saturation monitoring. Patients were to be withdrawn from the study if the oxygen saturation could not be maintained at 95% or greater with or without supplemental oxygen, if the respiratory rate could not be maintained at 8 breaths/min or greater, or if excessive sedation occurred. BODY.STATISTICAL ANALYSIS: The analyses of efficacy data were performed on the intent-to-treat population, defined as all randomized patients who received at least 1 dose of study medication. The pain intensity data collected after a patient received the first dose of study medication were included in the calculation of the primary efficacy end point, time-weighted SPID48. Pain intensity data collected within 1 hour after IV morphine dosing for inadequate analgesia were excluded from the derivation of the efficacy end points based on the pain assessment data. The pain intensity and pain relief scores collected just prior to each dose of rescue morphine was imputed for this 1-hour time interval. The last observation carried forward imputation method was used for any missing data points after termination because of reasons other than adverse event, and the worst observation carried forward imputation method was used for missing data points for patients who discontinued because of an adverse event. All statistical tests were 2-sided and were performed at the α = 0.05 significance level. Demographics and baseline characteristics were compared by a 2-sample t test for numeric variables and the Fisher exact test for categorical data. A parallel lines analysis of covariance model was used for the analysis of the primary efficacy end point and continuous secondary efficacy end points. This model included treatment and center factors and baseline pain intensity as a covariate. The least squares mean of each treatment and its 95% confidence interval were constructed. Ordinal categorical data were analyzed using the Cochran-Mantel-Haenszel test of general association with modified ridit scores. Dichotomous outcome data were analyzed by a 2-sample Z test on 2 proportions between treatment groups. For time-to-event data, Kaplan-Meier product limit estimators of cumulative rates of patients reaching the event (ie, termination due to inadequate analgesia and time to take first rescue medication) at follow-up time points were calculated. A log-rank test was used to compare 2 treatment groups. The Fisher exact test was used to compare the incidence of adverse events between treatment groups. Using an effect size of 0.55 for the primary efficacy end point, a sample size of 159 patients had 90% power to show statistical difference between 2 treatment groups. This calculation was based on a 2-sided 2-sample t test with a 2:1 sample size allocation ratio and a significance level of α = 0.05. Assuming a 10% nonevaluable rate, 180 patients were planned for randomization in this study. BODY.RESULTS: BODY.PATIENT DISPOSITION AND DEMOGRAPHICS: Of the 172 patients who received the study drug, 105 (61.0%) completed the 48-hour study period (Fig. 2), and 40 (23.2%) completed the 72-hour study period. One patient who was randomized to receive SSTS instead received a placebo system and therefore was included in the SSTS group for the analysis of efficacy data but was included in the placebo system group for the analysis of safety data. Demographics were similar for the 2 treatment groups, except for a significantly higher proportion of older patients (≥65 years) and female patients in the placebo system group (Table 1). Most of the surgeries were open lower abdominal/pelvic procedures (52%), followed by laparoscopic-assisted open procedures (36%) and open upper abdominal procedures (12%). While the protocol allowed spinal anesthesia, all patients received a general anesthetic in this study. FIGURE 2Patient disposition flow diagram. mcg indicates microgram. TABLE 1Demographics and Baseline Characteristics: Intent-to-Treat Population BODY.EFFICACY RESULTS: The primary end point, SPID48 score, was significantly higher in the SSTS group than in the placebo system group (least squares mean [SEM], 105.60 [10.14] vs 55.58 [13.11]; P = 0.001). Summed pain intensity difference and TOTPAR scores were also significantly higher in the SSTS group at all evaluation time points from 1 hour (SPID) and 2 hours (TOTPAR) until 72 hours. These summed scores were generated from the original pain intensity and pain relief scores at each time point, which are shown in Figures 3A and B, respectively. Analgesia resulting from use of IV morphine was not included in the summed pain scores (SPID or TOTPAR) because premorphine pain intensity scores were carried forward for 1 hour after morphine dosing for all these end points in order to be conservative in representing the analgesic effect of the active drug. Figures 3A and B, however, show the original pain intensity and pain relief values without any imputation for either IV morphine use or patient dropout; therefore, not surprisingly, the pain scores are similar after the first day for any patients remaining in the study. FIGURE 3Mean (SEM) of (A) pain intensity scores and (B) pain relief scores by evaluation time point (intent-to-treat population). To demonstrate onset of analgesia, the patient’s baseline pain intensity score must be taken into account as well as utilization of imputation to adjust for rescue analgesics. Therefore, PID to baseline scores by evaluation time point were evaluated following the initial dose of study drug, and the results from the first 4 hours are plotted in Figure 4. The SSTS group had significantly greater PID scores (ie, a greater drop in pain intensity compared with baseline) than in the placebo system group as early as 45 minutes after the first dose of study drug, and these differences were maintained for a majority of time points throughout the study. Following the initial dose of study drug and throughout the study, patients could dose every 20 minutes as needed; however, the average interdosing interval for the SSTS group throughout the 48-hour study was 100 minutes and for the placebo system group was 79 minutes (P < 0.05). FIGURE 4Least squares mean (SEM) of PID scores in the first 4 hours after dosing; *P < 0.05; ***P < 0.001. A lower proportion of patients in the SSTS group prematurely discontinued the study prior to 48 hours because of inadequate analgesia (SSTS: 17.4%; placebo system: 31.6%; P = 0.035), and over this same time period, a lower proportion of SSTS patients required IV morphine as rescue for inadequate analgesia (SSTS: 33.0%; placebo system: 66.7%; P < 0.001). The placebo system group had earlier discontinuations because of inadequate analgesia (P = 0.022; Fig. 5A) and earlier use of IV morphine as rescue (P < 0.001) compared with the SSTS group (Fig. 5B). The mean cumulative number of doses of IV morphine used was statistically lower in the SSTS group than in the placebo system group for all time periods (Table 2), although neither group used a significant amount. FIGURE 5Kaplan-Meier cumulative event rates for (A) time to termination from the study due to inadequate analgesia and (B) time to take first rescue medication due to inadequate analgesia (intent-to-treat population). Numbers below x axis indicate the number of patients followed at each time point. TABLE 2Intravenous Morphine Use for Inadequate Analgesia In the SSTS group, more patients reported success (ie, responded good or excellent) on the PGA at 24 hours (69.6% vs 42.1%; P < 0.001), 48 hours (67.8% vs 45.6%; P = 0.005), and 72 hours (67.0% vs 45.6%; P = 0.007) and more health care professionals reported success on the HPGA at 24 hours (70.4% vs 43.9%; P < 0.001), 48 hours (69.6% vs 49.1%; P = 0.009), and 72 hours (69.6% vs 47.4%; P = 0.005) than in the placebo system group. Patient EOC questionnaire results were similar in both treatment groups, except that patients in the SSTS group had better scores for questions related to pain control than patients in the placebo system group (Table 3). Nurses filled out 1 EOC questionnaire either after setting up the system in at least 10 patients or at the end of the study, whichever occurred first. The nurse EOC results were compared for nurses with less than 1 year of experience setting up IV PCA pumps versus nurses with more than 1 year of experience. Overall, both patients and nurses rated the ease of care of the system as greater than a 4 on the 0- to 5-point scale. There was no statistical difference between the 2 nursing groups for any EOC score. TABLE 3Ease-of-Care Questionnaire Results The average interdosing interval of 100 minutes for patients in the SSTS group resulted in sufentanil venous plasma levels of 71 and 69 pg/mL at 24 and 48 hours, respectively. BODY.SAFETY: All adverse events in the SSTS group were mild or moderate in severity, whereas 1 adverse event in the placebo group was rated by the clinical investigator as severe (abdominal pain). Overall, 23.7% of patients in the SSTS group and 25.9% of patients in the placebo system group had 1 or more adverse events considered possibly or probably related to study drug by the investigator. There were no statistically or clinically meaningful differences between treatment groups for the proportion of patients with any possible or probably related adverse event (Table 4). Sedation does not appear as an adverse event in Table 4, as only 1 patient (0.9%) had this adverse event reported (in the SSTS group). TABLE 4Possibly or Probably Related Adverse Events (>1% in Either Treatment Group) One patient in the SSTS group had a treatment-emergent serious adverse event of moderate atrial fibrillation that was considered to be unrelated to study drug by the site investigator. Twelve patients (7 SSTS, 5 placebo) discontinued the study because of an adverse event, most commonly back pain (3 patients) and nausea and vomiting (2 patients each). There were no statistically significant differences between treatment groups for mean changes from baseline to 48 hours or final evaluation for any laboratory variable. In addition, there were no statistically significant differences between treatment groups for mean changes from baseline in oxygen saturation. Average mean oxygen saturation values ranged from 96.72% to 98.10% for the SSTS-treated patients and 96.39% to 98.50% for placebo-treated patients at evaluation time points up through 72 hours. BODY.DISCUSSION: This phase 3 study demonstrates the efficacy and safety of the SSTS in the treatment of postoperative pain in patients following open abdominal surgery compared with placebo. Both pain intensity and pain relief assessments throughout the study demonstrated consistently greater pain control for the SSTS group compared with the placebo system group. Patients could initiate study drug dosing only if they had a pain intensity score of 5 or higher on the 11-point NRS, thereby limiting the study to patients suffering from moderate to severe pain. The SSTS group averaged a PID score of greater than 1.3 by 1 hour after dosing, demonstrating a rapid onset of clinically significant analgesia.10 This rapid onset of action is possible because of the high lipophilicity of sufentanil (octanol:buffer partition coefficient [OBPC] of 1757:1) as well as a 20% nonionized fraction (at pH of 7.4).11 Lipophilic, nonionized drug molecules have rapid uptake from sublingual tissues into the plasma as well as rapid uptake from the plasma to the μ-opioid effector site in the central nervous system (CNS) (plasma:CNS equilibration half-life t½ke0 = 6.2 minutes for sufentanil).12 Fentanyl is less lipophilic (OBPC of 816:1) and has a lower nonionized fraction of 9%.11 Morphine is not lipophilic (OBPC of 1:1) and, therefore, even when delivered IV, has a t½ke0 of 2.8 hours, and the active metabolite morphine-6-glucuronide has an even more delayed equilibration (t½ke0 = 6.4 hours).11,13 Minimal use of rescue IV morphine over the entire 72-hour time period was required because of inadequate analgesia for the SSTS group (a total of 1.9 doses = 3.8 mg). The placebo group used statistically more IV morphine (8 mg); however, this amount is surprisingly low. This can partially be attributed to the earlier and higher dropout rate of placebo patients (32%), thereby giving them less time in the study with which to utilize the IV morphine rescue dosing. It could also be true that the 2-mg IV dose of morphine, given its aforementioned slow CNS equilibration, was n This study had a relatively high placebo-response rate (46.6% completed 48 hours), which, considering the availability of rescue IV morphine, is fairly typical for placebo-controlled analgesia studies in general. It is possible that the novelty of the device attributed to this effect. Approximately 17% of patients in the SSTS group dropped out of the study because of inadequate analgesia, significantly fewer than the placebo patients, and the dropouts occurred later in the SSTS group. Given the nature of a placebo-controlled study, where factors that will increase the placebo-response rate are purposely diminished (eg, no adjuvant analgesics or local anesthetic field blocks) and patients are unsure if they are receiving active study drug and may tend to drop out to guarantee access to analgesics, this rate of dropout from the active group is also neither surprising nor uncommon for large phase 3 placebo-controlled analgesic studies. Patient and health care professional global assessments as well as the EOC questionnaire ratings all demonstrate that both patients and health care providers found the system to provide adequate analgesia with a user-friendly device. Regardless of whether patients were randomized to active or placebo, scores on ease of use of the system were high, with the exception of pain relief, which appropriately was lower in the placebo system group. In an open-label, randomized study of SSTS compared with IV PCA morphine in patients following either open abdominal surgery or major joint replacement surgery, patient and nurse EOC scores were higher for the SSTS compared with IV PCA for both the total EOC score as well as each subscale score.14 The safety and tolerability of the SSTS were measured using standard adverse event reporting and vital sign measurements. There were no differences for related adverse events or changes in vital signs between active and placebo groups. To reflect the real-world surgical population, the protocol did not limit enrollment by age or body mass index, resulting in more than a quarter of the patients ranging from 65 to 92 years of age and 44% of the population rated as obese (body mass index ≥30 kg/m2). The similar safety profile of SSTS compared with placebo is encouraging for the use of this product in postoperative patients who often present with multiple comorbidities. Furthermore, there is an inherent additional safety factor in a system that cannot have a prescribing or programming error as the result of a fixed-dose, fixed-lockout paradigm, as well as having a noninvasive route of administration. These phase 3 study data are in agreement with earlier phase 2 dose-finding studies, which demonstrated an adverse event profile similar to placebo with only pruritus statistically higher in the active versus placebo groups.7 The slightly higher number of enrolled patients 65 years or older and female patients in the placebo group could possibly have affected the adverse event profile in this group. The “as-needed” dosing of both the SSTS and the rescue IV morphine is specifically to allow tailoring of the drug dose for individual patient’s analgesic requirements, thereby adjusting for any demographic influence. However, we cannot fully dismiss that these demographic variables could have impacted the results of the study. The SSTS contains a number of security features, including a patient-specific RFID technology to minimize “proxy” dosing, which is an advantage over traditional IV PCA, which can allow family members to dose the patient, resulting in adverse events. Other features include a security tether, a tamper-resistant drug cartridge to limit health care worker diversion, and an electronic tablet count on both the dispensing device and the cartridge RFID label, which can be reconciled to identify possible diversion. Although health care providers should be observant for patient diversion of medication, this issue in hospitalized patients is limited compared with the much larger problem of diversion of outpatient-prescribed opioids. In summary, the SSTS is an investigational patient-administered opioid system for the management of moderate to severe acute pain in a hospital setting. The system is preprogrammed and noninvasive, overcoming some of the issues with IV PCA, and results from this study suggest it is an effective analgesic treatment for patients following major abdominal surgery and possesses an encouraging safety profile that would integrate well within a multimodal approach to acute pain management in the hospital setting.

TITLE: Preemptive analgesia in third molar impaction surgery ABSTRACT.INTRODUCTION:: We have evaluated efficacy of diclofenac sodium as pre-emptive analgesia agent in a prospective triple blind placebo controlled randomized clinical trial in a patients undergoing third molar impaction surgery. ABSTRACT.MATERIALS AND METHODS:: Randomization of groups was done by randomization software and two groups were constituted one group receiving placebo pre operatively and then the drug for next five days while the other group was given diclofenac sodium pre operatively and then for five days. ABSTRACT.RESULTS:: Results were achieved with help of measurement of outcome variables like postoperative tenderness, swelling and trismus on a visual analogous scale (VAS) and other personalized scale. Collected data shows that there is a significant reduction in the score of postop tenderness in experimental group (P = 0.00), while there is a minimal difference between score of postoperative swelling and tenderness (P > 0.04). ABSTRACT.CONCLUSION:: So, we can conclude that use of diclofenac sodium as a preemptive analgesic agent is beneficial for better pain control in third molar impaction surgery. BODY.I:NTRODUCTION Effective postoperative pain control is very important factor in our day to day practice. Effective pain control with the help of diclofenac sodium as preemptive analgesic agent in third molar impaction surgery provides better patient compliance following surgery, and prevents occurrence of chronic postoperative pain experience.[1] For ideal postoperative pain management we need to eliminate or minimize pain and discomfort to the patient. This should be achieved with simple, easily available and economical measure which has very minimum or no adverse effect. Effective postoperative pain control is achieved by Preventing the initial neural cascade which leads to hypersensitivity produced by noxious stimuli.[2345] Efficient analgesic agent before the onset of the noxious stimulus to prevent central sensitization and preventing typically painless sensations to be experienced as pain (allodynia).[2345] Various preemptive agents can be used for effective pain control.[67891011] We have used oral tablet of diclofenac sodium in 50 mg dose one hour before surgery which is given as a preemptive analgesic agent in experimental group. While in control group placebo it is given one hour before surgery. With the help of our prospective randomized triple blind placebo-controlled clinical trial, we have evaluated efficacy of diclofenac sodium as preemptive agent in cases of third molar impaction surgery. We have measured postoperative tenderness, swelling and trismus for five postoperative days. BODY.M:ATERIALS AND METHODS Sixty patient, both male and female attending the Dept. of of Oral and Maxillofacial Surgery, for removal of impacted third molars had been enrolled in this study. All patients were explained about the study in detail, and the possible complications of the surgery and the drug were discussed with the patient. informed consent was obtained from all patients included for the study. Ethical committee clearance was obtained before starting of study. The study comprise of two groups of 30 patients each. One group (control group) consist of patients receiving placebo in the preoperative setting (one hour prior to surgery) followed by diclofenac sodium in the postoperative setting three times daily for a period of five days. Second group (experimental group) Had patients receiving diclofenac sodium in the preoperative (one hour prior to surgery) and postoperative settings. Allocation of patients to each group was done by randomization using the software. The patient, the operating surgeon as well as the evaluating surgeon, all were blinded during the study process. All patients, were evaluated by the principal investigator. Each patient was assessed at 1 hour postoperatively followed by 3rd and 5th days for swelling, tenderness, trismus and any other complications like nerve injury, infection etc, these findings were recorded on separate form and taken for evaluation after the suture removal. BODY.CRITERIA FOR MEASUREMENT: Pain on a scale of 0 to 10, 0 being absence of pain and 10 being severe pain (VAS).[12] Swelling, recorded as a following scale: absence of swelling - 0, mild swelling - 1, moderate swelling - 2 and severe swelling - 3. Trismus, recorded as following scale: absence of trismus - 0, mouth opening >76% - 1, <75% mouth opening >51% - 2, <50% mouth opening >26% - 3 and mouth opening <25% - 4. BODY.OBSERVATIONS AND RESULTS: At each stage, tenderness, trismus and swelling (Figures 3, 4, 5) were compared between experimental and control group, (Figures 1, 2). Table 1 gives the mean and SD values of these variables for both groups. To compare the tenderness, trismus and swelling between experimental and control groups, non-parametric test “Mann-Whitney” was applied and its P-value was obtained. Throughout the discussion, the level of significance was fixed at 5%. Figure 1Mean rank of outcomes in control group Figure 2Mean rank of outcomes in experimental group Figure 3Comparison of rank of tenderness of control and experimental groups Figure 4Comparison of rank of trismus of control and experimental groups Figure 5Comparison of rank of swelling of control and experimental groups Table 1Comparison between experimental and control groups using Mann-Whitney test Table 1 shows significant difference in tenderness at 3rd and 5th post operative day between experimental and control groups (P-value <0.05). Trismus and swelling, at any stage, did not see any difference between experimental and control groups (P-value >0.05). Table 2 shows the comparison of tenderness, trismus and swelling between consecutive two post-operative follow ups at the 3rd and 5th day of surgery. Wilcoxon signed - rank test was used. Table 2Comparison between consecutive two follow-ups: Wilcoxon Signed-Rank Test Tables 1 and 2 give the comparison between 1st post-operative hour and 3rd post-operative day as well between 3rd and 5th post-operative day. Tenderness, trismus and swelling shows significant difference between two post-operative follow-ups (P-values are less than 0.05). BODY.D:ISCUSSION Results of the derived data is suggestive of better pain control in experimental group. Post-operative score of tenderness shows highly significant difference, between both the groups (P = 0.00) with suggestive low score in the experimental group. Derived data were also suggestive of no significant change (P > 0.04) in both the swelling and trismus. BODY.DEFINITIONS OF PREEMPTIVE ANALGESIA: Three different definitions have been used as the basis for the recent clinical trials. Preemptive analgesia has been defined as treatment that: (1) starts before surgery; (2) prevents the establishment of central sensitization caused by incisional injury (covers only the period of surgery) and (3) prevents the establishment of central sensitization caused by incisional and inflammatory injuries (covers the period of surgery and the initial post-operative period).[13] Prospective study design is a valid method for this kind of research work, so we have designed this study accordingly. There might be chances of biased results from patients as well as from investigator's side if they are aware of time period of consumption of diclofenac sodium but, in this study we have minimized all such bias by making patient, surgeon and investigator blinded and also with and with use of randomization of the patient was done to minimize bias using a software. Diclofenac sodium is used as preemptive analgesic agent because of its easy ability, economic effective pain control and and relatively safe drug with minimal reported allergy. The plasma half-life of diclofenac sodium is also 1–2 hours, so we can achieve ideal optimal concentration if we provide it in 1-hour preoperatively. visual analogue scale (VAS) scale is an accepted method for assessment of post-operative pain.[14] Comfort level of the patients are also superior in experimental group than control group as shown with help of derived VAS score. The mean scores of tenderness in our study at 1-hour post-operatively in the experimental group and control group are 1.86 and 2.16, respectively, with P-value of 0.099 which is less significant; however, the mean scores for 3 days post-operatively for the experimental group and control group are 3.5 and 5.1, respectively, with P-value of 0.001 which is highly significant. Score mean of tenderness on the 6th post-operative day for experimental group is 1.6 and for control group is 3.6 with P value of 0.001 which shows a highly significant difference. And for trismus mean score at 1-hour post-operatively for experimental group is 0.36 and control group 0.66 with P value of 0.02, which shows a significant difference. All other findings show no significance difference. These achieved results are correlative to the previous studies. Ong, et al.[7] in 2004 conducted a double-blind, randomized, placebo-controlled study where 34 patients had each of their identical-impacted mandibular third molars removed under local anesthesia on two occasions. Throughout the 12-h investigation period, patients reported significantly lower pain intensity scores in the ketorolac pre-treated sides when compared with the post-treated sides. Aoki et al.[11] in 2006 compared the efficacy of the selective cyclooxygenase-2 (COX-2) inhibitor meloxicam for treatment of postoperative oral surgical pain by assessing in a randomized-controlled trial. Patients undergoing unilateral mandibular 3rd molar extraction surgery were allocated to 3 groups, A, B and C. After oral premedication of meloxicam 10 mg in group A, ampiroxicam 27 mg in group B and placebo in group C., post-operative pain was evaluated at the clinic on the 1st, 7th and 14th postoperative day (POD) using a VAS and concluded that COX-2 inhibitor, meloxicam 10 mg, used for premedication reduced post-operative pain compared with control in oral surgery. Pozos-Guillen, et al.[15] in 2007 compared the efficacy of tramadol given before or immediately after surgical extraction of an impacted mandibular third molar under local anesthesia. In this prospective, randomized-controlled, double-blind pilot study, 3 groups of 20 patients each were included: tramadol preoperative, 100 mg intramuscularly (IM) 1 hour before surgery (group A); tramadol postoperative, 100 mg IM immediately after surgery (group B); and saline (group C). This study suggests the preemptive use of tramadol as an alternative for the acute pain treatment after the removal of an impacted mandibular third molar that is carried out under local anesthesia. Whereas some of the studies show contradictory results from our study. Sisk, et al.,[6] in 1990 conducted a clinical trial for comparison of preoperative and postoperative naproxen sodium for suppression of pain in dental surgery cases. In their trial, they had given naproxen sodium 550 mg 30 min postop in control group while in preemptive group naproxen sodium 550 mg 30 min preop was given. Results are not supportive of the use of preemptive analgesia. Kaczmarzyk, et al.[12] in 2010 conducted a prospective, randomized, double-blinded clinical trial preemptive effect of ketoprofen on postoperative pain following third molar surgery. Ninety six patients were placed into three groups: pre-group (ketoprofen 60 min preoperatively); post-group (ketoprofen 60 min postoperatively); and no-group (placebo) and resulted that initial onset of pain was significantly delayed only in the post-group. Ketoprofen administered after third molar surgery provides more effective pain control than ketoprofen administered before the surgery or placebo. BODY.C:ONCLUSION Use of diclofenac sodium orally in 50 mg dose one hour preoperatively as preemptive analgesic agent is economical, effective, easy and safe method of postoperative pain control in mandibular third molar impaction surgery cases.

TITLE: Prevalence of and factors affecting post-obturation pain following single visit root canal treatment in Indian population: A prospective, randomized clinical trial ABSTRACT.AIM:: This prospective randomized clinical study (1) investigated the prevalence of post-obturation pain after single visit root canal treatment and (2) evaluated the influence of factors affecting the pain experience. ABSTRACT.MATERIALS AND METHODS:: One thousand three hundred and twenty eight (1328) patients were included in this study. Conventional single visit root canal treatment was carried out. The chemicomechanical preparation of root canals was done by a rotary protaper system with a combination of hand instruments. Post-operative pain was recorded by each patient by using visual analogue scale in well-defined categories at three time intervals, 12 h, 24 h, and 48 h. The data were analyzed using Fisher's exact test. ABSTRACT.RESULTS:: The prevalence of post-obturation pain (severe) within 48 h after treatment was 4% (n = 54) but less as compared to the pain experienced after 12 h (9%) and 24 h (8.6%). The factors that significantly influenced post-obturation pain experience were: Age (Fishers exact test = 46.387, P = 0.0), gender (Fishers exact test = 23.730, P = 0.0), arch (Fishers exact test = 11.710, P = 0.001), and presence of pre-operative pain (Fishers exact test = 67.456, P = 0.0). ABSTRACT.CONCLUSION:: The presence of post-operative pain was low (4%). The important prognostic determinants of post-obturation pain were: Old age, female, mandibular teeth, and presence of pre-operative pain. The vital condition of the tooth does not affect the intensity and frequency of post-obturation pain. BODY.INTRODUCTION: The basic principles of root canal treatment are the eradication of root canal irritant, obturation of the root canal system and preservation of the natural dentition. The root canal procedure can be done in single visit or multiple visits.[1] Traditionally root canal treatment was performed in multiple visits, with medication between root canal preparation and obturation, which mainly aims to reduce or eliminate microorganisms and their by-products from the root canal system before obturation. However, with various advances in endodontics, single visit root canal treatment is commonly performed. However, despite advances in endodontic therapy, studies have shown that pain frequency and intensity vary after root canal treatment. There is no consistency in the literature, the frequency of postoperative pain is mentioned to be lower in single visit or multiple visits or to have similar results.[2] Post-operative pain after endodontic procedures is an undesirable occurrence for both patients and clinicians.[3] Pain affects patient's quality of life and the success of dentist patient relationship.[2] Patients might consider post-operative pain as a benchmark against which the clinician's skills are measured. It might undermine patient's confidence in their dentist or patient satisfaction with their treatment.[1] The occurrence of mild pain is relatively common even when the treatment has followed the highest standards and should be expected and anticipated by patients.[3] Numerous studies with post-operative pain after root canal treatment have been published, with the incidence of post-operative pain ranging from 1.9% to 48%, and they showed conflicting findings.[1] Pain associated with root canal therapy is a poor indicator of pathosis and an even more unreliable predictor of long-term success.[4] Prospective, randomized studies are generally believed to provide the highest level of support for evidence-based clinical practice. Thus, a prospective, randomized study was carried out to evaluate the incidence and severity of post-operative pain following single sitting root canal therapy in Indian population. The relationship of certain factors (age, gender, pulpal status, arch, and presence of pre-operative pain) with post-operative pain was also analyzed. BODY.MATERIALS AND METHODS: One thousand three hundred and twenty eight patients were included in the study. Of the 1328 patients, 593 (44.7%) were women and 735 (55.3%) were men. Of all the teeth 1097 (82.6%) were vital and 231 (17.4%) teeth were non-vital. Age range was 15-65 years. 606 (45.6%) treated teeth were in the maxilla and 722 (54.4%) teeth were in the mandible. All the teeth were molars, either first molar (783) or second molar (545). Single sitting root canal therapy was explained to the patient and oral and written informed consent was obtained. Patients who were pregnant, need retreatment, having complications during treatment (calcification, impossibility to achieve apical patency), taking antibiotics or steroids at the time of treatment, immuno-compromised or under 15-year-old were excluded from this study. BODY.TREATMENT PROCEDURE: Conventional RCT was carried out in single visit. After adequate anesthesia, rubber dam was applied and access opening was done. The working length of each canal was determined by an electronic apex locator and 2 or more angled radiographs. Canals were prepared with a combination of hand files (K file, Mani, Japan) and rotary protaper system (Dentsply Maillefer, Ballalgues, Switzerland) by using RC Prep (Premier Dental Product Co. King of Prussia, PA, USA). Intermittent copious irrigation was done with 5.25% sodium hypochlorite solution. The canals were obturated with protaper gutta percha points (Dentsply Maillefer) using AH plus sealer (Dentsply, Konstanz, Germany). Temporary restoration with cavit (ESPE, Germany) was given for 2 weeks. Then the post-obturation restoration was done with amalgam to maintain a good coronal seal. Post-operative pain was measured by using a visual analogue scale (VAS) of 0 (no symptom) to 3 (severe pain and/or swelling). VAS was taught to the participants as well as reporting the post-operative pain after 12 h, 24 h, and 48 h. Patients were contacted by telephone if they did not return the VAS form. The post-operative evaluation was recorded as 0 (no pain), 1 (slight pain/discomfort), 2 (moderate pain relived by analgesics) and 3 (severe pain/swelling not relieved by analgesics and required unscheduled visit). All patients were given a prescription for 50 mg of diclofenac potassium, with the dose of 1 tablet every 8 h taken only if they experienced moderate pain. Results were statistically analyzed using Fishers exact test. Differences were considered significant when the probabilities were equal to or less than 0.05. BODY.RESULTS: Results are shown in Tables 1-6: The overall incidence of post-operative pain after RCT during the follow-up period of 12 h, 24 h, and 48 h was assessed according to patient's record in the VAS [Table 1]. 1190 (89.6%) patients had no pain, 14 (1.1%) patients had mild pain, 5 (0.4%) patients had moderate pain, and 119 (9.0%) patients experienced severe pain after 12 h. After 48 h the number of patients with severe pain was drastically reduced to 54 (4%), and there was no patient with mild pain, and only 1 (0.1%) patient with moderate pain. Table 1Overall incidence of post-operative pain after 12 h, 24 h, and 48 h Post-operative pain related to the age: Differences were statistically significant (P = 0.0) between both the groups. Incidence of post-operative pain is high in group 2 (41-65 years) at all the three-time intervals [Table 2]. Table 2Incidence of post-operative pain in relation to age at 12 h, 24 h and 48 h Post-operative pain related to the gender: Statistically significant (P = 0.0) difference was present between both the genders. More number of women experienced severe pain as compared to men, at all the three-time intervals [Table 3]. Table 3Incidence of post-operative pain in relation to gender at 12 h, 24 h, and 48 h Post-operative pain related to the arch: In lower teeth post-operative pain was significantly higher (P = 0.001) at all the three time intervals [Table 4]. Table 4Incidence of post-operative pain in relation to arch at 12 h, 24 h, and 48 h Post-operative pain related to the vital status of the tooth: There was no statistically significant difference (P = 0.338) in post-operative pain, between both the groups at all the three time intervals [Table 5]. Table 5Incidence of post-operative pain in relation to the vitality at 12 h, 24 h, and 48 h Post-operative pain related to the pre-operative pain: In cases with reported presence of pre-operative pain, the presence of post-operative pain was significantly more (P = 0.0) [Table 6]. Table 6Incidence of post-operative pain in relation to the pre-operative pain at 12 h, 24 h, and 48 h BODY.DISCUSSION: One of the main problems in studying pain is the patient's subjective evaluation and its measurement. For this reason, design of the questionnaire is critical and must ensure that it will be fully understood by patients and easily interpreted by researchers.[5] In this report, a simple verbal categorization was used in the feedback form with 4 categories: 0 - no pain, 1 - mild pain, 2 - moderate pain, and 3 - severe pain. These categories were easily understood by the patients. It is well known that pain perception is a highly subjective and variable experience modulated by multiple physical and psychological factors.[4] Postobturation pain is considered to be related with several factors, including pre-operative pain, infection, retreatment, intracanal medications, and physical and chemical damage to periapical tissue. The lower incidence of post-operative pain in single visit root canal treatment might be attributed to immediate obturation, thereby to avoid passage of medications, repeated instrumentation and irrigation. The reported prevalence of post obturation pain ranges widely from 0% (at 30 days) to 65% (at 1 day), generally declines over time and should therefore be qualified by duration after last treatment episode.[6] In this investigation, overall incidence of post-operative (severe) pain in RCT was 9% after 12 h, which was reduced to 8.6% after 24 h and drastically to 4% after 48 h. Thus, under sound biological principles and by using contemporary scientifically based techniques, a low incidence of post-operative pain can be expected.[1] The pain was more frequent during the first 24 h of the observation period and quickly decreased thereafter. These findings were in agreement with those of some authors.[27–11] In this study, five variables (age, gender, arch, vitality, and presence of pre-operative pain) were considered separately to explore the potential influence on the prevalence of post-obturation pain. Except for one variable (i.e., vital status of the tooth), the remaining four variables were significantly associated with presence of post-obturation pain. Prevalence of post-operative pain is significantly higher (P = 0.0) in old patients (7.8%) as compared to young patients (0.8%), at all the three time intervals. This may be because of less pain tolerance, less blood flow and delayed healing. The prevalence of post-operative pain is very high in women (7.4%) as compared to men (1.8%) (P0 = 0.0). This finding is in agreement with Ryan et al.,[12] who presented gender (women) as a factor that significantly influenced post-operative pain. Various hypotheses have been proposed to explain women predominance in pain prevalence. A more legitimate explanation is based on emerging evidence that biological differences between genders may explain increased pain prevalence in women. There are two possible explanations:[6] Differences in pelvic and reproductive organs may provide an additional portal of entry of infection in females leading to possible local and distant hyperalgesia. Fluctuating female hormonal levels may be associated with changing levels of serotonin and noradrenaline leading to increased pain prevalence during the menstrual period and in women receiving hormonal replacement therapy or oral contraceptives. In mandibular molars post-obturation pain is significantly higher (6%) as compared to maxillary molars (2.2%) (P = 0.001). This might be because mandible has a dense trabeculae pattern, thus there is reduced blood flow and more localization of infection and inflammation, which might lead to delay healing. Analysis of post-obturation pain shows no statistically significant difference between vital (3.7%) and non-vital teeth (5.6%) (P = 0.338). This finding is in agreement with DiRenzo et al.,[4] Harrison et al.,[10] and Roane et al.[13] In cases with presence of pre-operative pain, frequency, and degree of postoperative pain were significantly more (P = 0.0) (10.5%) as compared to patients with absence of pre-operative pain (0.9%). This finding is in agreement with ElMubarak et al.,[1] and Siqueira et al.[3] This finding could be explained by the presence of infection of the root canal system and periapical region pre-operatively. This tissue being initially irritated may become secondarily irritated during treatment.[2] BODY.CONCLUSION: Thus, within the limitation of this study, it is concluded that post-obturation pain is likely to occur in first 24 h which further reduces as time passes. Thus, it is a strong indication that clinician should not overreact to early post-obturation pain by immediately initiating endodontic retreatment procedures or extraction of the involved tooth. Analysis of the influence of patient's age, gender, arch, vitality of the tooth, and presence of pre-operative pain, on prevalence of the post-operative pain showed that it is higher in the old age group (group 2: 41-65 years), women, mandibular teeth, and with presence of pre-operative pain. The vital condition of the tooth does not affect the intensity and frequency of post-obturation pain. Other factors reported to be significantly related to post-obturation pain such as apical periodontitis, retreatment, irrigating solution, intracanal medicament, tooth positions were not analyzed in this study.

TITLE: Early supported discharge after stroke in Bergen (ESD Stroke Bergen): three and six months results of a randomised controlled trial comparing two early supported discharge schemes with treatment as usual ABSTRACT.BACKGROUND: Stroke causes lasting disability and the burden of stroke is expected to increase substantially during the next decades. Optimal rehabilitation is therefore mandatory. Early supported discharge (ESD) has previously shown beneficial, but all major studies were carried out more than ten years ago. We wanted to implement and study the results of ESD in our community today with comparisons between ESD and treatment as usual, as well as between two different ESD models. ABSTRACT.METHODS: Patients with acute stroke were included during a three year period (2008–11) in a randomised controlled study comparing two different ESD models to treatment as usual. The two ESD models differed by the location of treatment: either in a day unit or in the patients’ homes. Patients in the ESD groups were followed by a multi-disciplinary ambulatory team in the stroke unit and discharged home as early as possible. The ESD models also comprised treatment by a multi-disciplinary community health team for up to five weeks and follow-up controls after 3 and 6 months. Primary outcome was modified Rankin Scale (mRS) at six months. ABSTRACT.RESULTS: Three-hundred-and-six patients were included. mRS scores and change scores were non-significantly better in the two ESD groups at 3 and 6 months. Within-group improvement from baseline to 3 months was significant in the ESD 1 (p = 0.042) and ESD 2 (p = 0.001) groups, but not in the controls. More patients in the pooled ESD groups were independent at 3 (p = 0.086) and 6 months (p = 0.122) compared to controls and there also was a significant difference in 3 month change score between them (p = 0.049). There were no differences between the two ESD groups. Length of stay in the stroke unit was 11 days in all groups. ABSTRACT.CONCLUSIONS: Patients in the ESD groups tended to be more independent than controls at 3 and 6 months, but no clear statistically significant differences were found. The added effect of supported discharge and improved follow-up seems to be rather modest. The improved stroke treatment of today may necessitate larger patient samples to demonstrate additional benefit of ESD. ABSTRACT.CLINICAL TRIAL REGISTRATION: Unique identifier: NCT00771771 BODY.BACKGROUND: Stroke is the most common cause of lasting disability in older age, leading to both personal and familial burdens as well as high societal costs [1,2]. The increasing incidence of stroke with higher age and decreasing stroke mortality [3], combined with an increasing number of people in the higher age groups [4], may eventually lead to substantially more persons suffering a stroke or having residual symptoms from previous stroke. Optimal rehabilitation is therefore important both for the individual stroke patient and regarding societal costs caused by stroke. Early supported discharge (ESD) is a rehabilitation concept emphasising early discharge from institution to home for patients after stroke, followed by rehabilitation while home-dwelling, and often supervised by a specialised multi-disciplinary team [5]. This has been studied in several randomised controlled trials previously and the results have been analysed in an updated Cochrane report from 2012 [5]. This meta-analysis suggested a beneficial effect of ESD for a selected group of stroke patients, but at the same time pointed towards the need for future studies in order to clarify which elements of ESD in the primary health care were important, and for more precise clarification of cost-benefit for different patient groups. The results from eleven trials where rehabilitation was offered either centre-based or home-based were reviewed in a recent meta-analysis, and the authors found a significant effect in favour of home-based rehabilitation at 6 weeks and 3–6 months [6]. An international consensus document was recently published with statements regarding team composition, model of team work, intervention, and success [7]. The present ESD Stroke Bergen study was established in order to compare the rehabilitation results after ESD to rehabilitation as usual and to investigate the effect of community treatment given in two different settings; either in a day unit (ESD 1) or in the patients’ homes (ESD 2). We therefore designed a randomised controlled trial with two different ESD arms and one control arm, and with two null hypotheses: (1) ESD does not improve functional outcome evaluated by mRS; and (2) day unit and home rehabilitation are equally effective. BODY.METHODS: BODY.STUDY DESIGN: The ESD Stroke Bergen study is an open randomised controlled trial with three study arms, two for ESD and one control arm. The study was conducted in collaboration between the Department of Physical Medicine and Rehabilitation (DPMR) and the Department of Neurology at Haukeland University Hospital, the University of Bergen (Department of Global Public Health and Primary Care; Physiotherapy and Lifestyle Epidemiology Research Groups) and the Municipality of Bergen, Norway. The full protocol for the study has been published previously and should be consulted for additional details [8]. The study was approved by the Western Norway Regional Committee for Medical Research Ethics and registered in ClinicalTrials.gov with registration number NCT00771771. BODY.PATIENT INCLUSION AND RANDOMISATION: All patients admitted to the stroke unit at the Department of Neurology, Haukeland University Hospital with suspected stroke were screened for inclusion. Inclusion criteria were living at home in the Municipality of Bergen prior to having a stroke, stroke within the previous seven days, being admitted to the stroke unit within the previous five days, and a National Institutes of Health Stroke Scale (NIHSS) score of 2–26. The scale used in this study was a 13 items Norwegian version (range 0–34) [9]. Patients with NIHSS score <2 were included if modified Rankin Scale (mRS) [10] score was ≥2. The patients had to be awake and able to agree to participation in the study by signing an informed consent, either themselves or by their relatives. There were no age limits. Exclusion criteria were serious psychiatric disorders, alcohol or substance abuse, other serious conditions of importance to the cerebral disorder or subsequent rehabilitation process, or poor knowledge of the Norwegian language. Patients found eligible by a stroke physician were informed by a designated nurse and asked to participate. Participants were randomised according to a computer-generated block randomisation list (six patients in each block; two for each study arm) and consecutively assigned to their groups in the same order as they were included into the study. The randomisation list was kept by a study coordinator and was not known to any persons in the stroke unit. The inclusion period lasted from 8 December 2008 to 20 December 2011, and 306 patients fulfilling the pre-specified criteria were included. BODY.DESCRIPTION OF STUDY ARMS: Patients in two of the three study arms were treated according to the ESD concept. They were followed by a designated multi-disciplinary ambulatory team consisting of a nurse, a physiotherapist and an occupational therapist from soon after admission to the stroke unit until shortly after discharge to home. This team originated from DPMR and served as a coordinating link between the patient, relatives, hospital personnel and the personnel in the primary health care. The team was particularly important in the discharge process and cooperated closely with the municipal health care in the planning and implementation of further treatment after discharge. The two ESD arms differed by the location of treatment: patients in the ESD 1 group received their treatment in a community day unit, whereas ESD 2 group patients stayed in their homes with home-visits from the community health team. Patients in the third study arm constituted a control group and were treated as usual without any intervention from the study, except appointments for testing at DPMR at 3 and 6 months. The treatment “as usual” mainly comprised institutional stay if necessary and/or physiotherapy as needed in the municipality (0–2 hours per week). Patients in all three study arms received language therapy as needed, regardless of allocated arm. The patients in the two ESD arms were discharged to their homes as soon as possible. Patients in need of a longer in-patient treatment period than offered by the stroke unit were discharged to a municipal institution or DPMR for a period before going home. All patients in the ESD arms were offered rehabilitative treatment by a multi-disciplinary community health team, consisting of a nurse, a physiotherapist and an occupational therapist. The scheduled treatment period was five weeks and maximally four hours per day five days a week, but many patients did not comply with this. Reasons included tiredness, low capacity and lack of motivation, as well as many patients being only minimally neurologically affected and therefore not needing this therapy. During the treatment period one or more persons from the community health team were present three days a week, and the last two the days of the week the patients trained by themselves after instructions from the team. Part of our study population was also studied by Gjelsvik et al. regarding physical tests and particularly balance, and in this subgroup mean total treatment time for the three days per week supervised by the community health team was 22 hours for ESD 1 and 16.6 hours for ESD 2 patients [11]. After the five week period ESD patients were strongly encouraged to continue training and social activities. They were also offered out-patient 3- and 6-months follow-ups at DPMR with a physician together with a member of the DPMR multi-disciplinary ambulatory team. BODY.STUDY OUTCOMES AND TEST PROCEDURES: The primary study outcome was mRS at 6 months. Secondary outcomes included mRS at 3 months, as well as NIHSS, Barthel ADL Index (BI) [12] and patient satisfaction (five-point Likert scale with 1 best) at 3 and 6 months. Baseline tests were done before study arm allocation and the individual patient’s group was therefore not known to the patients or the baseline testers. Also at 3 and 6 months the testers were blinded for study arm and the patients were instructed not to reveal this information. Background and clinical information relevant to the study were prospectively registered in a database (The Bergen NORSTROKE Registry) [13]. NIHSS was assessed immediately after admission and repeated regularly during the first week. The last NIHSS score recorded during the acute phase was used for the baseline analyses. mRS and BI were assessed on day 7, or earlier if the patient left the stroke unit sooner. Participants were re-tested at 3 and 6 months after inclusion at the DPMR out-patient clinic, and for the ESD 1 and 2 groups coupled to the corresponding follow-up. Control group patients were not followed-up by the study physician and advised to contact their general practitioner if they had medical questions of any kind. For various reasons some patients did not want to travel to the DPMR out-patient clinic for testing, and in these cases NIHSS, mRS, BI and patient satisfaction were scored by the testers coming home to the patients if permitted. To look for possible differences between the study arms, information on in-patient days in hospital or municipal institutions during the first six months after inclusion was collected for all participants. BODY.STATISTICAL METHODS AND SAMPLE SIZE: At study start at least 400 included patients were anticipated, but that had been revised to 350 when the study’s protocol was published [8]. This sample size of approximately 350 included patients was divided into two intervention arms and a control arm, each of 117 patients. Based on a previous relevant study of acute stroke patients [14] the expected proportions of patients with mRS ≤2 at 6 months were 65% for the ESD groups and 51.9% for the control group. This provided a power of 73% (one-sided analysis) to demonstrate a statistically significant difference between the groups. Demographics were analysed by analysis of variance (ANOVA) (age) and χ2-test (gender). Outcome differences between the groups at 3 and 6 months and within-group differences were analysed by nonparametric ANOVA using the Kruskal-Wallis test, and by χ2-test for dichotomised outcomes. Discharge destination and days in institution were analysed by χ2-test and Kruskal-Wallis test. All analyses were done as intention-to-treat and no imputation of missing data was made. The statistical programmes package Stata/SE 13.1 for Windows (StataCorp LP, Texas 77845, USA) was used for all data analysis. BODY.RESULTS: 1749 patients were screened for participation and 306 were finally included (Figure 1) reducing the power described above to 68%. The main reasons for exclusion were not living in Bergen (approximately 50%) or not having suffered a confirmed stroke (approximately 25%), whereas only 13 eligible patients declined to participate. Seventy-three and 82% of patients in ESD groups 1 and 2, respectively, received the allocated treatment. The patients’ main reasons for not receiving the treatment were being admitted to long-term municipal institution or declining because they did not want or did not need the allocated treatment. The drop-out rates at retest after 3/6 months were 20%/21% in the ESD 1 group and 14%/21% in ESD 2 group, versus 28%/33% in the control group (Figure 1).Figure 1 CONSORT flow-diagram showing patient flow from initial assessment for inclusion to six months testing in the Early supported discharge after stroke in Bergen study. The participants were 55.2% men (mean age 69.6 years) and 44.8% women (mean age 75.6 years). Age and gender were not significantly different between the three study groups (Table 1) and neither were baseline scores for mRS, BI and NIHSS (Table 2).Table 1 Baseline characteristics of 306 ESD Stroke Bergen study patients All ESD 1 group ESD 2 group Control group Variables N = 306 n = 103 n = 104 n = 99 p-value* Age; mean (range)   All 72.24 (27–98) 70.61 (29–91) 72.00 (27–92) 74.19 (32–98) 0.152   Male 69.55 (27–92) 69.27 (29–91) 69.41 (27–92) 70.02 (32–92) 0.947   Female 75.56 (38–98) 72.21 (38–90) 75.67 (44–92) 78.81 (44–98) 0.055 Gender; n (%)   Male 169 (55.2) 56 (54.4) 61 (58.7) 52 (52.5) 0.665   Female 137 (44.8) 47 (45.6) 43 (41.3) 47 (47.5) Abbreviation: ESD Early Supported Discharge. *Age analysed by ANOVA; gender analysed by χ2-test. Table 2 Descriptive statistics of modified Rankin Scale (mRS), Barthel Index (BI) and National Institutes of Health Stroke Scale (NIHSS)* at baseline, 3 and 6 months follow-ups and of patient satisfaction at 3 and 6 months in 306 ESD Stroke Bergen study patients Variables All groups ESD 1 group ESD 2 group Control group p-value† mRS; mean (SD) (n)   Baseline; all 2.59 (1.22) (306) 2.52 (1.29) (103) 2.59 (1.15) (104) 2.66 (1.23) (99) 0.711    Males 2.53 (1.22) (169) 2.52 (1.24) (56) 2.51 (1.16) (61) 2.56 (1.27) (52) 0.959    Females 2.66 (1.23) (137) 2.53 (1.36) (47) 2.70 (1.12) (43) 2.77 (1.18) (47) 0.651   3 months; all 2.45 (1.41) (242) 2.45 (1.46) (82) 2.30 (1.37) (89) 2.62 (1.40) (71) 0.316    Males #2.25 (1.34) (134) 2.32 (1.44) (44) 2.08 (1.16) (51) 2.38 (1.44) (39) 0.612    Females 2.69 (1.46) (108) 2.61 (1.48) (38) 2.61 (1.57) (38) 2.91 (1.30) (32) 0.530   6 months; all 2.52 (1.50) (229) 2.40 (1.53) (81) 2.46 (1.45) (82) 2.73 (1.52) (66) 0.410    Males §2.26 (1.42) (122) 2.17 (1.58) (42) ||2.09 (1.18) (45) 2.60 (1.50) (35) 0.248    Females 2.80 (1.54) (107) 2.64 (1.46) (39) 2.92 (1.62) (37) 2.87 (1.57) (31) 0.796   Change scores 0–3 months −0.22 (1.08) (242) −0.26 (0.93) (82) −0.36 (1.21) (89) 0.00 (1.07) (71) 0.063   Change scores 0–6 months −0.15 (1.21) (229) −0.23 (1.08) (81) −0.36 (1.21) (89) 0.00 (1.19) (66) 0.532 Barthel Index; median (IQR) (n)   Baseline 95 (40) (304) 100 (50) (102) 92.5 (35) (104) 95 (45) (98) 0.742   3 months 100 (15) (231) 100 (15) (78) 97.5 (10) (86) 100 (20) (67) 0.976   6 months 100 (15) (213) 100 (15) (76) 100 (10) (77) 100 (15) (60) 0.977   Change scores 0–3 months 0 (20) (229) 0 (20) (77) 0 (20) (86) 0 (15) (66) 0.642   Change scores 0–6 months 0 (20) (211) 0 (20) (75) 0 (20) (77) 0 (20) (59) 0.912 NIHSS; median (IQR) (n)   Baseline 3 (4) (304) 2 (4) (103) 3 (3) (104) 2 (4) (97) 0.593  3 months 2 (3) (226) 2 (3) (76) 2 (3.5) (84) 2 (3) (66) 0.925   6 months 2 (3) (205) 1 (3) (73) 1.5 (3.5) (76) 2 (3.5) (56) 0.718   Change scores 0–3 months −1 (3) (224) −1 (3) (76) −1 (3) (84) 0 (3) (64) 0.370   Change scores 0–6 months −1 (3) (203) −1 (3) (73) −1 (2) (76) −1 (3) (54) 0.615 Patient satisfaction; mean (SD) (n)   3 months 1.50 (0.99) (187) 1.32 (0.78) (62) 1.49 (0.91) (70) 1.71 (1.23) (55) 0.115   6 months 1.59 (1.08) (197) 1.62 (1.22) (73) 1.51 (0.98) (71) 1.68 (1.01) (53) 0.355 Abbreviations: ESD Early Supported Dischargem, SD Standard Deviation, IQR Interquartile Range. *mRS range from 0 (best) to 6; BI range from 0 to 100 (best); NIHSS range from 0 (best) to 34; patient satisfaction range from 1 (best) to 5. †All analyses: Kruskal-Wallis test; #Males better than females, p = 0.006; §Males better than females, p = 0.004; ||Males better than females, p = 0.011. The groups did not differ significantly at any point in time for mean values of the primary outcome mRS, nor for BI and NIHSS scores as well as all change scores (Table 2). However, the two intervention groups generally showed somewhat better scores than the control group. Within-group analyses from baseline to 3/6  Separate analyses for men and women demonstrated a significantly better mRS mean score in men than in women in all groups together at 3 (p = 0.006) and 6 months (p = 0.004), as well as in ESD group 2 separately at 6 months (p = 0.011). Patient satisfaction was rated better in ESD groups 1 and 2 at 3 months (1.32 and 1.49) compared to the control group (1.71), though not significantly (p = 0.115). mRS and BI scores dichotomised to independency (mRS ≤ 2; BI ≥ 95) or dependency (mRS > 2; BI < 95) demonstrated non-significantly better results for mRS in the intervention groups pooled together vs. the control group (p = 0.086 at 3 months; p = 0.122 at 6 months) (Table 3). Mean mRS scores were not significantly different between the pooled intervention groups and the control group at 3 (p = 0.204) and 6 months (p = 0.188). Change score for mRS at 3 months was significantly better in the pooled ESD groups than in the control group (p = 0.049), but with no difference at 6 months (p = 0.319) (Table 3).Table 3 Percentage of patients independent (BI ≥ 95 or mRS ≤ 2) and dependent (BI < 95 or mRS > 2) at baseline and after 3 and 6 months in the ESD arms combined and in the control arm; mRS and mRS change scores at 3 and 6 months in 306 ESD Stroke Bergen study patients Time ESD arms (1 + 2) Control arm p-value* Baseline   BI ≥ 95; n/N (%) 108/206 (52.4) 51/98 (52.0) 0.950   mRS ≤ 2; n/N (%) 109/207 (52.7) 49/99 (49.5) 0.605 3 months   BI ≥ 95; n/N (%) 113/164 (68.9) 42/67 (62.7) 0.362   mRS ≤ 2; n/N (%) 93/171 (54.4) 30/71 (42.3) 0.086   mRS; mean (SD) 2.37 (1.41) 2.62 (1.40) 0.204   mRS change score   0–3 months; mean (SD) −0.31 (1.08) 0 (1.07) 0.049 6 months   BI ≥ 95; n/N (%) 111/153 (72.5) 40/60 (66.7) 0.395   mRS ≤ 2; n/N (%) 90/163 (55.2) 29/66 (43.9) 0.122   mRS; mean (SD) 2.43 (1.49) 2.73 (1.52) 0.188   mRS change score   0–6 months; mean (SD) −0.21 (1.22) 0 (1.19) 0.319 Abbreviations: BI Barthel Index, mRS Modified Rankin Scale, ESD Early Supported Discharge, SD Standard Deviation. *Statistical tests: χ2-test (analyses of dichotomised BI and mRS) and Mann–Whitney U-test (mRS and mRS change score), p-value <0.05 indicated in bold. Subgroup analysis according to discharge destination from the stroke unit (directly home [n = 171]; to municipal institution [n = 78]; to DPMR [n = 47]; others/died [n = 10, results not shown]) showed significantly poorer mRS at 3 and 6 months for patients in the control group discharged to DPMR (mean mRS = 3.25/3.25) compared to the ESD 1 group (mean mRS = 2.76/2.50) and ESD 2 group (mean mRS = 2.33/2.21) (p = 0.041/0.037). Change scores were, however, not significantly different between the groups for 0–3 and 0–6 months. There were no differences in mRS or mRS change scores between the groups for patients discharged home or to a municipal institution (Table 4). In addition, the patients were divided into three groups as equally sized as possible, according to last baseline NIHSS score (0–1 [n = 88]; 2–4 [n = 130]; ≥5 [n = 86]), and mRS scores were analysed for differences between these groups. There were some significant differences as seen from Table 5, but they were not clinically meaningful.Table 4 Subgroup analyses: modified Rankin Scale (mRS) at baseline, 3 and 6 months and mRS change scores, according to primary discharge destination in 306 ESD Stroke Bergen study patients Discharge destination ESD 1 group ESD 2 group Control group p-value* Home (n=171); mean (SD) (n)   Baseline 1.72 (0.88) (54) 1.97 (0.94) (60) 1.89 (0.88) (57) 0.359   3 months 1.69 (1.15) (39) 1.74 (1.16) (50) 1.93 (1.00) (44) 0.461   6 months 1.66 (1.15) (41) 2.00 (1.30) (45) 1.94 (1.15) (36) 0.514   Change score 0–3 months −0.18 (0.94) (39) −0.26 (1.23) (50) 0.07 (1.09) (44) 0.252   Change score 0–6 months −0.10 (1.04) (41) −0.05 (1.33) (45) 0.06 (1.24) (36) 0.820 Municipal Institution (n = 78); mean (SD) (n)   Baseline 3.77 (0.75) (22) 3.56 (0.87) (25) 3.65 (0.84) (31) 0.588   3 months 3.50 (1.19) (20) 3.35 (1.30) (23) 3.83 (1.29) (18) 0.521   6 months 3.60 (1.19) (20) 3.41 (1.44) (22) 3.71 (1.52) (21) 0.858   Change score 0–3 months −0.25 (0.79) (20) −0.30 (1.18) (23) 0.00 (1.19) (18) 0.509   Change score 0–6 months −0.15 (0.81) (20) −0.05 (1.46) (22) 0.05 (1.28) (21) 0.881 DPMR (n = 47); mean (SD) (n)   Baseline 3.26 (1.05) (19) 3.22 (0.73) (18) 3.70 (0.67) (10) 0.378   3 months 2.76 (0.97) (17) 2.33 (0.72) (15) 3.25 (0.71) (8) 0.041   6 months 2.50 (1.29) (14) 2.21 (0.89) (14) 3.25 (0.71) (8) 0.037   Change score 0–3 months −0.41 (1.00) (17) −0.93 (0.96) (15) −0.50 (0.53) (8) 0.258   Change score 0–6 months −0.79 (1.37) (14) −1.00 (0.88) (14) −0.50 (0.53) (8) 0.492 Abbreviations: ESD Early Supported Discharge; SD standard deviation, DPMR Department of Physical Medicine and Rehabilitation. *All analyses: Kruskal-Wallis test, p-values <0.05 indicated in bold. Table 5 Subgroup analyses: modified Rankin Scale (mRS) at baseline, 3 and 6 months and mRS change scores, according to baseline NIHSS score in 306 ESD Stroke Bergen study patients Baseline NIHSS score ESD 1 group ESD 2 group Control group p-value* NIHSS 0–1 (n = 88); mean (SD) (n)   Baseline 1.64 (0.99) (28) 1.96 (1.02) (25) 1.83 (1.12) (35) 0.561   3 months 1.45 (0.96) (22) 2.05 (0.95) (22) 1.96 (1.15) (26) 0.109   6 months 1.32 (1.09) (22) 2.25 (0.79) (20) 2.00 (1.65) (20) 0.016   Change score 0–3 months −0.36 (0.85) (22) 0.05 (1.13) (22) 0.23 (1.34) (26) 0.187   Change score 0–6 months −0.27 (1.08) (22) 0.20 (1.11) (20) 0.20 (1.67) (20) 0.424 NIHSS 2–4 (n = 130); mean (SD) (n)   Baseline 2.35 (1.06) (48) 2.17 (0.93) (46) 2.50 (0.85) (36) 0.424   3 months 2.32 (1.34) (34) 1.74 (1.33) (38) 2.09 (0.90) (23) 0.059   6 months 2.40 (1.33) (35) 1.83 (1.42) (35) 2.25 (0.99) (24) 0.049   Change score 0–3 months −0.18 (1.00) (34) −0.47 (1.22) (38) −0.22 (0.80) (23) 0.202   Change score 0–6 months −0.11 (0.99) (35) −0.37 (1.44) (35) −0.08 (0.83) (24) 0.238 NIHSS ≥5 (n = 86); mean (SD) (n)   Baseline 3.74 (1.02) (27) 3.64 (0.74) (33) 3.88 (0.71) (26) 0.391   3 months 3.46 (1.33) (26) 3.24 (1.21) (29) 4.00 (1.21) (20) 0.114   6 months 3.38 (1.53) (24) 3.44 (1.37) (27) 3.95 (1.23) (20) 0.411   Change score 0–3 months −0.27 (0.92) (26) −0.52 (1.21) (29) 0.00 (0.97) (20) 0.168   Change score 0–6 months −0.38 (1.21) (24) −0.22 (1.40) (27) −0.05 (1.05) (20) 0.813 Abbreviations: NIHSS National Institutes of Health Stroke Scale, ESD Early Supported Discharge, SD standard deviation. *All analyses: Kruskal-Wallis test, p-values <0.05 indicated in bold. Post hoc we analysed for group differences in discharge destination from the stroke unit, length of continuous stay in hospital or municipal institution after the stroke as well as total days spent in the stroke unit, DPMR and municipal institution during the first six months after stroke. There were no differences between the groups (Table 6).Table 6 Discharge destination and days of in-patient treatment first six months after inclusion in 306 ESD Stroke Bergen study patients Variables All N = 306 ESD 1 group n = 103 ESD 2 group n = 104 Control group n = 99 p-value* Discharged from stroke unit to home; n (%) 171 (55.9) 54 (52.4) 60 (57.7) 57 (57.6) 0.687 Days in institution from stroke to first discharge home within first 6 months after inclusion; mean (SEM) 38.4 (2.9) 37.7 (5.1) 35.6 (4.6) 42.2 (5.6) 0.881 Total days in institution from stroke until 6 months after inclusion; mean (SEM) 45.5 (3.1) 46.0 (5.5) 42.8 (5.0) 47.7 (5.5) 0.930   Days in stroke unit 11.4 (0.4) 11.3 (0.6) 11.3 (0.7) 11.6 (0.8) 0.919   Days in DPMR 7.3 (1.2) 7.9 (2.0) 7.6 (1.9) 6.5 (2.3) 0.560   Days in municipal institution 24.0 (2.6) 23.4 (4.6) 21.5 (4.5) 27.4 (4.5) 0.111 Other 2.7 (0.4) 3.4 (0.7) 2.5 (0.5) 2.1 (0.6) 0.074 Abbreviations: ESD = Early Supported Discharge; SEM = standard error of the mean; DPMR = Department of Physical Medicine and Rehabilitation. *Analysed by χ2-test (discharge to home) and Kruskal-Wallis test (institution days). BODY.DISCUSSION: The primary research aim was to investigate whether two different ESD regimens were superior to treatment as usual. We found, in general, small differences in favour of the ESD groups for the outcomes mRS, NIHSS, BI, relevant change scores and patient satisfaction when examined by ANOVA, but mostly not statistically significant. We think that the main reason for these rather small differences in rehabilitation outcome between the early supported groups and the treatment as usual group must be ascribed to the quite different and superior treatment of stroke patients today, as compared to 10–15 years ago when most previous studies were carried out. Stroke prophylaxis is much improved, as well as acute stroke treatment with thrombolysis being used in more than 20% of stroke patients in our region. In addition, the rehabilitation services in general have improved during this period, making it difficult to demonstrate further improvement without quite large study groups. There is also a possibility that the outcomes mRS and BI, which are in the activities domain of the ICF, are too blunt to detect improvements in more specific physical functions or more qualitative outcome differences between the ESD and control treatment, e.g. regarding quality of life. Gjelsvik et al. reported the results of physical tests in a subgroup of our patients that was discharged directly from the stroke unit to their homes and found statistically significant differences between the groups for trunk control, self-report on walking and ADL [11]. The power calculation for the study was based on the results from a previous large Norwegian study carried out in Trondheim during the period 1995–1998 [14]. In this study the outcomes mRS and BI were dichotomised to dependency and independency and there was a substantial difference at 6 months (p = 0.017 for mRS; p = 0.056 for BI) between patients subjected to early supported discharge and controls. Since comparison between ESD and non-ESD models was a main objective in our study, some of our analyses were repeated with the ESD 1 and 2 groups pooled together vs. the treatment as usual group and the outcomes mRS and BI dichotomised (Table 3). These analyses demonstrated a non-significantly better result for mRS in the two ESD groups compared to the controls (p = 0.086 at 3 months; p = 0.122 at 6 months). The corresponding mRS change score difference at 3 months was marginally significant (p = 0.049). There were no differences for BI. In retrospect, such a large difference as in the Trondheim study could not be realistically expected in our patients now compared to their patients 15 years ago, due to the improved acute stroke treatment and pharmacological prophylaxis of today. Our study may therefore be considered as underpowered. We also did not reach neither the initially intended number of at least 400 included patients nor the revised number of 350, thereby further reducing study power. However, we recruited almost all eligible patients during the study period and a longer recruitment period was not realistically feasible. We performed subgroup analyses according to the severity of the initial neurological affection, based on the suggestion that ESD models are assumed most beneficial to stroke patients with a medium severe neurological affection [5]. The patients were divided into three groups according to last baseline NIHSS score, and mostly the three study arms improved about equally in these three NIHSS subgroups (Table 5). There were, however, some statistically significant differences between mRS scores that were clinically inconsistent and not accompanied by significant differences in corresponding change scores, and therefore considered to be without relevance. We also analysed the primary outcome mRS according to whether the patients were discharged directly to home, to a municipal institution, to DPMR or otherwise (other specialised institution or died in hospital). For patients discharged to DPMR before going home there was a significant difference with the ESD 1 and ESD 2 groups scoring better at 3 and 6 months compared to the control group. The corresponding change scores were on the other hand not significantly different and the clinical significance therefore unclear. There were no differences between the groups for the other discharge destinations. Post-hoc we also analysed the in-patient days for patients in the three study arms, but there were no statistically significant differences in this respect as seen from Table 6. Quite unexpectedly, there also was no difference between the two ESD arms and the control group for length of stay in the stroke unit (11.3, 11.3, and 11.6 days respectively). This demonstrates that today, at least in our region, all stroke patients are discharged from the stroke unit as soon as possible and there is therefore not more to gain in this respect. In fact, the determining factor for the patients’ length of stay in this study was predominantly their need for institutional stay after the discharge from the stroke unit. Patients going directly home spent 8.3 days in the stroke unit whereas those needing an institutional bed after discharge stayed for 15.4 days, with only minute differences between study arms (results not shown). The other main research question was whether the rehabilitation setting (day unit or home) was of importance for the clinical outcome, but we found no differences between the groups. Hillier et al. have previously reported on centre-based vs. home-based models in a meta-analysis from 2010 comprising 11 trials [6]. They found home-based treatment to be superior as measured by BI in the early period post-discharge, while the results at 6 months were conflicting. They concluded that the rehabilitation should be shifted towards more home-based services and specifically recommended that client preference should be used to determine treatment location in individual patients [6]. We found that men made a better recovery than women in the two intervention groups as judged by mRS. Similar findings have been reported repeatedly. Paolucci et al. reported in a case–control study from 2006 that men and women had a similar neurological recovery, whereas functional recovery was better in men [15]. Wyller et al. reported from Norway in 1997 that women seemed to be functionally more impaired than men [16]. Kim et al. in 2010 reported a similar difference [17]. A possible reason for men recovering better than women in our study may be a higher degree of social support, since substantially more women were living alone (results not shown). Besides suboptimal power a major challenge in this study was to obtain enduring compliance of included patients during the study period. They were generally old and many found travelling to the out-patient clinic demanding, necessitating testing in their homes. On this background we regard the follow-up rate in our study of about 80-85% for the two intervention arms as satisfactory. The figure of about 70% in the treatment as usual group may be explained by these patients feeling more weakly connected and committed to the study since they did not receive any treatment or medical follow-up from the project. This study has several clinical implications. Due to the future increase of people in high age groups and consequently increased anticipated disease burden, treatment and rehabilitation models outside specialist health care are strongly needed. To our best knowledge this is the first large randomised controlled trial examining ESD after acute stroke since the pivotal studies before and around 2000. The results indicate a slightly better improvement in the ESD groups compared to controls despite a generally much improved stroke treatment. On the other hand, the length of stay has already been cut strongly down making it difficult to further reduce in-patient time. Concurrently with the planning of the present study the Municipality of Bergen wanted to develop and strengthen their community rehabilitation, and this enabled a close cooperation between our hospital and the community during the study period. This collaboration has led to a permanently improved rehabilitation service in our community which will benefit patients beyond the stroke category. The final model for ESD rehabilitation, and possibly rehabilitation outside of institution in general, should probably not be a question of treatment either at home or in a day unit, but rather a combination based on clinical judgement by the community health team for the individual patient. This was also suggested by Hillier et al. [6]. In the Municipality of Bergen this model has now been implemented after the study period ended. Another constraint imposed by the RCT study was the duration of the community health team’s treatment period, which has also been modified after study completion and is now upwards limited to three months. There are, however, still improvements to be made. A recent qualitative study by Taule et al. [18] where eight stroke patients from the ESD 2 (home) group were interviewed indicates unmet needs concerning existential and emotional distress after stroke. BODY.CONCLUSIONS: The two ESD groups showed better functional improvement than the treatment as usual group, but not statistically significant. There was no difference between the two ESD groups. The main treatment difference between the intervention groups and the controls turned out to be the supported discharge with later follow-ups, since patients were discharged equally early in all three groups. Our interpretation: Improved prophylaxis and treatment during the last decade has led to reduced neurological disability after stroke, in parallel with reduced in-hospital time. The added effect of supported discharge and improved follow-up seems to be rather modest and larger patient samples are probably necessary to demonstrate a benefit with statistical significance. In addition, the outcome measure mRS is probably not optimal for measuring meaningful change.

TITLE: The effect of melatonin treatment on postural stability, muscle strength, and quality of life and sleep in postmenopausal women: a randomized controlled trial ABSTRACT.BACKGROUND: Melatonin is often used as a sleeping aid in elderly adults. As previous studies suggest a protective role of melatonin against osteoporosis, it is important to document its safety. Treatment should not cause any hangover effect that could potentially lead to falls and fractures. We therefore aimed to evaluate the effect of melatonin on balance- and muscle function. ABSTRACT.METHODS AND PATIENTS: In a double-blind placebo-controlled study, we randomized 81 postmenopausal women with osteopenia to receive 1 or 3 mg melatonin, or placebo nightly for 12 months. Postural balance as well as muscle function was measured. In addition, we assessed quality of life and sleep at baseline and after 12 months treatment. ABSTRACT.RESULTS: Compared to placebo, one-year treatment with melatonin did not affect postural balance or risk of falls. Furthermore, no significant changes between groups were observed in muscle strength in neither upper- nor lower extremities. Treatment did not affect quality of life or sleep. However, in the subgroup of women with sleep disturbances at baseline, a trend towards an improved sleep quality was seen (p = 0.08). ABSTRACT.CONCLUSION: Treatment with melatonin is safe in postmenopausal women with osteopenia. There is no hangover effect affecting balance- and muscle function following the intake of melatonin. In women with a good quality of sleep, melatonin has no effect, however in poor quality of sleep, small doses of melatonin trended towards improving the quality. ABSTRACT.TRIAL REGISTRATION: (# NCT01690000) BODY.INTRODUCTION: Melatonin in known for its regulation of circadian rhythm. It is produced in the pineal gland and is stimulated by darkness while inhibited by light [1]. In humans, the production decreases by age [2], and treatment with melatonin may be used as a sleeping aid in elderly people. In some European countries and the United States, small doses of melatonin are considered a dietary supplement. In other European countries, including Denmark, melatonin is still a prescription drug against primary insomnia recommended to patients >55 years of age in a dose of 2 mg taken at bedtime. Despite an unknown prevalence of the usage of melatonin, there is an increasing interest in melatonin in addition to its effects as a sleeping aid. Melatonin has also shown to be of importance for a number of other physiological functions as experimental and clinical studies have shown a stimulation of the immune system [3], protection against aging [4], cancer [5] and hypertension [6]. Moreover, melatonin may be of importance to bone, as it has been shown to upregulate osteoprotegerin (OPG) and suppress receptor activator of NK-κB ligand (RANKL) [7, 8]. Furthermore, studies have demonstrated an increased stimulation of the osteoblastic cell lineage with suppression of peroxisome proliferator-activated receptor gamma (PPARγ) and enhanced expression of bone morphogenic proteins (BMPs) 2 and 4 [9, 10]. In response to treatment with melatonin, a recent randomized, placebo-controlled study by our group demonstrated an increase in arealBMD in the femoral neck as assessed by DXA, and in volumetricBMD in the lunbar spine as assessed by quantitative computed tomography (QCT) in postmenopausal women with osteopenia (low bone mass with a T-score between -1 and -2.5 [11, 12]). In general, melatonin is considered a safe treatment option in the elderly. However, if melatonin is going to be used for long term prevention and treatment of osteoporosis (low bone mass with a T-score < −2.5 [11]), it is of specific importance to document its safety. Treatment should not cause any hangover effect (e.g. dizziness or drowsiness) resulting in decreased postural balance and muscle strength that could potentially lead to falls and fractures. Therefore, as a part of our double-blind randomized placebo-controlled trial on the effect of melatonin in postmenopausal women with osteopenia [12], we evaluated the effects of one-year treatment with melatonin in a daily dose of 1 or 3 mg administered at bedtime on postural balance and muscle function. Furthermore, quality of life and sleep were assessed to see if any effects are present in otherwise healthy women without known sleep disturbances. BODY.METHODS: BODY.STUDY POPULATION: We performed a double-blinded randomized placebo-controlled study. Inclusion criteria were postmenopausal women diagnosed with osteopenia (T-score −1 to–2.5 in either hip or spine). There were no requirements to quality of sleep prior to entering the study. We invited 202 women, and 150 responded positively, hereof 65 were excluded due to exclusions criteria or declining participation after further information. Ultimately, 85 women accepted the invitation, and the first 81 (aged 56–73 years) to accept were randomised to treatment or placebo [12]. The participants were recruited by letter from our outpatient clinic as all women were already diagnosed with osteopenia. The exclusions criteria were, in brief women with known medical conditions affecting the bone. This included p-creatinine >120 μmol/L, P-ionized calcium >1.32 mmol/L, intestinal malabsorption, impaired lever function, smokers and users of drugs with effects on calcium homeostasis. Furthermore, users of antiresorptives, hormone replacement therapy and alcohol abusers (>14 units/wk) were also excluded [12]. The patients were randomized to receiving either 1 or 3 mg of melatonin, or identical placebo daily for 12 months. The tablets were given orally at bedtime. Melatonin as well as identical placebo was manufactured by Skanderborg Pharmacy. As previously mentioned, melatonin is a prescription drug in several European countries recommended in doses of 2 mg. Therefore, to ensure no discomfort among the study subjects, besides the reports from the use of 2 mg, we chose doses close hereto. Furthermore, all participants received a daily supplement of 800 mg calcium and 20 μg vitamin D3. All participants provided informed consent prior to participation in the study. The study was performed in accordance with the Helsinki II declaration. The study was approved by the local ethic committee of central Denmark (#M2012-252-12), the Danish National Board of Health (EudraCt nr 2011-004670-28), the Danish Data Protecting Agency, and registered by ClinicalTrials.gov (# NCT01690000). The Good Clinical Practice (GCP) unit at Aarhus University Hospital, Denmark monitored the project. All study subjects provided a written consent before entering the study. BODY.POSTURAL STABILITY: We assessed postural stability by using a stadiometer (Good Balance Platform System™, Metitur Ltd. Finland) to measure body sway. The stadiometer is a triangular platform used to convert shifts in weight to digital data to obtain an assessment of maintenance of balance. The platform reports length (millimetres) and speed (millimetres/second) of the sway in medio-lateral and anterior-posterior direction. The interclass correlation coefficient was 0.87-0.96 [13]. Under four settings, we measured postural stability:Normal standing eyes open (EO). The participant is placed with the feet next to each other with 20 cm apart. The arms are in relaxed position hanging freely at each side and with a fixed gaze on a marked spot in eye level. Normal standing eyes closed (EC). The participant is placed in the same position as the previous exercise but with the eyes closed. Semi-tandem. The heel on the foot on the non-dominant hand side is placed alongside the big toe on the dominant hand’s side. Arms and gaze are in accordance with the first measure. Tandem. The non-dominant hand’s foot is placed in front of the dominant hand’s foot. Eyes and arms as described in measure 1. The exercises were performed barefoot. The duration of each exercise was 20 second and repeated three times. Data are presented as the velocity moment (VM mm2/s), which is calculated as 90 % of the product of the actual distance of movement in the medio-lateral and anterior-posterior direction from the center of pressure per second. We adjusted for the effect of body height, and vertical location of the center of body mass by Scaled Velocity Moment (SVM) = (VM/(height in cm)2) × 1802. The best measure for each exercise was chosen for further analysis (i.e. the lowest VM). BODY.MUSCLE STRENGTH: The maximum voluntary isometric muscle strength at the upper and lower extremity was measured with an adjustable dynamometer chair connected to a computer (Good Strength™, Metitur Ltd, Finland) [14]. The device has demonstrated high reliability coefficients (0.88-0.96) for both upper and lower extremities [15]. Recording time of each measure was 5 seconds. Upper extremity strength was assessed by hand grip, and elbow flexion and extension with the elbow in 90 dgr flexion from neutral position. Knee extension and flexion were measured in a 60 dgr and 90 dgr angle from fully extended leg. All measurements were performed while sitting in the chair. The trunk was supported with three belts to minimize transfer of strength from other sites. The excises were performed on the dominant hand’s side and repeated three times with a 30-second break between the recordings. The best performance was chosen for further analysis. Maximum strength was measured in newton (N). BODY.QUALITY OF LIFE: We used a Danish versions of the Short Form questionnaire 36 version 2 (SF-36) and WHO-Five Well Being Index (WHO-5) to assesses quality of life and well-being. The SF-36 questionnaire consist of 36 questions categorized into 8 subdomains describing physical functioning (PF), role-physical (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), role-emotional (RE), and mental health (MH). The subdomains are summed up to a mental component score (MCS) and a physical component score (PCS) and can be calculated using norm-based values according to the user’s manual [16]. The Who-5 index consists of five questions, and answers are scored from 0 (worst) to 5 (best). The scores are summed up, multiplied by four, and presented as a total percentage from 0–100. A well-being score below 50 % indicates a depressive affection [17]. Both questionnaires are well used and validated [18]. BODY.QUALITY OF SLEEP: A Danish version of Pittsburgh Sleep Quality Index (PSQI) [19] was used to assess the quality of sleep. The questionnaire comprises of 21 questions generated into seven components: Subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleep medication and daytime dysfunction. The subdomains are summed up to a global score; scoring five or below indicating a good quality of sleep while a score above five is associated with a poor quality of sleep. BODY.PHYSICAL ACTIVITY SCALE (PAS): We assessed physical activity by a Danish version of the Physical Activity Scale (PAS) questionnaire in which the participants reported time spend on sports, work, and leisure time on an average weekday [20]. The questionnaire is organized into nine questions regarding activity corresponding to a known metabolic equivalents (MET, 1 kcl/kg/h) level ranging from low activity to high intensity activity: sleep (0.9 MET), TV-viewing/reading (1.0 MET), sitting/working (1.5 METs), standing up (2.0 METs), light work (3.0 METs), light to moderate activity (4.0 METs) moderate activity (5.0 METs), moderate to high activity (6.0 METs) and high intensity activity (7 METs) [20]. Each question is imaged and described by examples of the specific activity. A total MET-score was calculated as minutes and hours spent on each activity multiplied by the assigned MET value, and adding the nine MET activity levels together. BODY.GENERAL QUESTIONNAIRE: Our participants filled out a questionnaire regarding life style including question about use of medication, previous fractures, and daily intake of dietary calcium. BODY.BLOOD PRESSURE: In calm settings, and with the study subject in a resting state, we measured blood pressure and heart rate. For the purpose, we used an automatically inflated brachial cuff connected to an oscillometric reading system measuring heart rate and blood pressure. BODY.CLINICAL VISITS: Questionnaires, and balance- and muscle function tests were performed at baseline and after 12 months treatment. At baseline and after 3, 6, 9, and 12 months of treatment the women came for a clinical visit answering question about falls, fractures and adverse advents, and to have their blood pressure measured. Balance- and muscle function were assessed during the morning or afternoon while all clinical visits were carried out in the morning. The same investigator conducted all visits and supervised all exercises to ensure a uniform instruction to all study subjects. BODY.STATISTICS: As this is a part of our randomized placebo-controlled study on women with osteopenia, sample size was calculated on the basis of our main outcome with a predicted 1.5 % change in BMD between the melatonin and placebo group. This ultimately led to a total of 72 study subjects (80 % statistical power and 5 % significance). In the present study, to show a 10 % change in knee extension 60° in healthy women (mean force 393 N, SD ±53 N) [21] a total number of 30 study subjects in each groups was required (80 % statistical power, 5 % significance). We analysed data using with the intention to treat approach as results from all randomized subjects with a follow-up value were included according to the treatment allocation. Differences between groups were tested by the Student’s t test or Wilcoxon Rank-Sum test, as appropriate. Serial changes of blood pressure values were analysed by variance for repeated measures (ANOVA). Interaction between group and time (gr. vs. time) were studied in order to determine whether treatment affected changes blood pressure differentially during the study. Area under the curve (AUC) was calculated in order to assess differences between groups in average blood pressure. We used Pearson Chi-square or Fischer’s Exact Test for categorical variable. Correlations between variables were tested by bivariate correlation analyses (r). Data are presented as mean with 95 % confidence interval (CI) or median with 25–75 % interquartile range (IQR). The significant level was p < 0.05. We used IBM SPSS Statistics 21 to perform all calculations. BODY.RESULTS: Forty-one women were allocated to placebo, while 40 were allocated to either 1 or 3 mg melatonin. Descriptive data are shown in Table 1. In general, randomization was well balanced with no significant differences between groups.Table 1Descriptive data at baseline. Mean (±standard deviation) or median (25–75 % interquartile range) Placebo N = 41Melatonin N = 40P-valueAge, years 62.9 (±4.7) 62.4 (±3.5) 0.46 BMI (kg/m2) 24.9 (21.4;26.6) 23.6 (21.3;27.5) 0.38 Weight (kg) 66.6 (60.0;76.0) 65.0 (58.3;75.5) 0.58 Systolic blood pressure (mmHg) 143 (130;149) 138 (128;144) 0.15 Diastolic blood pressure (mmHg) 83 (77;92) 85 (76;91) 0.76 Heart rate 68 (64;75) 70 (64;76) 0.94 Dietary calcium (mg/d) 950 (800;1125) 975 (800;1100) 0.66 Previous fracture after the age of 55 years, (N %) 6 (15 %) 8 (20 %) 0.57 Prescribed medication, N (%) 21 (51 %) 20 (50 %) 0.99 Simvstatin, N (%) 5 (12 %) 3 (8 %) 0.71 Antidepressives, N (%) 2 (5 %) 2 (5 %) 0.99 Benodiazepines, N (%) 1 (2 %) 0 0.99 Antihypertensives, N (%) 6 (14 %) 4 (10 %) 0.74 Levothyroxine, N (%) 1 (2 %) 1 (2 %) 0.99 Pittsburgh Sleep Quality Index > 5 20 (49 %) 9 (23 %) 0.02 During our one-year trial including 81 women, we recorded in total 36 adverse events (AE) and 12 serious adverse events (SAE). The events occurred at equal frequency in both groups. In all, we recorded two falls in each group. Two of the falls (one in each group) were caused by snow covering irregularities in the pavement. The other two falls were caused by sightseeing as the women overlooked irregularities in the pavement. Seventy-two women completed the study. Of the nine women dropping out during the trial, five were in the placebo group while four were in the melatonin group [12]. The treatment was well-tolerated and only 16 (40 %) of the women in the melatonin group guessed their randomization compared with 29 (74 %) in the placebo group. The few adverse events and the good-tolerance was the reason why they believed to receive placebo in any of the experimental settings. At baseline the placebo group had a reduced postural stability by normal standing EO compared to the melatonin group (Table 2). However, 12 months of treatment with melatonin did not affect postural stability compared with placebo.Table 2Postural balance (scaled velocity moment [m2/s]). Baseline and percentages changes after 12 months of treatment with melatonin or placebo (median with 25–75 % interquartile range Baseline (m2/s)P-valueChanges after 12 months (%)P-value*Normal standing, eyes open 0.04 0.11  Placebo 4.1 (2.9;6.4) −10.98 (−30.39;19.00)  Melatonin 3.3 (2.7;4.1) 6.30 (−22.83;43.43) Normal standing, eyes closed 0.31 0.29  Placebo 5.7 (3.6;8.5) −7.79 (−39.72;33.31)  Melatonin 4.7 (3.4; 6.7) 4.06 (−24.17;50.00) Semi tandem 0.58 0.67  Placebo 20.4 (15.6;33.1) −4.97 (−33.98;29.42)  Melatonin 20.5 (16.4; 28.7) −15.60 (−26.79;23.33) Tandem 0.48 0.55  Placebo 48.7 (34.1;63.3) −4.00 (−38.45;48.70)  Melatonin 43.3 (29.0; 73.3) 2.10 (−26.27;46.77) Significance is shown in bold *Percentages change between groups Table 3 shows the results of the muscle strength measures. No significant differences between groups were present at baseline. One-year treatment with melatonin did not affect upper or lower extremity performance in any of the groups.Table 3Maximum voluntary muscle strength (Newton [N]). Baseline data and percentage change after 12 months of treatment according to group allocation. Median with interquartile range (25–75 % percentiles) Muscle groupP-valueP-value*Baseline (Newton)Change after 12 months (%)Hand grip 0.79 0.58  Placebo 287 (257;311) −0.18 (−7.02;6.78)  Melatonin 281 (244;333) −0.75 (−10.40;7.59) Elbow extension 0.23 0.74  Placebo 116 (98;138) 6.21 (1.23;12.85)  Melatonin 111 (92;121) 5.20 (−3.51;15.28) Elbow flexion 0.18 0.37  Placebo 178 (156;192) 1.95 (−6.77;10.75)  Melatonin 165 (152;185) −1.45 (−9.12;6.91) Knee extension 90° 0.67 0.99  Placebo 309 (278;377) 0.33 (−2.68;5.44)  Melatonin 312 (275;357) 0.55 (−6.66;11.26) Knee flexion 90° 0.40 0.93  Placebo 160 (137;177) 0.26 (−7.13;16.89)  Melatonin 153 (121;177) 0.00 (−6.09;13.79) Knee extension 60° 0.26 0.33  Placebo 384 (326;433) 0.76 (−4.08;4.16)  Melatonin 370 (313;410) 1.73 (−5.94;10.49) Knee flexion 60° 0.44 0.54  Placebo 169 (139;203) 4.31 (−9.61;22.58)  Melatonin 163 (138;182) 0.99 (−2.53;9.66) *Significant changes between groups Stratifying for doses of melatonin did not change the results in either balance- nor muscle function tests (data not shown). Table 4 shows the results of the questionnaire data. According to quality of sleep index (PSQI), both groups had a good quality of sleep at baseline (5 in placebo group vs. 4.5 in melatonin group). No significant changes between groups were found in response to treatment. Restricting analyses to study subjects with a score above normal range, i.e. associated with a poor quality sleep/insomnia, this was reported more frequently by women in the placebo group compared with women in the melatonin group (49 vs. 23 %, respectively, p = 0.02) (Table 1). In response to treatment, the women in the melatonin group rated with insomnia reduced their score by −37.5 % (IQR: −64.5;−15.5) compared with −13.3 % (IQR: −32.1;9.4) in the placebo group (p = 0.08 for between group changes).Table 4Quality of life, sleep quality, and physical activity. Baseline data and changes (%) in response to 12 months of treatment with melatonin or placebo. Median scores with 25–75 % interquartile range BaselineP-valueChanges after 12 months (%)P-value*Physical component score 0.69 0.16  Placebo 54.9 (48.2;56.9) 1.80 (−3.79;7.11)  Melatonin 54.7 (50.9;57.4) −0.30 (−4.00;4.83) Mental component score 0.12 0.94  Placebo 56.6 (51.8;59.0) −0.78 (−5.88;4.47)  Melatonin 58.0 (55.6;59.5) − WHO-5 Well-being Index 0.63 0.27  Placebo 80.0 (72.0;84.0) 0.00 (−9.52;5.00)  Melatonin 80.0 (73.0;84.0) 0.00 (−5.00;5.56) Pittsburgh Sleep Quality Index 0.36 0.31  Placebo 5.0 (2.0;8.5) 0.00 (−32.14;38.33)  Melatonin 4.5 (3.0;5.0) 0.00 (−41.11;28.79) Pittsburgh Sleep Quality Index > 5 0.47 0.08  Placebo, N = 20 8.5 (6.0;9.8) −13.3 (−32.1;9.4)  Meltonin, N = 9 8.0 (6.0;8.5) −37.5 (−64.5;-15.5) Physical activity scalea 0.17 0.43  Placebo 43.8 (37.4;48.7) −0.16 (−8.46;8.29)  Melatonin 46.0 (40.9;51.3) −2.27 (−13.84;10.07) *Percentage change between groups aMetabolic equivalent (MET) score for 24 hours In the subgroup of women with insomnia, 58 % of the women guessed their randomization i.e. 86 % in the placebo group and 14 % in the melatonin group (p = 0.01). The percentage change in PSQI-score did not differ in women believing to receive melatonin compared to those believing to receive placebo (data not shown). There were no differences in quality of life between groups in any of the subdomains (data not shown), MCS or PCS at baseline or at follow up. In all the subdomains as well as MCS and PCS, both groups scored within the normal range compared to norm-based values. Who-5 index did not change over time between the groups (Table 4). A significant inverse correlation was present between changes in PCS and changes in PSQI (r = −0.36, p < 0.01) meaning that the better PCS, the better quality of sleep. No correlation was present between PSQI and other physical parameters such as PAS, muscle and balance function (data not shown). No changes between groups were observed when assessing physical activity (PAS). Heart rate and blood pressure did not differ between groups at baseline. Compared with placebo, melatonin caused a non-significantly decrease in systolic (5 mmHg, p = 0.12) and diastolic (2 mmHg, p = 0.40) blood pressure (Fig. 1). Heart rate did not change in response to treatment (data not shown).Fig. 1Effects of treatment with placebo or melatonin on blood pressure and heart rate. Mean ± SD BODY.DISCUSSION: In the present study we investigated the effects of one-year treatment with melatonin on balance- and muscle function as well as physical and mental well-being in postmenopausal women with osteopenia. Our study did not raise safety concerns regarding reduced postural balance- or muscle function. Furthermore, in postmenopausal women with a normal sleep quality, treatment with low doses of melatonin did not affect quality of life or sleep. In the subgroup of people with insomnia there was, however, a borderline significant improvement in quality of sleep in the melatonin group compared to placebo. To the best of our knowledge this is the first study to evaluate the effect of longer term treatment with small doses of melatonin on balance- and muscle function in postmenopausal women. Melatonin is commonly used as a sleep aid in the elderly. As melatonin increased bone mineral density [12] through previously mentioned possible mechanisms, it may also in the future play a role in the prevention and treatment of osteoporosis. It is therefore of importance to rule out any long term adverse effects that may increase the risk of falls and fractures. The manufacture of 2 mg melatonin describes the side effects in terms of hangover (dizziness and sleepiness during the day) as events occurring in 0.1–1 % of the patients [22]. The half-life of melatonin is dependent on the composition of the drug varying from app. 30 min to four hours, and may be affected by e.g. fasting state [22–25]. Administered just before bedtime, melatonin should be eliminated in the morning. However, as described by the manufactures of 2 mg melatonin, hangover during the day may still occur, which is probably caused by intersubject viability. In previous studies, discrepant results have been reported on the effects of melatonin on balance performance. In one study [26], an oral intake of 10 mg of melatonin was found to impair postural stability, whereas Otmani et al. [27] reported no detrimental effects of 2 mg short term treatment of melatonin on postural balance. Furthermore, the hangover effect of melatonin has previously been examined in relation to muscle strength in young adults showing no alteration in response to a single dose of 5 mg melatonin [28, 29]. In the present study we evaluated the effect of melatonin in response to one-year treatment. A potential adaption to side effects causing no changes in balance and muscle function cannot be completely ruled out. However, contradicting this postulate, we reported an equal frequency of falls in the groups throughout the trial. Furthermore, to avoid learning effect to the exercises, it is of rational not to repeat the measurements until one-year of treatment. In a previous study by Kotlarczyk et al. [30], the authors investigated the effect of 3 mg melatonin/d for 6 months in 18 healthy perimenopausal women. In line with our results, no effect of melatonin was seen in relation to the average quality of sleep. This is most likely explained by the fact that both studies comprised of subjects without known sleep disturbances, and the results are further supporting the findings by other investigators [31]. It is, however, well known that melatonin has a positive effect on sleep in patients with insomnia [32, 33]. Similar to these results we did find a trend towards a positive effect of melatonin on quality of sleep in the subgroup of people with pre-exiting poor quality of sleep. The lack of statistical significance is most likely explained by a too low statistical power. Concerning the self-rated quality of life questionnaires, we conclude that melatonin does not affect the outcome measures negatively. Our results are in accordance with Kotlarczyk et al. [30] who demonstrated a safe use of melatonin with no significant changes in domains, except physical, as assessed by Menopause-Specific quality of life (MENQOL) questionnaire. The physical domain increased in the study in response to melatonin. Our results showed a significantly inverse correlation between the physical component score (PCS) and quality of sleep indicating that the better the physical score, the better the sleep. Although an effect of melatonin in relation to quality of life has previously been shown in patients with known sleep disturbances [33] our study consisted of healthy women with a good quality of life and an increase was not expected. There have previously been demonstrated a reduction in nocturnal systolic and diastolic blood pressure after nightly therapeutic doses of 2–3 mg melatonin [6, 34, 35]. However, it line with previous results [30], we did not observe significant effects of melatonin on blood pressure. This may be explained by the fact that we did not measure blood pressure during night after ingestion of melatonin. There are several strengths to the study including its design as a double-blind randomized placebo- controlled trial. Furthermore, as to evaluate the effect of melatonin, the study subjects were in the relevant age group. There are however, also limitations to the study, as the women did not suffer from sleep disturbances prior to entering the study. However, a positive effect was not anticipated as our primary goal was to establish the safety aspects of longer term treatment with melatonin. In conclusion, melatonin in a daily dose of 1 or 3 mg is safe to use in postmenopausal women with osteopenia. There is no long term hangover effect causing a reduction in balance- and muscle function or quality of life. In women with poor quality of sleep, small doses of melatonin trended towards improving quality of sleep.

TITLE: Efficacy of Chuanxiong Ding Tong Herbal Formula Granule in the Treatment and Prophylactic of Migraine Patients: A Randomized, Double-Blind, Multicenter, Placebo-Controlled Trial Objective. To evaluate the efficacy of traditional Chinese herbal ChuanXiong Ding Tong herbal formula granule (CXDT-HFG) for migraine patients with “the Syndrome of Liver Wind and Blood Stasis.” Methods. 150 migraine patients were recruited and assigned randomly in a double-blind, placebo-controlled study to receive CXDT-HFG (n = 99) plus necessary analgesics, or placebo (n = 51) plus necessary analgesics for 16 weeks (12 weeks' intervention and 4 weeks' follow up). Outcome measures included migraine days, frequency of migraine attacks, analgesics consumption for acute treatment, and the proportion of responders as well as the visual analogue scale (VAS) scores and intensity for pain. Results. Compared with the placebo group, the CXDT-HFG group showed significant reduction in migraine days and attacks frequency at week 12 and follow-up period (P < 0.05) as well as in the reduction of VAS scores at follow-up period.There was significant difference in the proportion of responders between the two groups at follow-up period (P = 0.014). However there were no significant differences between the two groups in analgesics consumption (P > 0.05). Conclusion. CXDT-HFG was more effective than placebo in decreasing days of migraine attacks, frequency, VAS scores, and relieving pain intensity for migraine patients. BODY.1. INTRODUCTION: Migraine, a common and chronic headache, which is understood to be a neurovascular dysfunction, is characterized with recurrent headache attacks. The pain is located unilaterally, moderate to severe intensity, usually aggravated by physical activity, and usually lasts 4–72 hours. In addition, migraine attacks are often accompanied by nausea and/or vomiting, photophobia, phonophobia, etc. Migraine is high morbidity, procrastinating, and refractory, severely affecting the ability to work and quality of life [1–5]. The epidemiological study from the United States shows that the lifelong cumulative inDcidence of migraine is 7.4% in males and 21% in females [6]. The temporary functional disability, accompanying migraine [6], brings huge economic losses to the society. Migraine has been listed as one of the most serious, chronic, and dysfunctional diseases, which is equal to quadriplegia, mental disorders, and dementia by World Health Organization [7]. Due to its high incidence and high economic cost [2], the treatment and prevention of migraine has already attracted broad attention in the world in recent years. Migraine is divided into the period of acute episode and chronic remission [8] and its treatment and prevention requires standard drug therapy and regular management [9–12]. There are many drugs used to treat acute migraine, such as aspirin, acetaminophen, nonsteroidal, anti-inflammatory drugs (NSAIDs), dihydroergotamine, and the triptans. Meanwhile Beta-blockers, calcium channel blockers, tricyclic antidepressant, and antiepileptic drugs have been used for migraine prevention [4, 13, 14]. However, in clinical practice it has been shown that these drugs can lead to headache or increase the frequency of migraine attacks and other side effects [15, 16], and which, to some extent, limit their application to migraine sufferers. In the United States, Chinese herbal medicine, acupuncture and other natural therapy, have been licensed for use, and it is recognised clinical practice for herbal medicine to hold a place of importance in remedying headaches effectively and safely [17]. Clinical studies show that traditional acupuncture therapy can effectively reduce the migraine days and acute pain drug consumption, especially for migraine prevention, but no statistical differences are seen in the improvement of pain intensity [18, 19], thus sufferers still need drug treatment in the treatment and prevention of migraine. However, those studies on using Chinese herbal medicine to treat migraine with the method of multicenter, prospective, randomized, placebo controlled, double blind, are relatively few. According to the Guidelines for controlled trials of drugs in migraine (second edition) [20], this study chose migraine patients with “the Syndrome of Liver Wind and Blood Stasis” commonly seen in traditional Chinese medicine (TCM) clinic, and Chuanxiong Ding Tong herbal formula granule (CXDT-HFG) as treatment drug, so as to further evaluate the efficacy of Chinese herbal medicine in the treatment and prevention of migraine. BODY.2. MATERIALS AND METHODS: BODY.2.1. STUDY DESIGN: A multicenter, prospective, central-randomized, double-blind, placebo-controlled trial was conducted in this study. Migraine patients who met the inclusion criteria were randomly assigned into the experimental group and control group in a 2 : 1 ratio. The experimental group was treated with Chinese herbal medicine and the placebo group with placebo. Both groups were permitted to use the necessary analgesics during migraine acute attacks, as well as accepted regular management. The retrospective historical record of migraine attacks for nearly 3 months was defined as the baseline of the study. The study spans 16 weeks, including a treatment period of 12 weeks, and a follow-up period of 4 weeks. Headache diary was given to patients to record the details (migraine days, frequency, visual analogue scale (VAS) scores and acute medication, etc.) of migraine attacks during the trial period. These outcome measures such as migraine days, frequency of migraine attacks, VAS scores and intensity for pain were evaluated at 4 weeks, 8 weeks, 12 weeks, and follow-up period Figure1. This study was designed and carried out cooperatively by methodologists and statisticians of the China Academy of Chinese Medical Sciences in Beijing. And the study protocol conforms to the Helsinki Declaration [21] and the research regulations for Chinese clinical trials. The Ethics Committee of the Affiliated Dongzhimen Hospital of Beijing University of Chinese Medicine reviewed and approved the study protocol. All participants signed informed consent before enrolment. BODY.2.2. SETTING AND PARTICIPANTS: 150 migraine patients with “the Syndrome of Liver Wind and Blood Stasis” were recruited between January 2008 and June 2011 from outpatient departments in the following 8 hospitals: Dongzhimen Hospital affiliated to the Beijing University of Traditional Chinese Medicine, the Affiliated Hospital to Changchun University of Chinese Medicine, Beijing Tiantan Hospita affiliated to the Capital Medical University, Tai an Hospital of Traditional Chinese Medicine, Xiamen Hospital of Traditional Chinese Medicine, Hubei Hospital of Traditional Chinese Medicine, The First Subsidiary Hospital of Anhui College of Traditional Chinese Medicine, Xiyuan Hospital affiliated to the China Academy of Chinese Medical Sciences. All patients, with diagnosis of migraine with aura or without aura in accordance with the 2nd edition of the International Headache Society's International Classification of Headache Disorders (ICHD-II, IHS 2004) [8]. The study had a previous 3 months of retrospective record of migraine attacks (referred to as baseline) and 12-week double-blind, parallel group, placebo-controlled phase with trial drug treatment at day “0” of the double-blind phase, follow-up visit for 4 weeks after the experimental drug withdrawn (details will be described separately). BODY.2.3. DIAGNOSTIC CRITERIA: The diagnostic criteria for this study were as follows. (1) Diagnosis standard in Western medicine: migraine without aura (MO) or with a typical aura (MA) as defined by 2004 HIS [8]. (2) TCM Differentiation standard: in accordance with the guiding principles for the clinical study of new drugs for use in traditional Chinese medicine released in 2002, combined with the characteristics of migraine, the standard of “Liver Wind and Blood Stasis syndrome” as follows: headache in the left or right, repeated attacks, severe pain, lasting for several hours or days, accompanyed by nausea, vomiting, vertigo, etc.; dark red or dark purple tongue, or tongue has bruises, ecchymosis or stasis points, thin-white fur, wiry pulse. BODY.2.4. INCLUSION CRITERIA: The inclusion criteria for this study were as follows: (1) diagnosed as migraine without aura (MO) or with a typical aura (MA) according to the diagnostic criteria specified by the International Classification of Headache Disorders; (2) meet the diagnostic standard of “Liver Wind and Blood Stasis syndrome” in TCM; (3) age of first onset ≤50 years old; (4) the history of migraine >1 year; (5) with more than 6 times of migraine attacks in the previous 3 months; (6) age between 18 and 65 years old; (7) the patients voluntarily joined this study with informed consent. BODY.2.5. EXCLUSION CRITERIA: The exclusion criteria were as follows: (1) times of using analgesics for acute headache>10 times per month; (2) alcohol or other drug abuse; (3) primary disease of the liver, kidney, hematopoietic system, cardiovascular system, or cerebrovascular system; (4) psychiatric conditions; (5) hypersensitivity to the trial drug; (6) pregnancy and lactation. BODY.2.6. INTERVENTIONS: The patients in the experimental group would be provided with CXDT-HFG, while the control group would take placebo. Both of them were provided by Hua Run San-Jiu Pharmaceutical Co. LTD. Mix the drugs or placebo with warm-water, and take them twice daily (separately taken in the morning and evening). Both groups were permitted to use the necessary analgesics during an acute migraine attack as well as accepted regular management. The experimental drug CXDT-HFG is composed of Chuanxiong Rhizoma (Chuanxiong, 12 g), Cyathulae Radix (Chuan Niuxi, 10 g), Dioscoreae Hypoqlaucae Rhizoma (Chuan Bixie, 20 g), Chrysanthemi Flos (Juhua, 6 g), Uncaria rhynchophylla Pamuluscum Uncis (Gouteng, 20 g), Tribuli terrestris Fructus (Bai Jili, 10 g), Coicis Semen (Yi Yiren, 20 g), Amomi FructusRotundus (Bai Doukou, 6 g), Pinelliae Rhizoma Preparatum (Zhi Banxia, 6 g). The placebo of single Chinese medicine formula is consisted of dextrin, lactose, caramel pigment, and bitters. Caramel pigment is provided by Shanghai Love Food Industry Company and bitters are manufactured by Zhejiang Deere pharmaceutical factory. There are better similarities in some ways of dosage, shape, and predominant flavor between the experimental drug CXDT-HFG and placebo. Permitted medication and specification: generally the patients who have 2 days of headache attacks per week will be permitted to use acute drug treatment; patients should choose the analgesics commonly used without the effect of migraine prevention; the intensity of headache should be measured by VAS and patients should choose corresponding analgesics according to different pain degree. All analgesics used and their doses and effect should be recorded in the diary. Management standard of participants: (1) patients need to record the headache diary seriously so as to fully understand the time and characteristics of headache attacks. (2) Patients should have a regular lifestyle, the work and rest on schedule, quit smoking, and moderate alcohol consumption. (3) Regulate and refresh emotion and avoid disposition stimulation. (4) Light diet, avoid spicy, and greasy food, reduce the intake of chocolate, coffee, cheese, and other contour tyrosine foods and sausage, ham, hot dogs, and other bacon meats. (5) Patients need to do timely medical examination and treatment if the nature, the degree, and the frequency of headache have changed. BODY.2.7. OUTCOME MEASURES: The outcome measures included the changes of migraine days, attack frequency, analgesic consumption, and the proportion of responders (defined as the proportion of patients with a reduction of migraine days and times by at least 50% [18, 20]) from baseline to weeks 4, 8, 12 and follow-up period. The frequency of migraine attacks is the main evaluation index, which indicates improvement of the migraine to the maximum extent [22]. Thus the frequency of migraine attacks per 4 weeks should be the primary efficacy measure. The secondary outcome measure was the proportion of responders in attack frequency and migraine days during treatment and follow-up period compared with the baseline period. Inaddition, VAS scores and intensity for pain (mild, moderate, and severe) were assessed at weeks 4, 8, 12 and follow-up period (because VAS scores and pain intensity were not recorded in headache diary retrospectively for nearly 3 months, the 4 weeks' VAS scores and intensity would be seen as baseline at this point). Additionally, patients were required to record adverse events in their headache diaries. Patients filled in a headache diary on time and recorded the time, frequency, location (the forehead, top, temporal, and back of the head), VAS scores, intensity (mild, moderate, and severe), types of pain, analgesics consumption during migraine acute attacks, and concomitant symptom (nausea, vomiting, photophobia, phonophobia, etc.) in detail, as follows: (1) the days of migraine attacks refer to the onset of the migraine days (provided migraine attack in one day, it will be deemed to one day); (2) a migraine attack that was interrupted either by sleep or treatment but relapsed within 48 hours was required to be documented as a single attack. (3) VAS scores (quantitative index for pain): let the patient himself point out that most representative number of pain; (4) Pain Intensity on the basis of the Numeric Rating Scale (NRS-11) which has been widely used for the assessment of pain [23],was graded according to the number represent pain that patients point out from the scaleplate of VAS: 0 stands for no headache, score range 0~4 stands for mild headache, score range 4~7 stands for moderate headache, score range 7~10 stands for severe headache. BODY.2.8. STATISTICAL METHODS: Statistical analysis was performed with SPSS (13.0) program for Windows (SPSS, Chicago, IL, USA). Data was managed through the online facility established by the Traditional Chinese Medicine Clinical Foundation Institute of medicine of the China Academy of the Chinese Medical Sciences, and was analyzed on the full analysis set (FAS), and the per-protocol set (PPS) for adherence. The result of data analysis was mainly for PPS in this trial. The measurement data were expressed as mean ± standard deviation, to check the data of all groups with normal test and homogeneity of variance test. Data analysis was performed by nonparametric statistics to these unmatched normally distributed measurements, including age, course of disease, migraine days, and attacks frequency, etc., which were compared between groups using Mann-Whitney U test. Results were shown with 95% confidence interval (95% CI). Measurement data before and after treatment were compared using ANOVA for repeated measures. Headache intensity classification and the proportion of responders were analyzed by χ 2 test. The level of significance was set at 0.05, if P < 0.05, there were statistical differences. BODY.3. RESULTS: BODY.3.1. DROPOUTS: During the study, 22 patients dropped out, a rate of 14.7% (13 from the CXDT-HFG group 13.1% and 9 from the placebo group 17.6%). Among these, 1 patient was rejected for not meeting inclusion criteria (1 from the CXDT-HFG group), 15 patients dropped out during the treatment period (10 from the CXDT-HFG group, 5 from the placebo group) because of lack of efficacy, pregnancy, etc. And 4 patients dropped out for adverse events (2 from the CXDT-HFG group, 2 from the placebo group). The other 2 patients were lost to followup due to change of contact information (2 from the placebo group). The reasons for the dropouts in the 2 groups are detailed in Figure 1. BODY.3.2. CHARACTERISTICS OF DEMOGRAPHY AND BASELINE: The demographic and baseline parameters with the PPS population were shown in Table 1, which showed that the 2 groups were comparable at baseline. The Table 1 summarizes the main baseline characteristics of the 128 patients based on PPS. Participants of CXDT-HFG group had a mean age of 35.77 years and 73.3% (63) were women while the placebo group had a mean age of 34.58 years and 73.8% (31) were women. There were no statistical differences (P > 0.05) between the two groups in migraine days, attack frequency, course of disease, consumption of analgesics, all the baseline characteristics of the 2 groups were similar (Table 1). BODY.3.3. DAYS OF MIGRAINE ATTACKS: The baseline of migraine days in the CXDT-HFG group was 4.48 ± 4.12 days and 4.13 ± 4.33 days in the placebo group. At 12 weeks and follow-up period, the migraine days in the CXDT-HFG group decreased to 1.44 days and 1.06 days, respectively, whereas in the placebo group, the migraine days reduced to 1.93 days and 1.69 days, respectively. In other words, the mean reduction of migraine days in the CXDT-HFG group were 3.04 days, compared with 2.20 days in the placebo group at 12 weeks (95% CI, −4.13 to −1.93 versus 95% CI, −3.74 to −0.66; P = 0.033), and a mean reduction of 3.42 days in the CXDT-HFG group compared with 2.44 days in the placebo group at follow-up period (95% CI, −4.49 to −2.34 versus 95% CI, −3.97 to −0.90; P = 0.042). And there was significant difference in the proportion of patients with a reduction of migraine days by at least 50% between the CXDT-HFG group and the placebo group at follow-up period (83.7% versus 64.3%, P = 0.014) (Table 1; Figure 2). BODY.3.4. FREQUENCY OF MIGRAINE ATTACKS: The baseline of migraine attack frequency in the CXDT-HFG group is 3.82 ± 2.16 times and 3.91 ± 4.32 times in the placebo group. After 12 weeks' treatment, the frequency of migraine attacks of the CXDT-HFG group reduced 2.84 times, while the placebo group reduced 2.67 times (95% CI, −3.35 to −2.33 versus 95% CI, −4.11 to −1.24; P = 0.043). At follow-up period the attack frequency of the CXDT-HFG group reduced 3.08 times from baseline and the placebo group reduced 2.72 times (95% CI, −3.57 to −2.58 versus 95% CI, −4.17 to −1.28; P = 0.033). However, no significant difference was found in the proportion of patients with a reduction of migraine attack frequency by at least 50% between the CXDT-HFG group and placebo group (Table 1; Figure 3). BODY.3.5. VAS SCORES AND PAIN INTENSITY: The mean VAS scores have significantly decreased in the CXDT-HFG group from 4.96 ± 2.01 at 4 weeks to 1.66 ± 2.15 at follow-up period, whereas in the placebo group from 4.90 ± 2.09 at 4 weeks to 2.92 ± 2.51 at follow-up period (P = 0.005). According to VAS scores, the pain degree is divided into four levels with no pain, mild, moderate, and severe. At 12 weeks and follow-up period, there were significant differences between the 2 groups about the pain degree (P = 0.037, P = 0.013) (Table 2; Figures 5 and 6). BODY.3.6. ACUTE MEDICATION: The number of patients using acute pain drugs, such as aspirin, phenacetin, or ibuprofen, etc., has no significant difference between the 2 groups from baseline to follow-up period (P > 0.05) (Table 1; Figure 4). BODY.3.7. SAFETY AND TOLERABILITY: A total of 15 (11.72%) subjects in the randomized population experienced mild adverse events during the study period. Treatment-related adverse events were reported in 11.63% of the CXDT-HFG group (10 patients) and in 11.90% of the placebo group (5 patients). The main adverse events were as follows, maculopapula, eyelid edema, palpitations, abdominal pain and nausea, and they were resolved without sequelae after treatment was withdrawn. No clinically significant serious adverse events were reported in subjects on the aspects of liver and renal functions, blood, and urine routines before and after treatment. BODY.4. DISCUSSION: BODY.4.1. SUMMARY OF MAIN FINDINGS: This trial revealed Traditional Chinese herbal CXDT-HFG to be better than placebo in reducing the days of migraine attacks, frequency, and pain intensity at 12 weeks and follow-up period (P < 0.05) as well as in the reduction of VAS scores at follow-up period. And along with the extension of treatment time, the days and frequency of migraine attacks and pain intensity still improved in the CXDT-HFG group. Whereas no significant difference was found between the 2 groups in consumption of acute pain drug (Figures 2, 3, 4, 5, 6). At the follow-up period, we found that the placebo group presented rebound trend in migraine days and frequency, meanwhile the number of analgesics increased gradually. However the efficacy of the CXDT-HFG group continued to exist, and the decreasing trend was seen in the using of analgesics. Traditional Chinese herbal medicine has been practiced in China for thousands of years and vast experience has been accumulated for using medicinal herbs for clinical treatment of diseases. Although the biological mechanism of CXDT-HFG as traditional Chinese herbal medicine in improving the clinical consequence of migraine is not exactly clear, its analgesic effect and reduction of the days of migraine attacks, frequency, and pain intensity play an important role. First, researchers point out herbal medicines have held a place of importance in remedying headaches effectively and safely [17]. Second, according to the theory of TCM, Rhizoma Chuanxiong (Chuanxiong), originates from the plant Ligusticum chuanxiong Hort, which is used in TCM to “remove blood stasis and expel wind evil to relieve pain.” And Uncaria rhynchophylla (Gouteng) can clear liver heat and flatten rising liver yang as well as Chrysanthemums (Juhua). Modern chemical studies indicate that they have the effect of adjusting nerves, antioxidant, anti-inflammatory, sedation, and analgesic [24–26]. Mechanism research of Chinese herbs for the treatment and prophylaxis of migraine should be further studied. Overall, the study suggested that CXDT-HFG was efficient in reduction of the frequency of migraine attacks and in alleviating pain degree at week 12, there may be follow-up treatment and prophylaxis effect existing. BODY.4.2. STRENGTHS AND LIMITATIONS OF THE STUDY: Migraine is the patient's subjective feeling, so there may be selective bias in grouping as well as measuring bias and hybrid bias in clinical effect assessment. Thus blinded intervention is necessary [27]. This study is a randomized, double-blind, multicenter, placebo-controlled trial, which can reduce bias factors and improve the reliability and scientific validity of the clinical research. Migraine has the clinical characteristic of relapsing-remitting, and the study mainly chose the remission period for clinical research. Due to the long observation schedule and strict standard of cases inclusion/exclusion, the patients' compliance is poor and future research will be needed to strengthen the management and education of patients to improve the clinical research compliance, and control the rate of dropout. BODY.4.3. IMPLICATIONS FOR CLINICAL PRACTICE: Migraine is a recurrent disease and its acute episode and remission period have different pathogenesis characteristics and clinical manifestations. The United States migraine evidence-based guidelines [28] point out that the main goal of acute migraine attacks is to relieve the pain quickly and prevent recurrence; the prevention objective of migraine is to reduce the frequency of migraine attacks and relieve pain severity. The advantage of TCM for migraine lies in the preventative treatment during remission period. CXDT-HFG has certain clinical effect in the preventative treatment of migraine sufferers with “the syndrome of Liver Wind and Blood Stasis,” with low adverse reaction. So it is one kind of relatively safe TCM in clinical treatment and prophylaxis for migraine. BODY.4.4. UNANSWERED QUESTIONS AND FUTURE RESEARCH: The application of TCM must base on the theory of syndrome differentiation and treatment. Patients at different states of migraine need different therapeutic methods. In order to make the clinical efficacy of CXDT-HFG used repeatability, we need to expand sample size and do a larger sample, randomized, double-blind, parallel-controlled clinical research, in which we choose an effective drug for migraine which is accepted internationally as the comparison drug to evaluate the clinical effect characteristics of CXDT-HFG for treatment and prevention of migraine further accurately and objectively. BODY.5. CONCLUSION: CXDT-HFG could reduce the days and frequency of migraine attacks and relieve pain intensity, especially in the prevention of migraine. In addition further research could be conducted on the mechanism of CXDT-HFG for migraine prophylaxis.

TITLE: Rilonacept in the treatment of acute gouty arthritis: a randomized, controlled clinical trial using indomethacin as the active comparator ABSTRACT.INTRODUCTION: In phase-3 clinical trials, the interleukin (IL-1) blocker, rilonacept (IL-1 Trap), demonstrated efficacy for gout flare prevention during initiation of urate-lowering therapy. This trial evaluated rilonacept added to a standard-of-care, indomethacin, for treatment of acute gout flares. ABSTRACT.METHODS: Adults, aged 18-70 years, with gout presenting within 48 hours of flare onset and having at least moderate pain as well as swelling and tenderness in the index joint were randomized to subcutaneous (SC) rilonacept 320 mg at baseline plus oral indomethacin 50 mg TID for 3 days followed by 25 mg TID for up to 9 days (n = 74); SC placebo at baseline plus oral indomethacin as above (n = 76); or SC rilonacept 320 mg at baseline plus oral placebo (n = 75). The primary efficacy endpoint was change in pain in the index joint (patient-reported using a Likert scale (0 = none; 4 = extreme)) from baseline to the average of values at 24, 48 and 72 hours (composite time point) for rilonacept plus indomethacin versus indomethacin alone. Comparison of rilonacept monotherapy with indomethacin monotherapy was dependent on demonstration of significance for the primary endpoint. Safety evaluation included clinical laboratory and adverse event (AE) assessments. ABSTRACT.RESULTS: Patient characteristics were comparable among the groups; the population was predominantly male (94.1%), white (75.7%), with mean ± SD age of 50.3 ± 10.6 years. All treatment groups reported within-group pain reductions from baseline (P < 0.0001). Although primary endpoint pain reduction was greater with rilonacept plus indomethacin (-1.55 ± 0.92) relative to indomethacin alone (-1.40 ± 0.96), the difference was not statistically significant (P = 0.33), so formal comparison between monotherapy groups was not performed. Pain reduction over the 72-hour period with rilonacept alone (-0.69 ± 0.97) was less than that in the other groups, but pain reduction was similar among groups at 72 hours. Treatment with rilonacept was well-tolerated with no reported serious AEs related to rilonacept. Across all groups, the most frequent AEs were headache and dizziness. ABSTRACT.CONCLUSIONS: Although generally well-tolerated, rilonacept in combination with indomethacin and rilonacept alone did not provide additional pain relief over 72 hours relative to indomethacin alone in patients with acute gout flare. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov registration number NCT00855920. BODY.INTRODUCTION: A cardinal clinical feature of gout is recurrent acute inflammatory flares (acute gout flares) that result in debilitating joint pain and swelling. Gouty arthritis is mediated by monosodium urate monohydrate crystal deposition in and around the joint space due to hyperuricemia. Acute gout flares can be precipitated by a variety of factors including joint trauma, and putative remodeling of articular crystal deposits due to changes in serum urate concentrations, such as during the early months of initiation of uric acid-lowering therapy (ULT) [1,2]. The incidence and prevalence of gout are increasing, partly as a consequence of increased prevalence of comorbidities such as metabolic syndrome, type II diabetes, obesity, hypertension, and chronic kidney disease [3,4]. Gout is associated with a substantial economic burden due to high health care resource utilization and reduced work productivity [5-7], especially among patients who are refractory to conventional gout management strategies [8,9]. Because of their anti-inflammatory and analgesic characteristics, non-steroidal anti-inflammatory drugs (NSAIDs) are often used as first-line therapy for the treatment of acute gout flares [10,11]. Colchicine and systemic and locally injected corticosteroids are also appropriate options in many patients [10-12], with the corticosteroid prednisolone in particular showing equivalent efficacy to the NSAID naproxen [13]. However, colchicine is associated with risks of toxicity especially related to renal impairment and drug-drug interactions [14], and NSAIDs are also associated with clinically recognized risks of toxicities, especially related to their gastrointestinal and cardiovascular effects [15,16]. In a recent study, more than 90% of gout patients had a relative or absolute contraindication to NSAIDs, and up to 66% of patients had a contraindication to colchicine or a condition warranting colchicine dose reduction [17]. The presence of comorbid conditions in these patients included hypertension (88.7%), coronary artery disease (37.4%), chronic kidney disease (47.1%), and gastroesophageal disease (> 20%), with 65% of patients having multiple comorbidities [17]. Such risks increase among individuals with comorbidities and in those taking multiple medications, circumstances that are common in older adults [18,19]. Moreover, the intense pain of gout attacks is reduced with NSAID, colchicine, or corticosteroid therapy by only approximately 50% in 1 to 3 days in most clinical trials [12,13,20,21]. Consequently, there is a need for new approaches that provide increased efficacy and/or tolerability in the treatment of acute gouty arthritis. IL-1β is a major mediator of gouty inflammation and pain [22], and is now being increasingly evaluated for its role in acute and chronic gout. Of particular relevance is the observation that monosodium urate (MSU) crystals induce activation of the NLRP3 inflammasome, a protein complex expressed in macrophages and certain other cell types, which promotes caspase-1-driven release of mature IL-1β, with subsequent induction of numerous downstream inflammatory mediators that contribute to the clinical presentation of the signs and symptoms of gouty arthritis [23]. Neutrophils and mast cells also express proteases such as elastase and chymase, respectively, that activate pro-IL-1β [24]. Data from case reports and early-phase clinical trials of the IL-1 inhibitors anakinra and canakinumab confirmed the role of IL-1 inhibition as a treatment option for acute gout [25-30]. In particular, studies of the IL-1β-specific monoclonal antibody canakinumab for the treatment of an acute gout flare provided evidence of efficacy relative to a single intramuscular (IM) low dose of triamcinolone acetonide 40 mg [30,31]. Rilonacept is a fully-human, recombinant, soluble decoy receptor protein engineered from human IL-1 receptors and IgG1Fc that binds IL-1α and IL-1β, thus preventing their activation of cell surface receptors [32]. Since rilonacept was generated using so-called Trap Technology, it is also known as the IL-1 Trap [32]. The half-life of rilonacept is approximately 1 week [33]. Recent clinical trials of rilonacept in gout have demonstrated significant and marked efficacy vs placebo for prevention of acute gout flare over 4 months, among patients initiating ULT with allopurinol [34,35]. We therefore tested in the current study the hypothesis that a single subcutaneous (SC) administration of rilonacept, along with an oral NSAID, during the first 48 hours of an acute gout flare can reduce the pain of the gout attack compared to NSAID alone. Specifically, in this superiority study, we evaluated the efficacy and safety of SC rilonacept as add-on therapy to a standard-of-care NSAID regimen of oral indomethacin, rilonacept monotherapy, and indomethacin monotherapy, for the reduction of pain in patients experiencing an acute gout flare. BODY.MATERIALS AND METHODS: BODY.STUDY DESIGN AND PATIENT POPULATION: This phase 3, randomized, double blind, double-dummy, active- and placebo-controlled study was conducted at 60 study centers in North America. The study was performed in accordance with the revised Declaration of Helsinki, approval was obtained from the independent ethics committee (Copernicus Group IRB, One Triangle Drive, Suite 100, Research Triangle Park NC 27709), and all patients provided written informed consent. The study was open to male and female patients 18 to 70 years of age having a diagnosis of primary gout based on the American Rheumatism Association (1977 ARA preliminary criteria) for the classification of acute arthritis of primary gout [36]. Eligible patients must also have previously demonstrated symptomatic relief with NSAIDs for treatment of gout flare. Exclusion criteria included, but were not limited to treatment with short-acting NSAIDs within 48 hours of randomization, or other NSAIDs based on duration of action; use of systemic glucocorticoids within 4 weeks of randomization; use of colchicine at a dose exceeding 0.6 mg twice daily within 7 days of randomization; a history of NSAID intolerance or absolute contraindication; active or recurrent infections, and estimated creatinine clearance < 60 mL/minute using the Cockcroft-Gault method [37]. Since NSAID use was required in this study, a history of bleeding disorders, gastrointestinal bleeding or perforation, as well as poorly controlled hypertension and other cardiovascular risk factors were reasons for exclusion. Eligible patients remained in screening and were subsequently randomized to treatment when they presented with an acute flare within 48 hours of pain onset and met the following additional criteria: pain in the gouty index joint of at least moderate severity using a 5-point Likert scale (1 = none, 2 = mild, 3 = moderate, 4 = severe, 5 = extreme), a score of at least 1 on a scale of 0 to 3 for assessments of swelling and tenderness at the gouty index joint, and presentation of acute gout flare in three joints or fewer. The index joint was defined as the joint that was most painful at the time of randomization. BODY.RANDOMIZATION AND DOSING: Patients were randomly allocated 1:1:1 to treatment with either SC placebo at baseline plus oral indomethacin 50 mg three times daily for 3 days (and then 25 mg three times daily for up to 9 days); SC rilonacept 320 mg at baseline plus oral indomethacin as above; or SC rilonacept 320 mg at baseline plus oral placebo three times daily for 3 days and then oral placebo three times daily for up to 9 days. Randomization was stratified by baseline pain score (moderate, or severe or greater) and country. On-site study visits occurred at baseline (day 1), days 4 and 8, and at a safety follow up on day 31. BODY.ENDPOINTS: Patient self-assessment of pain and other gout symptoms were recorded using an electronic diary at baseline, 4, 8, 12, and 24 hours, and then daily until the flare ended. Pain was assessed using the 5-point Likert scale and an 11-point numerical rating scale (NRS; 0 = no pain to 10 = extreme pain). Based on index joint pain, patients were eligible for rescue medication at two time points. At 24 hours, patients with extreme pain or severe pain that had not decreased from baseline were eligible for rescue medication with blinded indomethacin 50 mg three times daily for 1 day (group not receiving indomethacin, that is, rilonacept only group) or blinded placebo rescue in the other two treatment groups receiving indomethacin. At 48 hours patients with either a) extreme pain alone or b) moderate or severe pain and pain reduction from baseline less than 20% were eligible to receive blinded rescue with indomethacin in the rilonacept-only group (50 mg three times daily for 1 day, followed by 25 mg three times daily for up to 9 days), or with placebo in the two groups receiving indomethacin; the duration of rescue was determined by the investigator. Patients receiving rescue medication were not required to be withdrawn. For all groups, efficacy data were set to missing after initiation of rescue medication. The last observation carried forward (LOCF) method was used to impute data after rescue medication for the analysis of covariance (ANCOVA) model, used for the primary analysis. The primary efficacy endpoint was the change in patient-reported pain (Likert scale) in the index joint from baseline to the average of the patient-reported pain values at 24 (day 2), 48 (day 3) and 72 hours (day 4) (composite time point). Secondary efficacy endpoints included the change from baseline in patient-reported pain in the index joint at days 2, 3, and 4. Additional exploratory analyses included proportion of patients requiring rescue medication. Blood samples were obtained for analysis of high sensitivity C-reactive protein (hs-CRP), an established marker of inflammation, at baseline and at study visits on days 4, 8, and 31. Safety and tolerability were evaluated based on the incidence of adverse events (AEs) up to and including the safety follow up visit, and the occurrence of clinically significant laboratory findings determined by the investigator. BODY.STATISTICAL ANALYSIS: For this superiority study, a pre-specified step-down sequential testing procedure was used for the primary efficacy endpoint that compared rilonacept alone vs indomethacin alone only if the comparison of rilonacept + indomethacin vs indomethacin alone was statistically significant (P < 0.05). ANCOVA was used for the sequential testing of endpoints with treatment and baseline pain score strata (moderate, or severe or greater) and country as fixed effects. The LOCF was used for imputation of missing data, and pain scores obtained after rescue medication were considered missing for the primary efficacy analysis. A sample size of 75 patients per group was calculated to provide at least 90% power for pairwise comparisons based on the following assumptions: a 1-point improvement from baseline for the control group; a 2-point change in the comparator group, and a constant SD of 0.85, for 2-sided alpha level of 0.05 for the comparisons. All analyses were performed using SAS Version 9 (SAS Institute, Cary, NC, USA). The full analysis set included all randomized patients who received any study medication and had at least one post-baseline assessment, and the safety set included all patients who received any study medication. BODY.RESULTS: Figure 1 shows the distribution and flow through the study of the 225 patients who were randomized to treatment. The demographic and clinical characteristics were generally similar among the treatment groups (Table 1) with a population that was predominantly male (94.1%) and white (75.7%), a mean (SD) age of 50.3 (10.6) years, and a body mass index of 33.1 (6.9), indicating obesity. Mean serum urate was 8.27 mg/dL, 15.8% of patients had visible tophi on examination, and 40.1% of patients had a history of ULT use. Prior use of analgesic medications (paracetamol or acetylsalicylic acid) as reported at baseline was low (8.2%). Figure 1Flow of patients through the study. SC, subcutaneous. Table 1Baseline demographic and clinical characteristics VariableSC placebo + oral indomethacin(n = 75)SC rilonacept + oral indomethacin(n = 74)SC rilonacept + oral placebo(n = 73)Age, years, mean (SD) 51.3 (10.9) 48.6 (10.0) 51.0 (10.8) Gender, n (%)  Male 71 (94.7) 71 (95.9) 67 (91.8)  Female 4 (5.3) 3 (4.1) 6 (8.2) Race, n (%)  White 54 (72.0) 54 (73.0) 60 (82.2)  Black or African American 15 (20.0) 15 (20.3) 11 (15.1)  Asian 5 (6.7) 2 (2.7) 2 (2.7)  Other 1 (1.3) 3 (4.1) 0 BMI, kg/m2, mean (SD) 32.1 (6.3) 33.5 (7.5) 33.7 (6.7) Pain severity, mean (SD)  Likert scale (0 to 4) 2.6 (0.7) 2.7 (0.7) 2.6 (0.7)  Numerical rating scale (0 to 10) 6.8 (2.2) 6.8 (2.0) 6.5 (2.3) Duration of disease, years, mean (SD) 8.8 (6.7) 11.0 (7.9) 10.2 (9.9) Prior number of gout flares per year, mean (SD) 4.8 (5.19) 5.5 (5.26) 5.2 (4.76) Duration of a typical gout flare, days, mean (SD) 5.8 (3.6) 7.1 (4.2) 6.8 (7.3) Tophi present, n (%) 10 (13.3) 12 (16.2) 13 (17.8) Serum uric acid, mg/dL, mean (SD) 7.9 (1.9) 8.2 (2.1) 8.3 (1.7) Prior medication use, n (%)  Urate-lowering therapy 28 (37.3) 34 (45.9) 27 (37.0)  Analgesics (paracetamol or acetylsalicylic acid) 2 (2.7) 9 (12.2) 7 (9.6) BMI, body mass index. All treatment groups were observed to have significant reductions in pain from baseline when averaged at 24, 48 and 72 hours (P < 0.0001) and assessed using the Likert scale. However, the mean reduction in pain with rilonacept plus indomethacin, 1.55 points, was not statistically significantly greater than the mean reduction in pain with indomethacin alone, which was 1.40 points (least squares mean difference -0.14, 95% CI -0.44, 0.15, P = 0.333) (Figure 2A). Since the difference was not significant, formal comparison between pain reduction in the rilonacept monotherapy group, which was 0.69 points, and the indomethacin monotherapy group, which was 1.40 points, was not required. However, a separate ad hoc analysis for this comparison showed that the difference between indomethacin monotherapy and rilonacept monotherapy significantly favored indomethacin (P < 0.0001). Similar results were observed with the NRS; significant reductions from baseline were observed in each of the treatment groups (P < 0.0001), with pain reductions of 3.87 in the rilonacept plus indomethacin group, 4.33 in the indomethacin monotherapy group, and 1.81 in the rilonacept monotherapy group (Figure 2B). While the NRS change with rilonacept plus indomethacin was similar to that of indomethacin alone (P = 0.2533), the ad hoc analysis significantly favored indomethacin monotherapy relative to rilonacept monotherapy (P < 0.0001). Figure 2Change in pain from baseline. Data are mean change in pain of the index joint from baseline to pain averaged for the 24-, 48-, and 72-hour assessments for (A) the primary endpoint using a Likert scale (0 = no pain to 4 = extreme pain), and (B) using a numerical rating scale (NRS; 0 = no pain to 10 = extreme pain). SC, subcutaneous. For the secondary endpoints of mean change in pain at 24, 48, and 72 hours (Figure 3), the trends in pain reduction among the three treatment groups were similar when pain was assessed using the Likert scale and the NRS; no significant differences were observed with rilonacept plus indomethacin relative to indomethacin monotherapy, but indomethacin monotherapy was significantly superior to rilonacept monotherapy at all time points (P < 0.05). Figure 3Mean change in pain of the index joint from baseline at 24, 48, and 72 hours. (A) Likert scale (0 = no pain to 4 = extreme pain). (B) Numerical rating scale (0 = no pain to 10 = extreme pain). SC, subcutaneous. The proportion of patients who reported having taken rescue medication at > 24 to 48 hours in the rilonacept plus indomethacin group (3.0%) was similar to the indomethacin monotherapy group (4.3%). All treatment groups were characterized by a general reduction from baseline in hs-CRP from initiation of treatment to the safety follow up at day 31 (Figure 4). At day 4, these reductions were significantly greater with rilonacept plus indomethacin (P < 0.0001) and rilonacept monotherapy (P = 0.0142) relative to indomethacin monotherapy. Figure 4Serum concentrations of high sensitivity C-reactive protein (hs-CRP) from baseline to day 31 (safety follow up). SC, subcutaneous. Overall, the incidence of AEs was higher in the groups treated with rilonacept relative to indomethacin alone (Table 2); AEs were generally of mild to moderate severity. There were three patients with five serious AEs reported (hypertensive cardiomyopathy, myocardial infarction, ulcerative colitis, tubulointerstitial nephritis, and pyoderma gangrenosum), all in the rilonacept plus indomethacin group, which were not considered by the investigator to be related to treatment with rilonacept. One death was due to hypertensive cardiomyopathy that was not considered by the investigator to be related to treatment with rilonacept. Similar proportions of patients in each treatment group withdrew due to AEs, and the most frequently reported AEs were headache and dizziness, each of which occurred with a similar incidence across the treatment groups (Table 2). Table 2Treatment-emergent adverse events through safety follow up (day 31): incidence ≥ 5% in any treatment group Number (%) of patientsAdverse event (AE)SC placebo + oral indomethacin(n = 77)1SC rilonacept + oral indomethacin(n = 73)1SC rilonacept + oral placebo(n = 75)Any AE 23 (29.9) 34 (46.6) 27 (36.0) Discontinuations due to AE 2 (2.6) 1 (1.4) 1(1.3) Serious AE 0 3 (4.1) 0 Headache 6 (7.8) 4 (5.5) 7 (9.3) Dizziness 4 (5.2) 3 (4.1) 2 (2.7) 1Numbers differ from patient disposition since one patient was administered the wrong drug. BODY.DISCUSSION: Whereas rilonacept has previously demonstrated efficacy in prevention of gout flare during initiation of ULT with allopurinol [34,3538,39] buttresses the current data. Although rilonacept monotherapy was not formally compared with the other treatment groups, the reduction in pain at 24 and 48 hours with this regimen was clearly less than that achieved with the other treatment regimens, and was demonstrated to be significantly inferior to indomethacin monotherapy in the ad hoc analysis. However, improvement in pain at 72 hours for the three groups was similar. This may reflect a delayed effect of rilonacept or could simply reflect the self-limiting natural course of an acute gout flare [40]. Several factors may account for the observed results. In this study, rilonacept was administered within 48 hours of onset of an acute gout flare in patients who were already experiencing substantial pain. First, it is possible that rilonacept may have been more effective had it been administered earlier relative to flare onset, since the ability of IL-1 inhibition to effect a reduction in pain by blocking the cascade of downstream inflammatory mediators may depend on the timing of treatment. Results of a clinical trial with colchicine indicated the success of treating acute gout flares within 12 hours of onset [12]. Secondly, after a single SC injection, Tmax for rilonacept is approximately 48 to 72 hours. Since the primary endpoint was the change from baseline to the average of the pain assessments at 24, 48 and 72 hours, it is possible that drug concentrations at the gouty joint were insufficient during the majority of the primary endpoint assessment period. Furthermore, the size of rilonacept (approximately 250 kilodaltons) may limit the rate of distribution from plasma to target tissues involved in a gout attack. It is possible that, had rilonacept been administered by the intravenous route, higher serum concentrations achieved soon after administration could have driven greater drug penetration to target tissues, and hence, greater efficacy in treating acute gout flares. These data appear to contrast with recent clinical trials of another IL-1 inhibitor, canakinumab, which demonstrated that IL-1 inhibition ameliorated inflammation and pain during an ongoing gout flare as compared to low-dose steroid injections [30,31]. Several reasons should be noted that may account for this difference, including that the canakinumab studies had several clinically relevant differences in study design and study populations. For example, the canakinumab studies were conducted in patients poorly responsive to, or unable to tolerate, NSAID and/or colchicine therapy. The duration of acute gout flare before therapy was allowed to be up to 5 days in each of the canakinumab studies [30,31] compared with only 48 hours in the current study of rilonacept. Prior to enrollment, patients in the current study were also specifically required to have tolerated NSAIDs and to have demonstrated symptomatic relief of gout flare pain with NSAIDs. The active comparator in the canakinumab studies was a corticosteroid, triamcinolone acetonide, administered as a single IM dose of 40 mg. This dose is lower than the single 60 mg IM dose previously suggested as being effective in two small clinical studies in acute gout [41,42], and although it was associated with partial pain relief in acute gout [30,31], the significance of the clinical effect is uncertain in the absence of comparison with placebo, or standard of care (colchicine or NSAIDs). It should also be noted that the indomethacin comparator in the current study has both potent analgesic as well as anti-inflammatory properties. Given the collective distinctions in study designs, the extent of the intrinsic differences that may have contributed to the differing outcomes between the soluble decoy receptor rilonacept and the monoclonal antibody canakinumab are unclear. Differences in efficacy among agents in another class of anti-cytokine therapy, TNF-α antagonists, have been demonstrated to be related to such factors in arthritic diseases other than gout [43,44]. It remains to be addressed whether doses of rilonacept higher than employed in this study, selection of a different patient population, or a different comparator or route of administration (intravenous or intra-articular), might be more effective for treatment of acute gout flares. Interestingly, despite the lack of incremental efficacy with rilonacept for pain reduction compared with indomethacin in an acute gout flare, it should be noted that rilonacept, both as monotherapy as well as in combination with indomethacin, did result in significantly greater early reductions in hs-CRP than indomethacin monotherapy (P < 0.05). These reductions likely reflect IL-1 inhibition by rilonacept, and are consistent with those reported for canakinumab in acute gout flares [30]. Treatment with rilonacept in this clinical setting was generally well-tolerated, with the frequency of withdrawals due to AEs similar across treatment groups. Although the combination therapy group had a higher incidence of serious AEs relative to both of the monotherapy groups, these AEs were not considered related to rilonacept. BODY.CONCLUSIONS: In conclusion, in contrast with previous studies that have demonstrated efficacy of rilonacept for prevention of acute gout flares in patients initiating ULT, addition of rilonacept to an indomethacin treatment regimen and use of rilonacept alone provided neither significant additional pain relief nor superior pain relief, compared with indomethacin alone over the 72-hour period after treatment initiation in acute gouty arthritis. BODY.ABBREVIATIONS: AE: adverse event; hs-CRP: high sensitivity C-reactive protein; IL-1: interleukin-1; IM: intramuscular; MSU: monosodium urate; NSAID: non-steroidal anti-inflammatory drug; SC: subcutaneous; TNF: tumor necrosis factor; ULT: urate-lowering therapy. BODY.COMPETING INTERESTS: RT has consulted for Regeneron, Takeda, Savient, Novartis, Prescription Solutions, Pfizer, Ardea, BioCryst, and Metabolex. HRS has consulted for Regeneron, Novartis, Ardea, Pfizer, Metabolex, and Westward, and has received a grant from Takeda. JC has consulted for Regeneron. HB has been an investigator for Regeneron, Ardea, Novartis, Takeda, Metabolex, and Savient, has consulted for Regeneron, Ardea, Takeda, and Savient, and has spoken for Takeda, and Savient. RE, JW, SK, and SW are employees and stock holders of Regeneron Pharmaceuticals, Inc. Regeneron holds patents related to the content of the manuscript. BODY.AUTHORS' CONTRIBUTIONS: RT, HRS, RE, JW, SK, SW made substantial contributions to the conception and design of the clinical trial. JC and HB were involved in the acquisition of data. All authors were involved in the analysis and/or interpretation of data. All authors were involved in drafting the manuscript and revising it critically for important intellectual content. All authors read and approved the final manuscript.

TITLE: Laryngoscope and a New Tracheal Tube Assist Lightwand Intubation in Difficult Airways due to Unstable Cervical SpineA New Tracheal Tube Assists Lightwand Intubation in Difficult Airway ABSTRACT.PURPOSE: The WEI Jet Endotracheal Tube (WEI JET) is a new tracheal tube that facilitates both oxygenation and ventilation during the process of intubation and assists tracheal intubation in patients with difficult airway. We evaluated the effectiveness and usefulness of the WEI JET in combination with lightwand under direct laryngoscopy in difficult tracheal intubation due to unstable cervical spine. ABSTRACT.METHODS: Ninety patients with unstable cervical spine disorders (ASA I-III) with general anaesthesia were included and randomly assigned to three groups, based on the device used for intubation: lightwand only, lightwand under direct laryngoscopy, lightwand with WEI JET under direct laryngoscopy. ABSTRACT.RESULTS: No statistically significant differences were detected among three groups with respect to demographic characteristics and C/L grade. There were statistically significant differences between three groups for overall intubation success rate (p = 0.015) and first attempt success rate (p = 0.000). The intubation time was significantly longer in the WEI group (110.8±18.3 s) than in the LW group (63.3±27.5 s, p = 0.000) and DL group (66.7±29.4 s, p = 0.000), but the lowest SpO2 in WEI group was significantly higher than other two groups (p<0.01). The WEI JET significantly reduced successful tracheal intubation attempts compared to the LW group (p = 0.043). The severity of sore throat was similar in three groups (p = 0.185). ABSTRACT.CONCLUSIONS: The combined use of WEI JET under direct laryngoscopy helps to assist tracheal intubation and improves oxygenation during intubation in patients with difficult airway secondary to unstable spine disorders. ABSTRACT.TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR-TRC-14005141 BODY.INTRODUCTION: Patients with cervical spine instability usually present with limited range of neck motion and a challenged airway for tracheal intubation. The operators (more and more emergency department and ICU doctors do the intubation) for tracheal intubation must consider two potentially competing factors, securing the airway while minimizing neck motion. Manual in-line stabilization (MILS) is recommended during direct laryngoscopy and intubation in patients with known or suspected cervical spine instability [1]. However, MILS often worsens direct laryngeal visualization and therefore raises possibility of difficult tracheal intubation [2]. The lightwand is a simple technique that has been proven to be safe and effective in difficult intubation [3–5]. It is usually difficult to intubate patients with unstable cervical spines by direct laryngoscopy. It was recommended the using of the lightwand in a presumed unstable cervical spine injury over the Macintosh laryngoscope [6]. A special tracheal tube designed by Huafeng Wei named WEI Jet Endotracheal Tube (WEI JET; Wei Medical LLC, Cherry Hill, NJ) applies supraglottic jet oxygenation and ventilation (SJOV) during tracheal intubation and assists tracheal intubation blindly in patients with difficult airway [7].We hypothesized that combined use of lightwand and WEI JET under direct laryngoscopy will maintain oxygenation/ventilation during tracheal intubation and assist tracheal intubation in patients with difficult airway due to unstable cervical spine. Thus, we compared the effectiveness and complication of tracheal intubation using regular endotracheal tube and lightwand only, regular endotracheal tube with lightwand under direct laryngoscope, and WEI JET with lightwand under direct laryngoscopy. BODY.MATERIALS AND METHODS: BODY.ETHICS STATEMENT: This study was approved by the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine. Written informed consent was obtained from all patients. BODY.PATIENTS AND STUDY DESIGN: Ninety patients with unstable cervical spine disorders (ASA I-III) under general anaesthesia were enrolled from July 2014 to September 2014. Patients with the following types of condition were excluded: age <18 years; had risk of gastric aspiration; increased intracranial pressure; obesity (BMI> 35); relevant drug allergy; abnormalities of the upper airway, polyps, tumors, abscesses, inflammation; or had foreign bodies in the upper airway. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see S1 CONSORT Checklist and S1 Protocol. Patients recruited in this study were randomly assigned by computer-generated randomization schedule. There groups were the LW group (intubations were performed only by lightwand and regular endotracheal tube), the DL group (lightwand used in conjunction with regular endotracheal tube under direct laryngoscopy), and the WEI group (lightwand used in conjunction with WEI JET under direct laryngoscopy). Each patient was placed in supine position with a neutral head position. MILS was created with a cervical immobilization collar (Stifneck Select, Leardal Medical GmbH, Germany). The appropriate size of MILS was determined by finger-sizing method following the manufacturer’s instructions. This device effectively downsizes mouth opening and limits neck extension. Patients were given 5 min of pre-oxygenation before anesthesia induction. Non-invasive blood pressure, SpO2, ECG were monitored and recorded every minute. Anaesthesia was induced with propofol target control infusion (4μg.ml−1) and remifentanil (3.5ng.ml−1) followed by succinylcholine (1mg.kg−1) [8]. After anesthesia induction, direct laryngoscopy was performed with a Macintosh size 3 blade, the trachea was intubated by one of four anesthesiologists, who was experienced with jet ventilation and had completed at least 50 successful tracheal intubations using lightwand before the study. The glottis view was described by Cormack and Lehane (C/L) and divided into four grades (1 = full view of the glottis; 2 = partial view of the glottis; 3 = only the epiglottis visible; and 4 = neither epiglottis nor glottis visible.), without applying external laryngeal pressure. More than two attempts required would be defined as a failure intubation. Then, the airway was managed by following the anesthesiologist’s former experience. In the LW group, tracheal intubations were performed only by using lightwand in which introduced a regular tracheal tube (7-mm ID tube in women, and an 7.5-mm ID tube in men). The tip of the tube/lightwand combination was bent to a 90° angle [9]. Room lights were dimmed, and the combination was introduced into the oral cavity and repositioned in the midline until its entry into the oropharynx. When the tip of lightwand was placed inside the glottis, a well-defined circumscribed glow could be seen in the anterior neck [10]. If esophageal intubation happened, the transmitted glow was diffused and the procedure was tried again. After removed the lightwand, proper tracheal tube placement was confirmed by end-tidal carbon dioxide monitoring. In the DL group, tracheal intubation was performed with lightwand in combination with regular endotracheal tube under direct laryngoscopy. A Macintosh size 3 blade was used for lightwand intubations. The blade was held in the non-dominant hand before insertion of the tracheal tube-lightwand combination. Tracheal intubation was performed under direct vision of the glottis (C/L Grade 1 or 2). For patients with Grade 3 glottis view, the lightwand was passed underneath the epiglottis. For Grade 4, tracheal intubation was accomplished with midline technique [11–12]. When an optimal transillumination was obtained, tracheal intubation was achieved. If transillumination was not seen in larynx, the procedure was tried again. In the WEI group, the WEI JET took the place of regular tracheal tube which combined with the direct laryngoscopy and lightwand. As shown in Fig. 1, The WEI JET contained two additional parts compared to a regular endotracheal tube: a catheter for end-tidal CO2 pressure (PetCO2) monitoring and a jet catheter for ventilation. The jet catheter with an ID 2.0 mm was built into the anterior wall of WEI JET. Jet ventilation was performed using a manual jet ventilator (Manujet III, VBM Medizintechnik GmbH, Germany) was connected to the proximal end of jet catheter. The operator adjusted the distal tip of the lightwand until the midline illumination was observed in the anterior neck, the maximum stable PetCO2 and chest rise were achieved [7–8]. The WEI JET was then slid into the trachea over the lightwand and the PetCO2 monitoring catheter. 10.1371/journal.pone.0120231.g001Fig 1BODY.ILLUSTRATION OF COMPONENTS AND ASSEMBLY OF THE WEI JET IN COMBINATION WITH LIGHTWAND.: Awake fiberoptic intubation has been recommended for unanticipated difficult airway [13]. Although experienced anesthesiologists make the procedure seem simple, fiberoptic intubation is a difficult technique to learn and master, and if rarely used, competence is difficult to maintain [14]. In addition, it requires patient cooperation and ready-to-use devices, which cannot be achieved sometime. This technique requires more time than direct laryngoscopy, even when performed by an expert [15]. Thus, alternative management techniques should be available. The primary endpoints were the rate of successful intubation and the time taken to intubate. The intubation time was defined as from insertion of the intubation device between the teeth to the first breath of controlled mechanical ventilation and detection of positive PetCO2. The secondary endpoints were the number of intubation attempts and the lowest SpO2 during intubation. After the procedure, patients were asked about the severity of sore throat using a VAS (0 = no pain, 10 = worst imaginable pain) at discharge from the post-anesthesia recovery room. BODY.STATISTICAL ANALYSIS: The sample size was calculated based on the hypothesis that a combination of WEI JET, lightwand and direct laryngoscope will increase intubation successful rate during the intubation. The sample size was also calculated based on a pilot study of 30 patients.We calculated that the overall intubation success rate was 80% for lightwand, 90% for direct laryngoscopy in combination with lightwand and 100% for the WEI JET. Based on these figures, accepting a two-tailed α error of 5% and β error of 20%, we calculated that 27 patients would be required for each group. Assuming the potential for patients to drop out of the study, the total sample size was increased to 90 patients. All data were reported as mean ± SD, absolute number (n), or percentages. Statistical analyses were performed with the Statistical Package for Social Sciences Software (SPSS 18.0 for windows; SPSS Inc., Chicago, IL, USA). The intubation time, lowest SpO2 during intubation, VAS score and number of intubation attempts were analysed using ANOVA for three group comparisons. Bonferroni correction was performed between-group comparisons, as appropriate. Kruskal-Wallis was used to analyse C/L grade. Fisher’s exact test was used to analyse rate of successful intubation. A P-value < 0.05 was considered to be statistically significant. BODY.RESULTS: A total of 90 patients were recruited into the study. No statistically significant difference was detected between groups in demographic characteristics, glottis view grade under direct laryngoscopy (Table 1). Patients were not conditioned with significant respiratory disease which might contribute to SpO2 baseline. 10.1371/journal.pone.0120231.t001Table 1BODY.CHARACTERISTICS OF PATIENTS IN GROUP LW, GROUP DL AND GROUP WEI.: CharacteristicGroup LWGroup DLGroup WEIP valuePatients,n 24 29 30 - Male, n (%) 8(33.3) 9(31.0) 9(30.0) 0.460 Age (yrs) 42.3±16.2 37.9±12.1 40.9±11.1 0.268 Weight (kg) 58.5±10.4 61.8±8.7 60.6±6.9 0.224 Height (cm) 162.0±7.0 164.8±7.9 163.7±6.7 0.268 BMI (kg/m2) 22.2±3.1 22.7±2.8 22.6±2.2 0.526 Mouth opening (cm) 2.8±0.33 3.0±0.37 2.9±0.33 0.450 C/L Grade, n (%) 0.551 1 0(0) 0(0) 0(0) - 2 3(12.5) 3(10.3) 2(6.7) - 3 21(87.5) 26(89.7) 27(90) - 4 0(0) 0(0) 1(3.3) - Patients in WEI group were successfully intubated on the first attempt. In contrast, five patients needed second attempt in the LW group and four in the DL group. Patients in all groups were successfully intubated except for six patients who were excluded due to intubation failure in the LW group and one in the DL group. There were statistically significant differences between three groups for overall intubation success rate (p = 0.015) and first attempt success rate (p = 0.000). With regard to the overall intubation success rate and first attempt success rate, there were significant difference between LW group and WEI group (p = 0.024, p = 0.000, respectively)(Table 2). 10.1371/journal.pone.0120231.t002Table 2BODY.COMPARISON OF MONITORED VALUES IN GROUP LW, GROUP DL AND GROUP WEI.: ParameterGroup LWGroup DLGroup WEIp(overall)n = 24n = 29n = 30Overall intubation success rate, n (%) 24(80) a 29(96.7) b 30(100)* 0.015 Success at first attempt, n (%) 19(63.3) a 25(83.3) b 30(100)** 0.000 Intubation time (s) 63.3±27.5 66.7±29.4 110.8±18.3** †† 0.000 Number of insertion attempts 1.21±0.4 1.14±0.4 1.0±0* 0.041 Lowest SpO2 during intubation (%) 96.6±2.8 98.4±2.1** 100±0** †† 0.000 Sore throat,VAS score 2.0±0.8 2.3±0.7 2.4±0.6 0.185 aIntubation success rate: calculated using total randomized number of patients in group LW (n = 30), including the six patients in whom intubation failed after two attempts. bIntubation success rate: calculated using total randomized number of patients in group DL (n = 30), including the one patient in whom intubation failed after two attempts. Significant comparisons are detailed using the following footnotes: *p<0.05 **p<0.01 compared with Group LW † p<0.05 †† p<0.01 compared with Group DL. Although the intubation time was significantly longer in the WEI group (110.8±18.3 s) than the LW group (63.3±27.5 s, p = 0.000) or DL group (66.7±29.4 s, p = 0.000), the lowest SpO2 in WEI group was significantly higher than other two groups. The WEI JET significantly reduced successful tracheal intubation attempts compared to the LW group (1.0±0 VS. 1.21±0.4, p = 0.043). There was no difference in time taken for intubation and number of intubation attempts between LW group and DL group (Table 2). There was no incidence of dental trauma in either group and the severity of sore throat was similar among three groups (p = 0.185) (Table 2). BODY.DISCUSSION: When a difficult airway is expected or unstable cervical spine is suspected, awake flexible fiberoptic intubation with awake positioning is recommended as a safe and sure way of airway control. However, awake fiberoptic intubation usually requires patient cooperation [16]. The failure rate at the first attempt is high (approximately 53.3%), which means it requires great skills acquired from extensive experience [17]. In addition, it may be unrealistic in emergency situations due to the longer time required for intubation using awake fiberoptic approach. Considering all these shortcomings of awake fiberoptic intubation, new techniques or approaches are needed to manage the challenging tracheal intubation in patient with unstable cervical spine, especially in the emergent situation. Neurologic injury during intubation may happen because of cervical spine movement in these patients [18–19]. Lightwand was recommended for its simple but effective procedure and less linear motion compared to direct Macintosh laryngoscopy [20]. This study demonstrated that glottic visualization failed (C/L grade 3 or 4) in 90% patients with the cervical immobilization under direct laryngoscopy. The overall intubation success rate was only 80% (24/30) in the LW group, being a semi-blind technique [12]. One study including 950 patients showed that all lightwand failures were resolved with direct laryngoscopy. Similarly, all failures of direct laryngoscopy were resolved with lightwand [5 The epiglottis is always in contact with the posterior pharyngeal wall and consequently makes passing underneath the epiglottis is difficult for the lightwand [21]. When using direct laryngoscopy to aid lightwand, the epiglottis was lifted off the posterior pharyngeal wall and consequently leads to unhindered passage of lightwand. Biehl and Bourke revealed that the lightwand could improve the view in the hypopharynx, and transillumination could assist a blind tracheal intubation with direct laryngoscopy [22]. Despite the above mentioned advantage on combination of lightwand with direct laryngoscope, there is no approach of oxygenation and ventilation during the process of tracheal intubation. Utilizing the open feature of jet ventilation, the striking advantage of WEI JET is to provide supraglottic or infraglottic jet oxygenation and ventilation during the process of tracheal intubation under direct laryngoscopy and facilitate tracheal intubation in grade III glottis view [7,23]. This new technique obviously can prolong the period of tracheal intubation without hypoxia, minimizing hypoxia related complications. Indeed, we demonstrated that all 30 patients were successfully intubated at fist time using WEI JET and lightwand guidance under direct laryngoscopy. In comparison with other two groups, the WEI JET reduced the number of tracheal intubation attempts, significantly, with significantly increased successful rate and decreased risk of hypoxia. Although many new airway devices have been developed that can be used for difficult tracheal intubation. However, few devices can provide oxygenation and ventilation during the process of tracheal intubation. The WEI JET is a special tracheal tube which can supply oxygenation during intubation without the need of mask ventilation and assist tracheal intubation blindly. It had been demonstrated that the SaO2 of apnoeic pig was maintained above 95% for at least 20 min without mask ventilation in a previous animal study. In this study, breath sounds, chest rise, and monitoring of PetCO2 helped guide blind intubation [23]. In addition, the direct laryngoscopy could improve the view in the hypopharynx, and could assist in guiding the tip of the lightwand to pass underneath the epiglottis. This combined approach may be particularly useful when an unanticipated C/L grade 3 or 4 laryngoscopic view is encountered. It can be assumed that using both instruments together would lead to a higher success rate of tracheal intubation with reduced hypoxia-related complications, should one device alone fail. For example, the lightwand has been combined successfully with other intubating techniques including intubation through a laryngeal mask airway (LMA) [24–25]. Although the WEI JET took longer time for tracheal intubation than the lightwand in the study, but the ability to provide oxygenation/ventilation make the tracheal intubation smoother and safer. We found no difference in airway complications among all three groups. There are still limitations in our study. First, our study is lack of a group which the WEI JET combined with direct laryngoscopy only, which means without lightwand. However, previous studies have demonstrated the advantages of WEI JET under direct laryngoscopy [23]. Second, observation bias may exist in the recordings for the unblinded design of this study. Third, all tracheal intubations were performed by an experienced anesthesiologist who is familiar with these techniques, therefore our results may not be suitable to inexperienced person. BODY.CONCLUSIONS: The WEI JET combined with lightwand under direct laryngoscopy can help to assist tracheal intubation and improve oxygenation during tracheal intubation in patients with difficult airway secondary to unstable cervical spine. BODY.SUPPORTING INFORMATION: S1 ProtocolBODY.THE TRIAL STUDY PROTOCOL IN ENGLISH.: (DOC) Click here for additional data file. S2 ProtocolBODY.THE TRIAL STUDY PROTOCOL IN CHINESE.: (DOC) Click here for additional data file. S1 CONSORT ChecklistBODY.THE CONSORT CHECKLIST.: (DOC) Click here for additional data file. S1 DatasetBODY.THE ORIGINAL DATA OF THE THREE GROUPS.: (XLS) Click here for additional data file. S1 FigBODY.THE CONSORT FLOWCHART.: (TIF) Click here for additional data file.

TITLE: The effect of alpha-linolenic acid supplementation on ADHD symptoms in children: a randomized controlled double-blind study Background: Attention deficit-hyperactivity disorder (ADHD) is the most common neuro-developmental disorder in childhood. Its pharmacologic treatment mostly includes methylphenidate, yet many parents seek alternative, “natural,” therapeutic options, commonly omega-3 fatty acids. Previous studies of supplementation with fish oil or long-chain omega-3 fatty acids to children with ADHD yielded mixed results. The use of alpha-linolenic acid (ALA), a medium-chained, plant-based omega-3 fatty acid (18:3 n-3), has not been sufficiently examined in this population. Methods: Forty untreated children with ADHD, aged 6–16 years, were randomized to receive either 2 g/day of oil containing 1 g ALA or placebo, for 8 weeks. Before and after supplementation, the children underwent a physician assessment of ADHD symptoms and a computerized continuous performance functions test. The children’s parents and teachers filled out Conners’ and DSM questionnaires. Results: Seventeen (42.5%) children completed the study, eight in the supplementation group, nine in the placebo group. Main drop-out reasons were capsule size, poor compliance, and a sense of lack of effect. No significant difference was found in any of the measured variables tested before and after supplementation, in both study groups. No between-group difference was found in the changes of the various measures of ADHD symptoms throughout the study period. Conclusion: Supplementation of 2 g/day of oil containing 1 g ALA did not significantly reduce symptoms in children with ADHD. Future studies in this field should consider an alternative method to deliver the oil, a higher dose, and a larger sample size. BODY.INTRODUCTION: Attention-deficit hyperactivity disorder (ADHD) is a childhood onset disorder with a relatively high global prevalence, ranging from 2.2–17.8% (Skounti et al., 2007). ADHD is considered the most common neuro-behavioral disorder of childhood, and one of the most prevalent chronic health conditions affecting school-aged children (Wolraich et al., 2011). The pharmacologic treatment of ADHD mostly includes methylphenidate (Wolraich et al., 2011). Surprisingly, one study from Israel found that the prevalence of methylphenidate prescription use in the population was only 2.5% – a much smaller rate than the estimated prevalence of ADHD in Israel (Vinker et al., 2006). A possible explanation as to why a large proportion of children with ADHD are not adequately treated could be the stigmatization of methylphenidate. Indeed, many parents continuously seek alternative, “natural,” therapeutic options other than methylphenidate (Berger et al., 2008). Omega-3 fatty acids are among the most common dietary supplements used in children with ADHD. The basis for this treatment stems from studies that identified low levels of omega-3 fatty acids in plasma phospholipids or red blood cell membranes of children with ADHD (Mitchell et al., 1987; Stevens et al., 1995; Antalis et al., 2006). Several trials of supplementation with fish oil or long-chain omega-3 fatty acids [mostly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] to children with ADHD have been conducted, and yielded mixed results. Systematic reviews and meta-analyses on this topic summarized that there is currently no consensus that omega-3 fatty acids influence ADHD symptoms (Richardson, 2006; Chalon, 2009; Raz and Gabis, 2009; Bloch and Qawasmi, 2011; Gillies et al., 2012). One possible mechanism suggested for the common lack of effect is a relatively poor incorporation of these fatty acids into the brain. In a pioneering study, Vaisman et al. (2008) showed that consumption of EPA and DHA that were incorporated into phospholipids, resulted in higher circulating levels and better executive functioning in children with ADHD, as compared with fish oil. The authors identified significant correlations between the chemical changes and the clinical effects. Another supplementation study with long chain omega-3 fatty acids conjugated to phosphatidylserine, in order to improve their absorption, also showed improved behavior of children with ADHD (Manor et al., 2012). Fewer trials examined the effect of the plant-based alpha-linolenic acid (ALA), the parent medium-chain omega-3 fatty acid, on ADHD symptoms. In one published study, 200 mg ALA administered as flaxseed oil together with vitamin C were given to children with ADHD (Joshi et al., 2006). The authors noted increased levels of circulating EPA and DHA, with improvements in parent-rated ADHD symptoms of impulsivity, restlessness, inattention, and self-control. Yet the lack of control group in this study, the relying on parent report only, and the concomitant addition of vitamin C, prevent from drawing clear conclusions on the effect of ALA alone. In another study, children with ADHD received either a supplement containing 120 mg of ALA and 480 mg of linoleic acid and, or vitamin C as placebo, for 7 weeks (Raz et al., 2009). Treatment effects were measured using questionnaires and a computerized test of attention, and did not differ significantly between groups. Of note, these authors administered a relatively low dose of ALA, while also supplementing with a higher dose of the parent fatty acid of the omega-6 family which could inhibit conversion of ALA to the long chain omega-3 fatty acids through competition on the same enzymes. It still remains unknown whether supplementation of a higher dose of ALA could affect ADHD symptoms more than placebo. The aim of the current study was to examine if supplementation with an ALA-rich sage oil can improve symptoms in children and adolescents diagnosed with ADHD. BODY.MATERIALS AND METHODS: BODY.PARTICIPANTS: The study population included 40 children and adolescents aged 6–16 years, recently diagnosed with ADHD, who were drug naïve and untreated, from two ambulatory ADHD specialty clinics in Israel. Exclusion criteria were refusal to undergo any or all of the testing procedures or to take the designated supplement; a history of chronic health conditions other than ADHD; or use of any chronic medications or dietary supplements, specifically methylphenidate or fatty acid/fish oil supplements. The study was approved by the Institutional Review Board of Hadassah Medical Center, Jerusalem, Israel, conducted according to the Declaration of Helsinki, and registered in a clinical trials registry before recruitment (#NCT00874536). At least one parent signed an informed consent form, and each participant verbally agreed to participate. BODY.INTERVENTION: The study participants were randomized 1:1 to receive either 2 g/day of sage oil or an identical appearing lactose placebo in gel capsules. Participants were instructed to consume two gel capsules daily. The composition of sage oil varies slightly by crop year, and is 50–54% ALA, 20–23% oleic acid, 16–18% linoleic acid, 6–7% palmitic acid, and 2–3% stearic acid (Tulukcu et al., 2012). This corresponds to a supplementation of 1 g/day of ALA, a dose that had been shown previously in adults to elevate the concentrations of omega-3 fatty acids (Barceló-Coblijn et al., 2008). The supplementation period lasted 8 weeks. BODY.RANDOMIZATION: Both types of capsules were supplied in identical amounts in solid plastic bottles. The bottles were numbered consecutively and coded by a person uninvolved in the study, and each participant received three bottles that contained all pills necessary for the study duration. Each ADHD clinic received half of the bottles, numbered consecutively. The children that agreed to participate in the study received their designated bottles in consecutive order. All study participants, parents, teachers, and study personnel were blinded to the allocation until completion of all data collection. BODY.OUTCOMES AND DATA COLLECTION: The primary outcomes at study end were ADHD symptoms, as assessed by validated questionnaires and a computerized continuous performance test (CPT, see below). Each child met the criteria for ADHD according to Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV-TR criteria (American Psychiatric Association, 2000), as assessed by a physician certified in ADHD diagnosis and treatment. The diagnostic procedure included an interview with the child and parents, filing of DSM-based ADHD diagnostic questionnaires, and a medical and neurological examination. Parents and teachers filled the appropriate Conners’ rating scales (Conners, 1997a,b). We used the MOXO-CPT, which is a standardized computerized test designed to diagnose ADHD-related symptoms (Berger et al., 2009). The total duration of the test is 15 min, and it is composed of eight levels of 53 trials each. In each trial, a stimulus in the form of a cartoon picture (designated as “target” or “non-target”) is presented for 500, 1000, or 3000 ms, followed by a “void” period of the same duration, during which the tested individual should respond by pressing the keyboard “space” bar as quickly as possible. In each level, 33 target and 20 non-target stimuli are presented. The tested participant is instructed not to respond to any other stimuli except the target, and not to press any other key but the space bar, and only once. The response timing and accuracy is measured after each stimulus. The test includes distracting stimuli that are presented to the tested participant. These distractors are short animated video clips, containing visual, and auditory features. Six different distractors are included, each of them could appear as only visual, only auditory, or as a combination of both. Each distractor is presented for a different duration ranging from 3.5–14.8 s, with a fixed interval of 0.5 s between two distractors. Distractor onset is not synchronized with target/non-target onset, and could therefore appear either during the stimulus events or during the void period. The burden of the distracting stimuli increases during the test. The MOXO-CPT measures four performance indices: attention, timing, impulsivity, and hyperactivity. The attention index corresponds to the number of correct space bar keystrokes in response to a target stimulus. This index is considered a pure measure of sustained attention, because it measures correct responses independently of the response time. The timing index is the number of correct responses given quickly, still within the period that the target stimulus is present on screen. The impulsivity index is the number of responses performed following a non-target stimulus. The hyperactivity index is the total number of commission responses that are not coded as impulsive responses (e.g., multiple keystrokes in response to a target stimulus, responses performed in the void period after a non-target stimulus, random key pressing). Other measures of the MOXO-CPT are described in detail elsewhere (Berger et al., 2013; Cassuto et al., 2013). The same assessment tools were used after 8 weeks of supplementation in both groups. At this final assessment visit, missing pills in the bottles were counted, to assess adherence. BODY.STATISTICAL ANALYSIS: The differences in questionnaire scores and CPT results between baseline to end-of-study values within each group were compared by the Wilcoxon signed-ranks test. The differences in clinical data, questionnaire scores, and CPT results between study groups at baseline and at trial end were compared by the Mann–Whitney U Test. The between-group differences in the changes of questionnaire scores and CPT results were also compared by the Mann–Whitney U Test. Proportions were compared using Fisher’s exact test. Statistical significance was defined as a two-sided p-value <0.05. BODY.RESULTS: Twenty children were included in each group, and received their designated supplement. After 8 weeks, only 17 participants remained in the study, and underwent the post-supplementation assessment: nine in placebo group (six males, three females, mean age 10.9 ± 2.3 years) and eight in the oil group (four males, four females, mean age 11.1 ± 3.0 years). Reasons for dropout were difficulty to take the capsules due to size or taste (n=7), a subjective sense of lack of effect (n = 4), loss of contact (n = 5), or lack of interest to perform the second assessment (n = 7). None of the participants complained about the most common intolerance to fish oils, namely a “fishy” smell and aftertaste. BODY.PARENT QUESTIONNAIRES: Table 1 presents data from parent questionnaires, regarding their perception of their children’s behavior, before and after the supplementation period. There were significant differences between study groups at baseline in two of the three scales, indicating worse ADHD behavior in the omega-3 group. There were also significant differences between groups at trial end in all scales, again in the direction of worse behavior. However, there were no significant between-group differences in the changes from pre- to post- supplementation values in any of the measured parameters. Table 1Data from parent Conners’ questionnaires at baseline and study end in both groups. Placebo (n = 9)Omega-3 (n = 8)p-value between changes***PrePostp-valuePrePostp-valueParent Conners’ ADHD Index 62[47–70] 62[46–64] 0.19 76*[71–90] 79**[54–89] 0.68 0.79 Parent Conners’ Global Index 68[49–80] 71[42–76] 0.50 85*[78–90] 77**[72–90] 0.46 0.42 Parent Conners’ DSM-IV: total 65[50–79] 62[45–69] 0.04 78[63–89] 87**[64–90] 0.89 0.22 Data is presented as median and [range].*Significantly different (p< 0.05) from the corresponding baseline value of the placebo group.**Significantly different (p< 0.05) from the corresponding post-supplementation value of the placebo group.***p-value of the comparison between the pre-post changes in each group. BODY.TEACHER QUESTIONNAIRES: Table 2 presents data from teacher questionnaires, regarding their perception of the children’s classroom behavior, before and after the supplementation period. There were no significant differences between study groups at baseline or at trial end in any of the tested parameters. Additionally, there were no significant between-group differences in the changes from pre- to post- supplementation values in any of the measures. Table 2Data from teacher Conners’ questionnaires at baseline and study end in both groups. Placebo (n = 9)Omega-3 (n = 8)p-value between changes*PrePostp-valuePrePostp-valueTeacher Conners’ ADHD index 59[59–75] 61[59–69] 0.27 69[53–89] 69[58–87] 0.67 0.26 Teacher Conners’ Global Index 63[60–64] 64[60–85] 0.65 65[58–68] 72[56–90] 0.25 0.76 Teacher Conners’ DSM-IV: total 62[57–75] 61[57–67] 0.18 63[46-90] 66[56–90] 0.28 0.17 Data is presented as median and [range].*p-value of the comparison between the pre-post changes in each group. BODY.DSM CRITERIA: Scores of DSM-criteria questionnaires also did not differ between placebo and omega-3 groups. Respective scores at baseline were 11 (range 6–17) and 13 (range 6–17), p = 0.29, with no measurable change in any of the values at trial end [11 (range 6–17) and 13 (range 6–17), p = 0.47]. BODY.COMPUTERIZED CONTINUOUS PERFORMANCE TEST: Table 3 presents data from the MOXO-CPT that the children undertook before and after the supplementation period. There were no significant differences between study groups at baseline or at trial end in any of the tested parameters. Additionally, there were no significant between-group differences in the changes from pre- to post- supplementation values in any of the measures. Table 3Data from the MOXO performance test at baseline and study end in both groups. Placebo (n = 9)Omega-3 (n = 8)p-value between changes*MeasurePrePostp-valuePrePostp-valueTiming 167[114–253] 158[54–208] 0.87 125[23–194] 178[130–214] 0.14 0.20 Reaction time 0.47[0.39–0.60] 0.54[0.10–0.65] 0.50 0.54[0.05–0.67] 0.52[0.42–0.66] 0.89 0.88 Impulsivity 14[2–130] 7[1–96] 0.73 7[0–23] 21[5–157] 0.14 0.20 Attention 230[166–264] 244[58–263] 1.00 178[24–258] 242[196–256] 0.22 0.53 Hyperactivity 48[7–293] 29[0–435] 0.50 20[4–151] 61[10–569] 0.35 0.27 Data is presented as median and [range].*p-value of the comparison between the pre-post changes in each group. BODY.DISCUSSION: The aim of this study was to examine if supplementation with an oil rich in ALA can improve behavior and function in children and adolescents with ADHD. Using several validated questionnaires and a CPT, we found no evidence for a significant effect in any direction. Although we did identify significantly higher post-supplementation scores in the parent Conners’ questionnaires in the omega group, these were attributed to baseline differences; there were no significant differences between the changes in these questionnaire scores between groups. A decrease in the parent Conners’ DSM-IV questionnaire score was seen in the placebo group only, with no other index of improvement in any other measure in this group. We believe that this does not truly reflect clinical improvement, and is probably a random finding resulting from multiple comparisons. Several previous studies of fish oil/long chain omega-3 supplementation to children with ADHD have been performed, with recent meta-analyses showing no significant clinical effect (Bloch and Qawasmi, 2011; Gillies et al., 2012). It should be noted that one meta-analysis on this topic did identify a small, statistically significant effect – but which is clinically much lesser than that obtained by methylphenidate and other medications (Bloch and Qawasmi, 2011). Therefore, from a clinical point of view, there is currently no evidence to support choosing omega-3 fatty acids over methylphenidate for ADHD treatment. The authors explained the discrepancy between their own findings and the null effect seen in most individual trials, by the small sample sizes used in the single studies. They calculated that in order to obtain sufficient statistical power to identify the small effect of omega-3 in ADHD compared to placebo, clinical trials would require a sample of approximately 330 children. The authors stated that the omega-3 fatty acid supplementation trials examining childhood ADHD used 26–117 participants only. Conversely, the Cochrane meta-analyses (Gillies et al., 2012), which included 13 trials with 1011 participants overall, concluded that there is little evidence that omega-3 fatty acid supplementation provides any benefit for the symptoms of ADHD in children and adolescents. To our knowledge, only two studies that utilized plant-based omega-3 fatty acids in ADHD were published to-date (Joshi et al., 2006; Raz et al., 2009). Both used relatively small amounts of ALA (200 and 120 mg, respectively). In the study by Joshi et al. (2006), increased levels of circulating EPA and DHA were found, demonstrating that even a much lower dose than that used in our study (yet when combined with vitamin C), is sufficient to increase concentrations of omega-3 fatty acids in the body. The improvement seen in most measures in that study, such as total hyperactivity score, restlessness, impulsiveness, and inattentiveness, was very large, around 1 SD. Nevertheless, the lack of placebo-control group and the addition of vitamin C, prevent from isolating the individual effect of ALA per se. Collectively, there is still a wide gap in our knowledge regarding the effects of omega-3 fatty acids in ADHD, which is even greater in the field of plant-based ALA. The main reasons are the large variations in the types of fatty acids used (omega-3 with or without omega-6, fish- or plant-based oils, etc.), in their dosages, in the proportions between EPA and DHA, and in the different characteristics and numbers of participants in each study. We recognize that our trial has several limitations. Firstly, we had a relatively small sample size with a high dropout rate, attributed both to the reported inconvenience of consuming 2 g of oil per day, and to the subjective feeling of a lack of effect by the participants or their parents. Recruiting a larger sample size of newly diagnosed, non-medicated children for such a trial may be a challenge, as many parents do request an immediate and efficient treatment once their child is initially diagnosed with ADHD. Secondly, we chose a wide age range of 6.5–16 years, which might have caused a wide variation in test results, especially in the computerized test. Baseline data of the DSM questionnaire also showed a wide range of disturbance, as reflected by the range of DSM scores seen. We a priori chose a wide age range and included participants from both sexes in order to increase generalizability, yet eventually this might have been a drawback. Lastly, the dose used might have still been too low to increase the amount of brain EPA and DHA. While this dose has been shown to elevate omega-3 fatty acids concentrations in red blood cells in adults (Barceló-Coblijn et al., 2008), we would expect at least the same effect in children and adolescents; however, examining the brain content of these fatty acids in children following supplementation is currently not possible. The strengths of the study were its randomized, placebo-controlled design; the choice of non-medicated, otherwise healthy children; and the concomitant use of several validated tools that directly assessed the child, as well as parent and teacher perceptions of his behavior. In summary, in this study, supplementation of 1 g/day of ALA using an ALA-rich oil to children and adolescents with ADHD did not improve any behavioral measure, as tested by several validated questionnaires and a computerized CPT. We do acknowledge that a major limitation to the trial was the relatively small sample size, attributed to a relatively high dropout rate. Nevertheless, our findings are in concert with many other studies which used both ALA and long-chain omega-3 fatty acids. Given the high dropout rate, and in light of previous research and anticipated effect size, we recommend recruiting much larger numbers of participants, and possibly using a higher ALA dose in future similar studies. BODY.AUTHOR CONTRIBUTIONS: Gal Dubnov-Raz contributed to study design, conductance, data analysis, and drafted the first manuscript. Zaher Khoury contributed to study conductance and data analysis. Ilana Wright and Itai Berger contributed to study design, conductance, and data analysis. Raanan Raz contributed to study design and data analysis. All authors contributed significantly to the manuscript text, and approved its final version. BODY.CONFLICT OF INTEREST STATEMENT: The Associate Editor Yael Leitner declares that, despite being affiliated to the same institution as author Gal Dubnov-Raz, the review process was handled objectively and no conflict of interest exists. Itai Berger serves on the scientific advisory board of Neuro-Tech Solutions Ltd. All other authors declare no conflicts of interest.

TITLE: Combined Diosmectite and Mesalazine Treatment for Mild-to-Moderate Ulcerative Colitis: A Randomized, Placebo-Controlled Study ABSTRACT.BACKGROUND: The relapse rate of ulcerative colitis (UC) is high. The efficacy of combined diosmectite and mesalazine treatment for active mild-to-moderate UC was investigated. ABSTRACT.MATERIAL/METHODS: A total of 120 patients with UC were enrolled in this randomized, single-blind, placebo-controlled study. Sixty patients were assigned to the Diosmectite group (diosmectite and mesalazine) and 60 were assigned to Placebo group (placebo and mesalazine). In the induction phase, the primary end point was the clinical remission rate at 8 weeks; secondary end points were clinical response, endothelial mucosal healing, Mayo score, erythrocyte sedimentation rate, C-reactive protein levels, and defecation frequency. In the maintenance phase, the primary end point was clinical remission at 52 weeks; secondary end points were clinical response, endothelial mucosal healing, Mayo score, erythrocyte sedimentation rate, and defecation frequency. ABSTRACT.RESULTS: At 8 weeks, the Diosmectite group had a significantly higher clinical remission rate (68.3% vs. 50%) and mucosal healing rate (66.7% vs. 48.3%) compared with the Placebo group. There were no significant differences in clinical response rates, Mayo score, erythrocyte sedimentation rate, C-reactive protein, or defecation frequency. At 52 weeks, the Diosmectite group had a significantly higher clinical remission rate (61.7% vs. 40%) and mucosal healing rate (60% vs. 38.3%) compared with the Placebo group. Defecation frequency was lower, but this was not significant. ABSTRACT.CONCLUSIONS: Combined diosmectite and mesalazine treatment successfully induced and maintained the treatment of active mild-to-moderate UC as indicated by higher rates of clinical remission and mucosal healing. BODY.BACKGROUND: Ulcerative colitis (UC) is a chronic, non-specific, inflammatory bowel disease (IBD) characterized by ulcerative intestinal mucosa, bloody stools, diarrhea, and abdominal pain. Etiology and pathogenesis of UC remain to be elucidated and current treatment strategies lack specificity. At present, no curative treatment is available, resulting in prolonged disease, recurrences, and long-term diarrhea that affect overall health and quality of life [1,2]. Aminosalicylates are the primary drugs used for the treatment of UC. These drugs are also used for remission induction and maintenance treatment [3,4]. Despite constant administration of 5-aminosalicylate, mesalazine, or sulfasalazine (SASP), the proportion of patients having relapses remains high. A recent Norwegian study reported that the 1-year relapse rate of UC was approximately 50%, even with the administration of SASP or mesalazine [5]. In addition, follow-up visits performed in 1161 patients with UC in Denmark during a 2–2.5 year period revealed that only 50% of the patients being treated with mesalazine had sustained remission [6]. A study in Japan showed that the relapse rate was 30% when using long-term mesalazine (4.0 g/d) treatment compared with 48% when using short-term treatment [7]. Lastly, a study performed in a Korean population showed that 49.6% of patients with UC treated with SASP or mesalazine experienced relapses of the disease [8]. Although mesalazine is well tolerated and its efficacy in maintenance treatment of mild-to-moderate UC is superior to that of placebo [9], the long-term clinical remission rate and endoscopic mucosal healing rate of UC require improvements. Diosmectite is a natural silicate that has been used widely for the treatment of diarrhea [10,11]. Animal studies have also shown that administration of diosmectite can effectively treat infectious diarrhea [12]. Diosmectite absorbs intestinal bacteria, proteins and toxins, and increases colonic mucin levels. Administration of diosmectite has successfully treated rats with hapten trinitrobenzene sulphonic acid (TNBS)-induced colitis. In these animals, diosmectite treatment alleviated diarrheal symptoms and pathological intestinal injury, and improved biochemical indices. Clinical studies have also indicated that diosmectite treatment significantly alleviates clinical diarrheal symptoms, as well as endoscopic and pathological damages in pediatric patients with UC [13]. For adult patients with UC whose clinical symptoms failed to be substantially improved following treatment with mesalazine for 4 weeks, the average clinical activity index was lowered by additional administration of diosmectite for 30 days with maintenance mesalazine treatment [14]. Therefore, the objective of the present study was to further investigate the clinical efficacy of diosmectite and mesalazine treatment in patients with UC. The effects of this combination on the prevention of relapses were investigated, introducing a new treatment protocol that could yield higher rates of clinical remission and endoscopic mucosal healing. BODY.MATERIAL AND METHODS: BODY.PATIENTS: A total of 120 patients diagnosed with active UC at the Jinan Military General Hospital, Peking University First Hospital and Shandong University between April 2010 and April 2012 were prospectively enrolled. All patients presented the Montreal classification criteria of IBD [15]. Inclusion criteria were: 1) provided a written informed consent; 2) age 18–65 years old; 3) continual or recurrent mucosal bloody stools and abdominal pain, without any pathogenic microorganisms being detected in stool cultures for 2 or more times; 4) discontinuation of any drugs that may influence UC within 1 week prior to the study; 5) diagnosed with active mild-to-moderate UC by enteroscopy and pathological examination 1 week prior to the study; and 6) a Mayo score <10 [16]. Exclusion criteria were: 1) a Mayo score >10; 2) history of allergies to salicylates; 3) alanine aminotransferase (ALT) levels ≥1.5-fold the upper limit of normal (ULN) and/or creatinine levels ≥ULN; 4) co-morbidities such as acute pancreatitis, leukopenia, pericarditis, or myocarditis; 5) concurrent gastrointestinal diseases such as Behçet’s disease, intestinal tuberculosis, or any other disease that could influence the implementation of this treatment protocol and/or influence the action and efficacy evaluation of treatment; or 6) patients treated with concomitant azathioprine/6-mercaptopurine. Patients were withdrawn from the study if: 1) they failed to take drugs for 2 consecutive days within a 4-week period; and 2) there was concomitant administration of any other drugs for the treatment of UC or the use of any other drugs and treatments during the study that could affect the clinical observations of the study. Criteria for study termination were: 1) serious adverse reactions, exacerbations, or complications; 2) development of other diseases for which treatment could affect the clinical observation of the study drugs or concurrent participation in clinical trials with other drugs; or 3) pregnancy (Figure 1). BODY.STUDY DESIGN: This was a prospective, randomized, placebo-controlled, single-blind study. The study was approved by the medical ethics committee of all 3 sites. All patients provided written informed consent. Patients were randomized using a computer-generated random number table and assigned to 1 of 2 groups: the Diosmectite group (diosmectite and mesalazine, n=60) and the Placebo group (placebo and mesalazine, n=60). Patients in the Diosmectite group received diosmectite (France Beaufour Ipsen Industrie, Tianjin, China) 3 g tid (3 times a day) 1 h before meals, combined with mesalazine (Ethypharm, France, registration certificate number: H20100063) 1 g qid (4 times a day) after meals for 8 weeks, then 0.5 g qid after meals for 44 weeks. Patients in the Placebo group received placebo (Pharmacy School of Shandong University) 3 g tid 1 h before meals, combined with mesalazine 1 g qid after meals for 8 weeks, then 0.5 g qid after meals for 44 weeks. BODY.STUDY MEASURES: The improved Mayo scoring system was used to evaluate clinical efficacy (Table 1) [16]. Clinical response was defined as a decrease in the Mayo score of at least 3 points and at least 30%, with an accompanying decrease in the subscore for rectal bleeding of at least 1 point or absolute rectal-bleeding subscore of 0 or 1. Clinical remission was defined as a total Mayo score of ≤2 with no individual subscore >1 point, or an absolute rectal bleeding subscore of 0 or 1. Mucosal healing was defined as an absolute endoscopy Mayo subscore of 0 (normal or inactive disease) or 1 (mild disease: erythema, decreased vascular pattern, mild friability) (Table 1) [17]. In the induction phase (weeks 0–8), the primary end point was clinical remission within 8 weeks after beginning treatment. The secondary end points included clinical responses, endoscopic mucosal healing, Mayo score, erythrocyte sedimentation rate, C-reactive protein (CRP) levels, presence of bloody stools, and defecation frequency. In the maintenance phase (weeks 9–52), the primary end point was clinical remission within 52 weeks after beginning treatment. The secondary end points included clinical responses, endoscopic mucosal healing, Mayo score, erythrocyte sedimentation rate, CRP levels, the presence of bloody stools, and defecation frequency. The following laboratory tests were performed in all patients of both groups: 1) CRP and erythrocyte sedimentation rate before treatment and at weeks 2, 4, 8, 26, and 52; and 2) serum levels of potassium, sodium, chlorine, creatinine, blood urea nitrogen, ALT, and total bilirubin before treatment and at weeks 8, 26, and 52. Colonoscopy was performed in all patients to observe lesions of the intestinal mucosa including erythema, vascular texture, tissue fragility, erosion, and hemorrhage before treatment and at weeks 8, 26, and 52. The occurrence of adverse events (headaches, nausea, increased stool frequency, abnormal ALT) were recorded during the follow-up visits, including the time elapsed after beginning treatment, severity, duration, and measures taken. BODY.STATISTICAL ANALYSIS: SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. All analyses were performed on an intention-to-treat basis. Continuous data are presented as means ± standard deviation (SD), and were analyzed using paired-samples t-tests for before/after treatment analyses, or independent samples t-tests for comparisons between groups (Mayo scores, erythrocyte sedimentation rates, C-reactive protein, and defecation frequencies). Categorical data are presented as absolute counts and proportions, and were analyzed using χ2 tests between the 2 groups after treatment (clinical response rates, clinical remission rates, and endoscopic mucosal healing rates). P-values <0.05 were considered statistically significant. BODY.RESULTS: A total of 120 patients were prospectively enrolled in this randomized, single-blind, placebo-controlled study (Figure 1). The cohort included 68 males and 52 females aged 18–65 years. Forty patients were newly diagnosed with UC and 80 were patients with chronic relapsing UC. The baseline clinical features of patients in both groups are presented in Table 2. Disease course ranged from 6 months to 2 years, and the average body weight was 56.5 kg. There were 62 patients with proctitis, 58 of which had left colitis. Seventy patients presented with mild UC and 50 with moderate UC. Sixty patients were randomly assigned to the Diosmectite group (diosmectite and mesalazine) and 60 patients were assigned to the Placebo group (placebo and mesalazine). After 8 weeks of treatment, a clinical response was observed in 55 of the 60 Diosmectite group patients (91.7%), 41 patients (68.3%) achieved clinical remission, 40 (66.7%) patients achieved endoscopic mucosal healing, and there was no improvement in 5 patients (Table 3). The average Mayo score decreased from 5.1±1.3 before treatment to 1.6±1.1 after treatment (P<0.05); the average erythrocyte sedimentation rate decreased from 30.1±12.3 mm/h before treatment to 11.5±5.7 mm/h (P<0.01); mean CRP levels decreased from 21.6±11.2 mg/l before treatment to 5.2±3.6 mg/l after treatment (P<0.01); and the mean defecation frequency decreased from 4.7±2.3 times per day before treatment to 1.0±1.3 times per day after treatment (P<0.05) (Table 3). In the Placebo group, 50 of the 60 patients (83.3%) showed a clinical response, 30 achieved clinical remission (50%), 29 achieved endoscopic mucosal healing (48.3%), and there was no improvement in 10. Mean Mayo score decreased from 5.3±1.4 before treatment to 2.2±1.9 after treatment (P<0.05); the average erythrocyte sedimentation rate decreased from 31.7±11.2 mm/h before treatment to 20.4±9.9 mm/h (P<0.05); mean CRP levels decreased from 22.6±10.8 mg/l before treatment to 9.0±6.9 mg/l (P<0.05); and average defecation frequency decreased from 4.5±1.4 times per day before treatment to 2.0±1.3 times per day (P<0.05) (Table 3). There was no significant difference in clinical responses between the Diosmectite group and the Placebo group (P>0.05). However, rates of clinical remission and endoscopic mucosal healing were significantly higher in the Diosmectite group compared with the Placebo group. There was no significant difference in Mayo scores, erythrocyte sedimentation rate, CRP levels, or defecation frequency between the 2 groups after treatment (Table 3). After 26 weeks of treatment, 38 (63.3%) patients in the Diosmectite group achieved clinical remission (Figure 2A) and 37 (61.7%) achieved endoscopic mucosal healing (Figure 2B). Defecation frequency was 1.0±0.8 times per day. In the Placebo group, 26 patients (43.3%) achieved clinical remission (Figure 2A) and 25 (41.7%) achieved endoscopic mucosal healing (Figure 2B). Defecation frequency was 2.0±0.9 times per day. Clinical efficacy comparisons showed that the clinical remission and endoscopic mucosal healing rate of the Diosmectite group were significantly higher than in the Placebo group (P=0.028 and P=0.028, respectively). The defecation frequency of the Diosmectite group was lower than in the Placebo group, but the difference was not significant (P=0.80). After 52 weeks of treatment, 37 patients (61.7%) in the Diosmectite group achieved clinical remission (Figure 2A), and 36 (60%) achieved endoscopic mucosal healing (Figure 2B). The defecation frequency was 1.0±0.7 times per day. In the Placebo group, 24 patients (40%) achieved clinical remission (Figure 2A) and 23 patients (38.3%) achieved endoscopic mucosal healing (Figure 2B). The defecation frequency was 2.0±1.2 times per day. The clinical remission rate and endoscopic mucosal healing rate in the Diosmectite group were significantly higher (61.7% and 60%, respectively) than in the Placebo group (40% and 38.3%, respectively, P=0.018 and P=0.018, respectively). The defecation frequency of the Diosmectite group was lower than in the Placebo group, but the difference was not significant (P=0.80). Both groups showed a decrease in clinical remission rate and mucosal healing (Figure 2A, 2B), but rates of remission and mucosal healing were significantly higher at all time points in the Diosmectite group compared with the Placebo group. Adverse reactions were recorded for all patients (Table 4). In the Diosmectite group, 1 patient had headaches and 1 patient had nausea. In the Placebo group, 3 patients had headaches, two patients had nausea, 3 patients had increased defecation frequency of more than 3 times per day, and 1 patient had elevated ALT levels that returned to normal following discontinuation of study drugs (Table 4). BODY.DISCUSSION: The objective of the present study was to investigate the efficacy of combined diosmectite and mesalazine treatment for UC compared with placebo and mesalazine using a randomized, single-blind approach. The combined treatment group showed higher rates of clinical remission and endoscopic mucosal healing as early as 8 weeks after beginning treatment, and these improvements were sustained for 52 weeks. Many studies have shown that diosmectite can effectively treat various types of pediatric and adult diarrhea, such as those caused by acute and chronic diseases or bacterial and non-bacterial infections [11]. This effect appears to be related to its pharmacological, structural, and antimicrobial properties [18]. Indeed, diosmectite is a mineral clay with a multilayer structure consisting of di-tetrahedron silicon oxide and octahedron aluminum oxide. Diosmectite presents an extensive superficial area (approximately 100 m2/g) that allows it to cover and adhere to the intestinal mucosal surface of the entire enteric cavity. It absorbs bacteria, viruses, and toxins and negatively affects pathogenesis of UC [19]. Diosmectite binds to Clostridium difficile toxins A and B, as well as Clostridium perfringens toxins and endotoxins [20]. Moreover, it binds to mucosa and mucin, and reinforces the mucosal barrier. In doing so, it protects already damaged intestinal mucosa and prevents further histopathological changes induced by bacterial infections. Diosmectite increases mucosal production and intestinal mucosal thickness, prolongs mucosa half-life, maintains normal intestinal secretions, reduces the loss of water and electrolytes, and reinforces the intestinal mucosa barrier function [21]. Importantly, diosmectite does not enter into the bloodstream, and is therefore safe and well-tolerated, with minimal adverse effects. Interestingly, a study has shown that diosmectite-zinc oxide modulates the expression of pro-inflammatory cytokines and tight junction protein in early-weaned pigs [21]. Another mineral clay, montmorillonite, relieves diarrhea, improves barrier dysfunction and expression of inflammatory cytokines, and increases tight junction protein expression, and it is possible that diosmectite acts in a similar manner [22]. Another possible mechanism is that it facilitates intestinal barrier repair by reducing inflammatory cell infiltration of the mucous membrane [12]. It has been demonstrated that diosmectite is more efficient in repairing damaged mucosal membranes than SASP, significantly improving the expression of colonic mucin 2 and protecting the mucosal barrier [12]. Previous studies have indicated that diosmectite might be an effective, additional treatment for UC patients. For instance, Olives et al. reported that diosmectite significantly relieves clinical diarrheal symptoms and reduces histopathological damages in children and adults [13]. In addition, combined mesalazine and diosmectite treatment decreased the inflammatory response in an early-phase study involving 25 patients with active-stage mild-to-moderate UC [14]. In the present study, after 8 weeks, CRP levels and erythrocyte sedimentation rate were significantly decreased. These data suggest that the combined treatment could relieve the inflammatory response in patients with active UC, since these indices are associated with UC disease activity [23,24]. There was also a significant reduction in defecation frequency, and clinical remission and endoscopic mucosal healing rates were significantly higher in the combined treatment group compared with the placebo group. These findings are important because improved mucosal healing can improve quality of life and prevent relapse [25,26]. As such, these improvements were still observed at 26 and 52 weeks of treatment. It has been proposed that mesalazine prevents 5-aminosalicylate from being absorbed in the upper gastrointestinal tract, and ensures that sufficient 5-aminosalicylate reaches the diseased colon at the distal end, allowing for effective and localized treatment [9]. In turn, 5-aminosalicylate may inhibit prostaglandin synthesis and inflammation of the intestinal mucosa. In contrast, diosmectite acts to absorb endotoxins and antigens in the enteric cavity, partially inhibiting the inflammatory response, increasing permeability, and protecting the intestinal mucosal barrier [12,18–20,27]. It is hypothesize that the combination of diosmectite and mesalazine facilitates and promotes the repair of damaged intestinal mucosa. In this study, there were no serious adverse reactions induced by the combined diosmectite and mesalazine treatment. This indicates that the combination is safe and well-tolerated. The present study is not without limitations. This was a single-blind study. A double-blind study, similar to a study previously reported investigating vedolizumab in induction and maintenance therapy of UC [28], may provide more conclusive findings. Other anti-inflammatory drugs exist for the treatment of IBD [29], and other combinations might be tried and compared. These limitations indicate the need for future research. BODY.CONCLUSIONS: Combined treatment with diosmectite and mesalazine was successful as both induction (8 weeks) and maintenance (52 weeks) therapies for patients with active-stage mild-to-moderate UC. These patients had higher rates of clinical remission and mucosal healing at 8 weeks and maintenance at 52 weeks of treatment compared with patients who received placebo and mesalazine treatment.

TITLE: Survivorship care plans have a negative impact on long-term quality of life and anxiety through more threatening illness perceptions in gynecological cancer patients: the ROGY care trial ABSTRACT.PURPOSE: Prior results from the registration system oncological gynecology (ROGY) care trial showed that survivorship care plans (SCPs) increased threatening illness perceptions in gynecological cancer survivors, but it remained unclear whether this would result in poorer physical and psychosocial outcomes. The aim of the current study is to assess the direct and indirect effects of SCPs on health-related quality of life (HRQoL) and anxiety and depression, through illness perceptions. ABSTRACT.METHODS: Twelve hospitals in the South of the Netherlands were randomized to providing ‘SCP care’ or ‘usual care.’ Newly diagnosed endometrial and ovarian cancer patients completed questionnaires after initial treatment (endometrial, 221 [75%]; ovarian, 174 [71%]) and after 6, 12, and 24 months. SCPs were automatically generated after initial treatment by the oncology providers through the web-based ROGY. Illness perceptions were measured after initial treatment and HRQoL and anxiety and depression after 6, 12, and 24 months. ABSTRACT.RESULTS: Structural equation models showed that endometrial cancer patients who experienced more symptoms or concern due to the SCP reported worse social functioning (β = − 0.82; p = 0.01) and more fatigue, insomnia, pain, and anxiety (β = 0.58–0.86, p < 0.05) within 12 months after treatment. Ovarian cancer patients who had lower trust that the treatment would cure their disease due to the SCP reported worse emotional functioning 6 months after treatment (β = 0.27, p = 0.02). ABSTRACT.CONCLUSIONS: Current results show that SCPs may have negative effects on HRQoL and anxiety in patients who experience more threatening illness perceptions due to the SCP. We should be aware of the potential negative consequences of SCPs. Trial Registration clinicaltrials.gov Identifier: NCT01185626. BODY.BACKGROUND: Over the last decade, survivorship care plans (SCPs) have been recommended as a standard of care for all cancer patients. SCPs contain written information to support patients in their physical and psychological challenges in life after treatment [1]. To date, a limited number of randomized controlled trials (RCTs) have been conducted to assess the impact of SCPs on patient reported outcomes [2–7]. As opposed to observational and qualitative studies [8, 9], RCTs failed to identify beneficial effects of SCPs on patient satisfaction with information provision and care, quality of life, or distress [2–5, 7]. However, SCPs may be beneficial for underserved patient populations [6]. The registration system oncological gynecology (ROGY) care trial was the first pragmatic cluster-randomized controlled trial that assessed the impact of automatically generated SCPs, and did find a negative effect on illness perceptions [5, 7]. Illness perceptions are generally defined as a patient’s belief about the disease through cognitive representations, including the perceived impact on life, duration of the illness, experienced symptoms and treatment trust, and also emotional representations, including concern, emotional impact, and personal control over illness [10]. In the ROGY care trial, SCPs caused more threatening illness perceptions: they increased experienced symptoms, emotional impact and concern in endometrial cancer patients [5], and led to lower trust that the treatment would help to cure the disease in ovarian cancer patients [7]. Previous studies in cancer patients show that more threatening illness perceptions are associated with poorer quality of life and more psychological morbidities [11–17] in accordance with Leventhals’ common-sense model of self-regulation (CSM). CSM presumes that individuals who are confronted with a health threat (i.e., cancer diagnosis) form illness perceptions, which impact physical and psychosocial outcomes through coping responses [10, 18]. To support emotional coping, psychological interventions have been developed that aim to decrease psychological distress after an event such as a cancer diagnosis [19]. Exposure therapies, such as psychological debriefing do not seem to decrease psychological morbidity, but may even worsen it due to exacerbation of the symptoms [20]. Similarly, SCPs containing extensive information on the disease and potential side-effects may exacerbate psychological distress and symptoms experienced among cancer patients. Consequently, prior results of the ROGY care trial suggest that SCPs may intervene in the pathway of the CSM by causing more threatening illness perceptions [5, 7], which may in turn affect physical and psychosocial outcomes. However, no evidence exists on the possible causal relationships between SCP provision, illness perceptions, and physical and psychosocial outcomes. It is important to consider the potential negative consequences of threatening illness perceptions due to SCPs before widespread implementation is decided upon. The aim of the current study is to assess whether SCPs have a negative effect on long-term health-related quality of life (HRQoL), anxiety and depression in patients who experience more threatening illness perceptions due to the SCP. Illness perception scales that have earlier shown to be affected by SCPs [5, 7] (i.e., increased experienced symptoms, concern and emotional impact in endometrial cancer, and lower treatment trust in ovarian cancer) are included in the current analysis. We hypothesize that SCPs have a negative impact on HRQoL, anxiety and depression through more threatening illness perceptions. BODY.METHODS: BODY.DESIGN: The ROGY care trial among endometrial and ovarian cancer patients aimed to assess the longitudinal impact of automatically generated SCPs on patient reported outcomes. A cluster-randomized design was used to avoid potential contamination between the trial arms. Twelve hospitals in the Netherlands were randomly allocated to either ‘usual care’ or ‘SCP care.’ The trial was centrally approved by a Medical Research Ethics Committee [21] and was registered as NCT01185626 in clinicaltrials.gov. BODY.PARTICIPANTS AND RECRUITMENT: All newly diagnosed women with endometrial cancer as a primary tumor between April 2011 and October 2012, or ovarian cancer between April 2011 and March 2014, were invited to participate shortly after initial treatment, by means of a letter and an informed consent form, sent directly to the patients’ home address by their own gynecologist. After consent, questionnaires were sent after treatment and follow-up questionnaires were sent at 6, 12, and 24 months after treatment (“Appendix”). Because of the pragmatic nature of the trial, exclusion criteria (i.e., borderline ovarian tumor, undergoing palliative care or unable to complete a Dutch questionnaire) were limited [21]. Earlier analysis showed that 73% of endometrial and 66% of ovarian cancer patients in the SCP care arm reported receipt of an SCP [22]. In the current analysis, all patients of both trial arms were included (intention-to-treat) to reflect real-life clinical practice in which not all patients receive an SCP. BODY.RANDOMIZATION AND BLINDING: To prevent imbalance between the trial arms, stratified randomization was used according to whether a hospital has a Gynecologic Oncology Center, and the annual number of endometrial and ovarian cancer patients diagnosed in each hospital. Randomization was performed via a table of random numbers, by an independent researcher blinded to the identity of the hospitals. Patients, but not oncology providers or researchers assessing the outcomes, were blinded to trial assignment [21]. BODY.SCP CARE VERSUS USUAL CARE: In ‘usual care’ hospitals, standard care was provided in accordance to the Dutch follow-up guidelines (http://www.oncoline.nl). In most hospitals, verbal information and the generic brochures of the Dutch Cancer Society were provided [21]. None of the hospitals provided SCPs as developed for this study. In the ‘SCP care’ hospitals, all oncology providers (gynecologist/gynecologic oncologist and oncology nurses, N = 24) attended an instruction evening devoted to when and how SCPs should be provided. They were instructed to provide an SCP to patients at the consultation where the results of histopathology and (adjuvant) treatment plan were discussed, mostly 7–14 days after surgery. If applicable (i.e., if there were any changes in the cancer, treatment or oncology provider), an updated version of the SCP could optionally be discussed in a follow-up consultation. Practical guidelines were given on the components of the SCP that should minimally be discussed with each patient during the consultation (i.e., diagnosis, prognosis, treatment(s), most important side-effects). Because of the pragmatic approach of the trial, care providers in the ‘SCP care’ arm were free to choose whether the gynecologist/gynecologic oncologist, or oncology nurse provided the SCP fitting their clinical practice [21]. BODY.SURVIVORSHIP CARE PLAN: The SCP was based on the Dutch translation of the National Academy of Medicine (NAM) SCP template [1], adjusted to the local situation [21] by a group of gynecologists/gynecologic oncologists, oncology nurses, a radiotherapist, medical oncologist, primary care physician, and patients [21]. The SCP consisted of information on diagnostic tests, type of cancer, stage, grade, and treatments received, and contact details of the hospital and medical specialists. In addition, the SCP contained a tailored follow-up care plan, including detailed information on the most common short- and long-term effects of the treatments received, effects on social and sexual life, possible signs of recurrence and secondary tumors, and information on rehabilitation, psychosocial support, and supportive care services [21]. Texts of the SCP were based on pilot-tested patient education material from the Dutch Cancer Society. In addition, the SCP was pilot-tested on patients with a low/intermediate educational level to ensure that the SCP was understandable. BODY.MEASURES: Age, socio-economic status (SES), and clinical data, such as cancer type, cancer stage, and date of diagnosis, were obtained from the Netherlands Cancer Registry (NCR). The NCR routinely collects data on newly diagnosed cancer patients in all hospitals in the Netherlands [23]. SES was based on postal code of the residence area of the patient, combining aggregated individual fiscal data on the economic value of the home and household incomes [24]. SES was categorized into low, medium, or high. Additional socio-demographic information (i.e., marital status, employment status, and comorbidities) was assessed in the first questionnaire. Marital status (‘married/living together’ vs ‘divorced/widowed/never married’) and employment status (‘having a paid job’ vs ‘not having a paid job’) were dichotomized. Comorbidity was assessed by the adapted Self-administered Comorbidity Questionnaire (SCQ), and categorized into no comorbidities, one comorbidity, or more than one comorbidity [25]. The Brief Illness Perception Questionnaire (B-IPQ) was used to assess illness perceptions after initial treatment [26]. The B-IPQ includes eight single-item scales (impact of disease on life, perceived duration of illness, personal control over illness, trust that the treatment would help to cure the illness, experienced symptoms, concern about the illness, understanding of the illness, and emotional impact of the illness). Only the scales that have earlier shown to be affected by SCPs in our trial [5, 7] were used in the analysis, including the amount of symptoms experienced, concerns about the illness, emotional impact of the illness with respect to endometrial cancer, and trust that the treatment would help to cure with respect to ovarian cancer [27]. The latter scale was reversed to ascertain that all B-IPQ scales were one-directional: a higher score indicates more threatening illness perceptions. Test–retest reliability (Pearson correlations 0.42–0.75) was fair [26]. The EORTC QLQ-C30 (version 3.0) was used to assess HRQoL 6, 12, and 24 months after diagnosis [28]. It contains five functional scales on physical, role, cognitive, emotional and social functioning, a global QoL scale; three symptom scales on fatigue, nausea and vomiting, and pain; and six single items. Response scales included: ‘Not at all,’ ‘A bit,’ ‘Quite a bit,’ and ‘Very much,’ except for the global QoL scale, which ranges from ‘Very poor’ to ‘Excellent.’ Latent variables of the scales were defined by the items of each scale [29]. Higher scores on global quality of life and the function scales indicate a better HRQoL, while higher scores on the symptom scales indicate more symptoms. Test–retest reliability was good (Pearson correlations = 0.82–0.91) [30]. Internal consistency of the multi-item scales (Cronbach’s alphas 0.71–0.92) in our study was good. The Hospital Anxiety and Depression Scale (HADS) was used to assess symptoms of anxiety and depression 6, 12, and 24 months after diagnosis [31]. The HADS assesses separate anxiety and depression scales, which both consist of seven items. All items were scored on a 0–3-point scale, with higher scores indicating more symptoms. Test–retest reliability of the scales (Pearson correlations = 0.86–0.88) was good [31]. Internal consistency of the scales (Crohnbach’s alphas 0.71–0.77) in our study was good. BODY.STATISTICAL ANALYSIS: Statistical analyses were conducted using Statistical Analysis System (SAS) version 9.4. (SAS Institute, Cary, NC, 1999). Differences in characteristics of patients between the trial arms for endometrial and ovarian cancer were compared using independent samples t tests for normally distributed continuous variables, Mann–Whitney U tests for not normally distributed variables and Chi-square tests for categorical variables. Differences in baseline B-IPQ scores between the trial arms were assessed using Chi-square tests of categorical variables defined by the 25th, 50th, 75th, and 100th percentile scores of the separate B-IPQ scales. Pearson’s correlation coefficients were computed to assess the correlations between the illness perception scales at baseline and outcomes (HRQoL, anxiety and depression) 6, 12, and 24 months after initial treatment, for endometrial and ovarian cancer separately. P values smaller than .05 were considered to be statistically significant. Structural equation models (SEM) were used to test the hypothesized causal relationships between trial allocation (SCPs), illness perceptions and the HRQoL, and anxiety and depression scales, with a linear equation system. SEMs are used to assess unobservable ‘latent’ variables by using observed variables, and to assess the relationships between those (observed and latent) variables [32]. Both direct and indirect effects of trial allocation on outcome variables were assessed using the effpart statement in the CALIS procedure in SAS. Direct effects would indicate an effect of SCPs on the outcome scales in all patients, while indirect effects would indicate an effect of SCPs on the outcome scales in patients who have altered illness perceptions due to the SCP. Statistical power was sufficient to detect indirect effects, but low to detect direct effects [33]. The CALIS procedure was used to define the model paths (i.e., hypothesized relationships between variables). First, simple mediation models were built to assess the direct and indirect effects of trial allocation on the separate outcome scales (HRQoL, anxiety and depression scales) 6, 12, and 24 months after initial treatment. Mediators were the separate B-IPQ scales measured after initial treatment that have earlier shown to be associated with trial allocation (i.e., increased experienced symptoms, concern and emotional impact in endometrial cancer, and lower treatment trust in ovarian cancer; Fig. 1). Models were defined for endometrial and ovarian cancer separately. The paths in each simple mediation model were defined as trial allocation ---> [BIPQ item score after diagnosis] ---> [outcome variable at time-point X]. When outcome scales consisted of multiple items, a latent variable was defined by the items of that scale. When standardized factor loadings of scale items were low (β < 0.6), they were removed from the model to obtain a better model fit [34]. When (semi-)complete separation of the outcome scales occurred, no SEM could be determined [35]. Second, the full SEMs were built by entering all significant paths (p < 0.05) of the simple mediation models into one model, for endometrial and ovarian separately, and for each time-point separately. Third, the insignificant paths (p > 0.05) were removed from the model to obtain a good model fit. Finally, the covariates that were significantly associated with any of the outcome scales were entered into the model. For all SEM models, full information maximum likelihood (FIML) was used, which handles missing data within the model without needing to impute data [36]. Model fit was assessed with the χ2 statistic, adjusted goodness-of-fit-index (AGFI), Bentler’s Comparative Fit Index (CFI), standardized root mean squared residual (SRMR), and root mean squared error approximation (RMSEA) [37]. Standardized beta coefficients were used to interpret the models, and range from − 1 to 1, in which coefficients closer to zero indicate smaller effects. An increase of 1 standard deviation of the independent variable corresponds to an increase in standard deviation of the dependent variable by the standardized beta coefficient [38]. Standardized beta coefficients of indirect effects can be considered small (0.05–0.10), moderate (0.1–0.25), or large (> 0.25) [38]. Fig. 1Bar charts of illness perceptions after diagnosis, SCP care (SCP) versus usual care (UC). Note only the illness perception items were included that have earlier been associated with trial allocation. High, med-high, med-low, and low illness perception categories were defined by the 25th, 50th, 75th, and 100th percentile scores of each B-IPQ scale separately BODY.RESULTS: Table 1 shows the clinical and socio-demographic baseline characteristics for both endometrial and ovarian cancer patients in the SCP care and usual care conditions. In endometrial cancer, patients in the ‘SCP care’ took more time after diagnosis to complete the questionnaires than patients in the ‘Usual care’ arm (p < 0.01). No differences in baseline characteristics were found between the trial arms in ovarian cancer patients. Table 1Baseline clinical and socio-demographic characteristics endometrial and ovarian cancer patients, SCP care versus usual care Endometrial cancerOvarian cancerSCP care (N = 119)Usual care (N = 102)P valueSCP care (n = 61)Usual care (n = 113)P value*Age at survey  Mean (SD) 67.4 (9.1) 67.8 (8.9) 0.71 63.6 (11.2) 64.3 (10.7) 0.67 SESa, n (%)  High 43 (36) 36 (35) 0.60 25 (41) 44 (39) 0.12  Intermediate 49 (41) 42 (41) 15 (25) 44 (39)  Low 21 (18) 22 (22) 12 (20) 18 (16)  Unknown 6 (5) 2 (2) 7 (6) Months since diagnosis, n (%)  Median 2.2 1.5 < 0.01 3.0 2.4 0.31  < 1 12 (10) 24 (24) 8 (13) 26 (23)  1–2 40 (34) 46 (45) 18 (30) 39 (34)  2–3 33 (28) 20 (20) 8 (13) 17 (15)  > 3 34 (29) 12 (12) 27 (44) 32 (28) Comorbidity, n (%)  None 19 (16) 18 (18) 0.53 21 (34) 28 (25) 0.18  1 32 (27) 20 (20) 12 (20) 36 (32)  2 or more 64 (54) 62 (61) 26 (43) 48 (36)  Unknown 4 (3) 2 (2) 2 (3) 8 (7) Marital statusb, n (%)  Partner 85 (71) 76 (75) 0.74 48 (79) 82 (73) 0.39  No partner 31 (26) 25 (25) 13 (21) 31 (27)  Unknown 3 (3) 1 (1) 0 (0) 0 (0) Employed, n (%)  Yes 22 (18) 15 (15) 0.40 20 (33) 31 (27) 0.44  No 85 (71) 79 (77) 41 (67) 83 (73)  Unknown 12 (10) 8 (8) 0 (0) 0 (0) FIGO stage, n (%)  I 102 (85) 89 (87) 0.34 21 (34) 31 (27) 0.63  II 6 (5) 1 (1) 7 (11) 9 (8)  II 9 (8) 9 (8) 23 (38) 50 (44)  IV 2 (2) 3 (3) 10 (16) 20 (18)  Unknown 0 (0) 0 (0) 0 (0) 3 (3) Treatment, n (%)  Surgery 117 (99) 97 (98) 0.46 54 (88) 104 (93) 0.33  Chemotherapy 6 (5) 12 (12) 0.06 44 (72) 92 (82) 0.13  Radiotherapy 44 (37) 37 (37) 0.99 P values < 0.05 are in bold aSocio-economic status (SES) was based on postal code of the residence area of the patient bMarital status included: partner married/living together; no partner divorced/widowed/never married. The numbers may not always add up to 100, because percentages have been rounded off to whole numbers Figure 1 shows the differences in illness perceptions between the SCP care and usual care arms. Significantly more endometrial patients in the SCP care arm compared to the usual care arm reported high experienced symptoms (18% vs 9%, p = 0.02) and high concerns about the illness (16% vs 7%, p = 0.02). No significant differences between the trial arms were found in emotional impact of the disease (19% vs 14%, p = 0.27) in endometrial cancer, or low trust that the treatment would help to cure the illness (16% vs 14%, p = 0.60) in ovarian cancer. However i,n earlier multilevel linear mixed model analyses, SCPs significantly increased threatening illness perceptions on these scales [5, 7]. Table 2 shows the correlations between illness perception scales after diagnosis and HRQoL, anxiety and depression after 6, 12, and 24 months, corrected for multiple testing (Bonferroni correction, α < 0.003). Consistent with our hypothesis, in both endometrial and ovarian cancer, significant moderate negative Pearson’s correlations were found between B-IPQ items and functioning scales (r = − 0.25 to − 0.41, p < 0.003). Significant moderate positive Pearson’s correlations were found between B-IPQ items and symptom scales (r = 0.27–0.41, < 0.003), and between B-IPQ scales and anxiety and depression (r = 0.28–0.46, < 0.003). Table 2Correlations between illness perception scales after diagnosis and HRQoL, anxiety and depression after 6, 12, and 24 months Endometrial cancerOvarian cancerIllness perceptions after treatmentSymptoms experiencedConcernsEmotional impactLower treatment trustMonths after treatment, outcome variables 6 18 24 6 18 24 6 18 24 6 18 24 N 15814712815814712815814712812410175Global quality of life − 0.24 − 0.25* − 0.21 − 0.25* − 0.29* − 0.34* − 0.29* − 0.29* − 0.29* − 0.42* − 0.40* − 0.36* Function scales  Physical functioning − 0.22 − 0.19 − 0.24 − 0.14 − 0.21 − 0.19 − 0.18 − 0.22 − 0.18 − 0.24 − 0.16 − 0.16  Role functioning − 0.06 − 0.20 − 0.28* − 0.09 − 0.15 − 0.36* − 0.22 − 0.13 − 0.25 − 0.27 − 0.18 − 0.25  Emotional functioning − 0.34* − 0.21 − 0.32* − 0.32* − 0.24 − 0.38* − 0.38* − 0.29* − 0.41* − 0.28* − 0.15 − 0.27  Cognitive functioning − 0.17 − 0.07 − 0.25 − 0.14 − 0.17 − 0.28* − 0.17 − 0.06 − 0.22 − 0.19 − 0.29 − 0.34  Social functioning − 0.41* − 0.28* − 0.32* − 0.23 − 0.29* − 0.38* − 0.33* − 0.27* − 0.27* − 0.16 − 0.07 − 0.26 Symptom scales  Fatigue 0.36* 0.28* 0.34* 0.26 0.26 0.37 0.35* 0.31* 0.32* 0.26 0.14 0.29  Nausea and vomiting 0.13 0.21 0.24 0.06 0.05 0.14 0.15 0.09 0.12 0.14 0.15 0.33  Pain 0.23 0.17 0.26 0.20 0.23 0.39* 0.22 0.20 0.26 0.28* 0.19 0.33  Dyspnea 0.18 0.19 0.11 0.07 0.14 0.14 0.15 0.15 0.20 0.20 0.02 0.23  Insomnia 0.32* 0.17 0.25 0.23 0.19* 0.28* 0.30* 0.22 0.26 0.31* 0.20 0.18  Appetite loss 0.28* 0.16 0.23 0.18 0.03 0.28* 0.27* 0.14 0.24 0.22 0.09 0.44*  Constipation 0.04 0.03 0.09 0.11 0.02 0.22 0.14 0.08 0.22 0.21 0.21 0.14  Diarrhea 0.18 0.11 0.13 0.15 0.11 0.05 0.21 0.08 0.14 0.06 < 0.01 0.13  Financial difficulties 0.30* 0.20* 0.20 0.17 0.08 0.11 0.21 0.10 0.08 0.16 0.09 − 0.08 Anxiety 0.30* 0.33* 0.34* 0.41* 0.45* 0.46* 0.46* 0.47* 0.45* 0.39* 0.31 0.24 Depression 0.39* 0.28* 0.23* 0.35* 0.35* 0.36* 0.39* 0.33* 0.34* 0.40* 0.25 0.38* Pearson’s correlations were estimated between illness perceptions measured after diagnosis, and outcome variables after diagnosis and 6, 12, and 24 months after diagnosis. Only the illness perception items were included that have earlier been associated with trial allocation *p < 0.003 (adjusted for Bonferroni correction 0.05/1) The SEM models showed no direct effects of SCPs on HRQoL, anxiety and depression scales. However, indirect effects through illness perceptions were observed. In endometrial cancer, SCPs indirectly increased fatigue, insomnia, and anxiety after 6 months (standardized, β = 0.58, SE = 0.09, p < 0.01; β = 0.69, SE = 0.08, p < 0.01; β = 0.58, SE = 0.09, p = 0.01), through more experienced symptoms (standardized, β = 0.21, SE = 0.09, p = 0.02; Fig. 2). Model fit was reasonable to good (AGFI = 0.87; CFI = 0.93; SRMR = 0.07; RMSEA = 0.047 [95% CI = 0.03–0.06]; χ2 = 87.2, p < 0.01) and effect sizes of the indirect effects are moderate (standardized, β = 0.12, SE = 0.06, p = 0.03; β = 0.15, SE = 0.06, p = 0.02; β = 0.12, SE = 0.05, p = 0.03) [38]. Further, SCPs indirectly decreased social functioning after 12 months (standardized, β = − 0.82, SE = 0.06, p < 0.01), and increased fatigue and pain after 12 months (standardized, β = 0.84, SE = 0.05, p < 0.01; β = 0.86, SE = 0.05, p < 0.01), through more concern (standardized, β = 0.25, SE = 0.09, p < 0.01; Fig. 2). Model fit was good (AGFI = 0.90; CFI = 0.98; SRMR = 0.046; RMSEA = 0.04 [95% CI = 0.00–0.06]; χ2 = 34.4, p = 0.08) and the effect sizes of the indirect effects were moderate (standardized, β = − 0.20, SE = 0.07, p < 0.01; β = 0.21, SE = 0.07, p < 0.01; β = 0.22, SE = 0.08, p < 0.01) [38]. In ovarian cancer, SCPs indirectly decreased emotional functioning after 6 months (standardized, β = − 0.66, SE = 0.20 p < 0.01), through lower trust that the treatment would help to cure the disease (standardized, β = 0.27, SE = 0.12, p < 0.05) (Fig. 2). Model fit was good (AGFI = 0.90; CFI = 0.96; SRMR = 0.06; RMSEA = 0.06 [95% CI = 0.01–0.09]; χ2 = 23.1, p = 0.04), and the effect size of the indirect effect was moderate (standardized, β = − 0.18, SE = 0.08, p = 0.02) [38]. No significant paths in the simple mediation models were found for outcomes after 24 months, for both endometrial and ovarian cancer. Fig. 2Path diagrams of the final structural equation models, outcomes 6 and 12 months after treatment. Note only the significant paths between the intervention (SCP), illness perception items, and outcome scales were entered in this model to obtain good model fit. Standardized coefficients are shown. Standardized beta coefficients were used to interpret the models, and range from − 1 to 1, in which coefficients closer to zero indicate smaller effects. Error terms and covariates in the model (age, FIGO stage, number of comorbidities) have been removed from the figure. *p < 0.05; **p < 0.01 BODY.DISCUSSION: The current study among endometrial and ovarian cancer patients shows that SCPs have a negative impact on long-term HRQoL and anxiety in patients who experience more threatening illness perceptions due to the SCP. Endometrial cancer patients who experience more symptoms or concerns due to the SCP, report worse social functioning and more fatigue, insomnia, pain, and anxiety in the year following treatment. Ovarian cancer patients who have lower trust that the treatment would cure their disease, due to the SCP, report worse emotional functioning 6 months after initial treatment. Earlier findings from the ROGY Care trial already showed that SCPs increased threatening illness perceptions: higher experienced symptoms, concern and emotional impact in endometrial cancer patients, and decreased trust that the treatment would help to cure the disease in ovarian cancer patients [5, 7]. However, it was yet unclear whether these threatening illness perceptions would deteriorate long-term physical and psychosocial outcomes. Our study confirms earlier findings in cancer patients that more threatening illness perceptions are associated with worse physical and psychosocial outcomes [11–17]. Consequently, our analyses confirmed that threatening illness perceptions due to the SCP led to worse HRQoL and more anxiety. Although no direct effects of SCPs were found, our results support our hypothesis that SCPs have a negative impact on HRQoL and anxiety through more threatening illness perceptions, consistent with Leventhal’s CSM [10]. Illness perceptions that mediated between SCP provision and HRQoL and anxiety were experienced symptoms and concern in endometrial cancer, and low treatment trust in ovarian cancer patients. Possibly, endometrial cancer patients, who are often diagnosed with low cancer stages, may perceive their cancer as a more serious condition due to information provided in the SCP (i.e., the diagnosis and treatments received, possible long-term and late effects, and chance of recurrence) than would otherwise be communicated by the oncology provider. The overall perception of a more serious condition in endometrial cancer patients may have caused higher symptom awareness, more anxiety, and the belief that one is unable to participate in social activities [16]. In ovarian cancer patients, who are more often diagnosed at advanced stages, the SCP led to lower treatment trust, possibly due to information on chance of recurrence in the SCP. Although this information may be realistic, it led to decreased emotional functioning after 6 months, meaning that patients felt more tense, worried, irritable, or depressed. Indeed, fear of recurrence has earlier been found to be most strongly associated with emotional functioning, of all EORTC QLQ-C30 functioning scales [39]. A limitation of the current study is that not all patients in the SCP arm reported receipt of an SCP [22]. A process evaluation of the ROGY care trial showed that ovarian cancer patients, older patients, and patients who have a distressed (type D) personality less often received an SCP [22]. We performed intention-to-treat analysis to reflect real-life clinical practice in which not all patients receive an SCP. Therefore, our results possibly underestimate the effects of SCPs on HRQoL, anxiety and depression in the total population, as patients with a type D personality may be more likely to experience threatening illness perceptions due to the SCP [40]. Further, as shown earlier [7], ovarian cancer patients with higher cancer stages were more often lost to follow-up due to death or ill-health, and were therefore not included in our longitudinal analyses. Therefore, current results in ovarian cancer may represent the healthier patient with lower cancer stages. However, we aimed to minimize selection bias by limiting exclusion criteria and our response rates were relatively high. Our SEM analyses violated the well-known recommendation of Baron and Kenny’s that a significant relationship between the independent variable (SCPs) and outcome (HRQoL, anxiety and depression) is required, in order to evaluate mediation effects [41]. However, this recommendation has since been criticized [33, 42]. Kenny and Judd argued that sample sizes needed to detect direct effects between independent and dependent variables, should be much larger than to detect indirect effects through mediation [33]. Therefore, our sample sizes of endometrial and ovarian cancer separately, were too small to detect direct effects of SCPs on the outcome scales. Possibly, direct effects of SCPs on the outcome scales would be found in larger sample sizes. An alternative explanation of our findings is the presence of a suppressing mediator, such as coping, which may ameliorate the indirect impact of SCPs on physical and psychological outcomes, while at the same time illness perceptions deteriorate the indirect impact of SCPs on outcomes. Therefore, there may be indirect effects but no direct effects of SCPs on HRQoL and anxiety [42], which would indicate that the impact of SCPs works differently across coping styles. In earlier publications of the ROGY care trial [5, 7], we argued that information provided in an SCP could be perceived as threatening but may also be realistic. Providing patients with honest and realistic information may be considered best to prepare patients for potential consequences of the cancer and cancer treatments, or would encourage patients to find social support to cope with the disease [43]. However, the current study shows that realistic information is not self-evidently beneficial for all patients. Instead, patients may attain worse expectations about negative outcomes such as side-effects or a recurrence, which has shown to potentially cause clinical worsening (“nocebo effect”) [44]. Similarly, psychological interventions that expose individuals to facts and rethinking of an event (i.e., cancer diagnosis and treatments) may not necessarily decrease psychological distress but rather exacerbate symptoms [20 BODY.CONCLUSION: In conclusion, no beneficial effects of SCPs on satisfaction with information provision and care in both endometrial and ovarian cancer patients were shown as primary outcomes of our trial [5, 7]. The current study highlights that SCPs may even have negative effects on HRQoL and anxiety in patients who experience more threatening illness perceptions due to the SCP. Therefore, we should be aware of the potential negative consequences of SCPs in some patients. A more tailored approach such as personalized SCPs fitting individual patient’s information needs should be further explored.

TITLE: Clinical and radiographic assessment of periapical pathology in single versus multivisit root canal treatment: An in vivo study ABSTRACT.OBJECTIVE:: The objective of the study was to compare and evaluate the clinical and radiographic outcome of single- versus multivisit endodontic treatment in teeth with periapical pathology at the end of 1, 3, and 6 months. ABSTRACT.MATERIALS AND METHODS:: Sixty single- and multi-rooted teeth indicated for root canal treatment with periapical pathology were included in the study. The teeth were assigned randomly into two groups Group I and Group II (n = 30 each), which were further subdivided into subgroup IA, subgroup IB and subgroup IIA, subgroup IIB (n = 15 each), respectively. Group I was medicated with ApexCal paste and obturated using the standardized protocol in second visit 7–10 days later, whereas Group II was obturated at the first visit. In subgroup IA and subgroup IIA, obturation was done using Apexit Plus sealer, whereas, in subgroup IB and subgroup IIB, AH Plus sealer was used. Patients were recalled at intervals of 1, 3, and 6 months to evaluate teeth for periapical healing. ABSTRACT.RESULTS:: Kruskal–Wallis and one-way ANOVA test showed no significant difference between Groups I and II, whereas Wilcoxon signed-rank test showed improvement in all the subgroups with highly significant P value (≤0.001). ABSTRACT.CONCLUSION:: Single-visit root canal treatment can be considered as a viable option for treatment of teeth with periapical pathology. BODY.INTRODUCTION: It has been established beyond doubt that periapical lesion is caused by bacteria in the root canal space.[1] Microbiological goal of endodontic treatment of teeth is to reduce the microbial bioburden to levels compatible with periradicular tissue healing and prevent microbial recolonization of the treated canal.[2] This is accomplished by thorough chemomechanical treatment of root canal followed by three-dimensional obturation.[3] Traditionally, root canal treatment was performed in multiple visits, with medication between root canal preparation and obturation, which mainly aims to reduce or eliminate microorganisms and their by-products from the root canal system before obturation. Multiple visit root canal treatment is well accepted as a safe and common therapy.[1] However, in recent years, there is a growing concern about the necessity of multiple appointments in endodontic treatment because no significant differences in antimicrobial efficacies have been reported between the single and multiple visit treatments.[4] Single-visit root canal treatment has become common practice and offers several advantages such as a reduced flare-up rate, good patient acceptance, and practice management considerations.[1] It is believed to eliminate the remaining microorganisms or to render them harmless by entombing them by complete obturation immediately after preparing and irrigating the canal space at the same visit.[5] Despite the fact that various practices are widely adopted, it does not indicate that the practices are biologically sound and/or appropriate. The argument for single-visit treatment relies heavily on convenience, patient acceptance, and reduced postoperative pain. The issue is very controversial, and opinions vary greatly as to the relative risks and benefits of single versus multiple visit root canal treatment. The direct evidence comparing healing rates following single and multiple visit root canal treatment should provide insight as to which regimen is more effective.[6] The purpose of the present study is to compare and evaluate the clinical symptoms and radiographic evidence of periapical healing after root canal therapy of teeth with periapical pathology completed in single and multiple visits. BODY.MATERIALS AND METHODS: This study was conducted on sixty single- and multi-rooted teeth indicated for root canal treatment. Approval for the scientific research on human subjects was obtained from the Institutional Ethical Committee. Definite guidelines were set for all the patients. Only those who fulfilled the inclusion criteria were included in this study. All patients were informed about the aims and design of the study, and written consent was obtained before their inclusion. The primary inclusion criteria included patients aged above 18 years, single and multi-rooted teeth with radiographic evidence of periapical pathology (periapical index score [PAI] ≥3). The exclusion criteria included patients with any systemic diseases, pregnant patients, patients who had been taking antibiotics, nonsteroidal antiinflammatory drugs, or corticosteroids before time of treatment, patients who need antibiotic premedication for dental treatment, if the tooth had been previously accessed, grossly decayed teeth, teeth with calcified canals, weeping canals, and teeth requiring re-treatment. Detailed history and thorough clinical examination were done before starting the treatment. Preoperative intraoral periapical radiograph was taken using a paralleling kit (Flow Dental, New York) before proceeding with actual procedure. The procedure was started with rubber dam isolation, caries excavation, if present, followed by access cavity preparation under 2% lignocaine hydrochloride (Cadila Pharmaceuticals Ltd., India) with 1:200,000 adrenaline. The pulp was extirpated, and a glide path was made using 10 and 15 no. K files (Mani Inc., Japan). Working length determination was done using Apex ID apex locator (SybronEndo, Orange, CA, USA) and confirmed using radiovisiography. The patients were divided into two groups: BODY.GROUP I: Group I consisted of thirty single- or multi-rooted teeth which were treated in multiple visits following a standardized protocol. It was further subdivided into two subgroups. BODY.SUBGROUP IA: In this subgroup, cleaning and shaping was carried out using HERO Shaper file system (Micro-mega, Besancon, France) in crown down manner. Copious irrigation was done with saline, 3% sodium hypochlorite (DentPro, India), and 2% chlorhexidine (Dentochlor, Ammdent, India) during and after instrumentation with canal prep (Ammdent, India) used as a lubricant. ApexCal medicament (Ivoclar-Vivadent, Schaan, Liechtenstein) was used into the root canal and temporarily restored with Cavitemp (Ammdent, India) and scheduled for a second visit 7–10 days later. At the second appointment, ApexCal was removed with copious irrigation. Corresponding 4% master cone radiograph was taken before obturation to confirm that it was at the correct working length. Canals were dried with paper points and obturated with gutta-percha cones (META Biomed Co., Ltd., Korea) and Apexit Plus (Ivoclar-Vivadent, Schaan, Liechtenstein) as a sealer, using lateral compaction technique. Access cavity was sealed using direct composite restoration, and immediate postobturation intraoral periapical radiograph was taken. BODY.SUBGROUP IB: In this subgroup, cleaning and shaping was done in the same manner as that of subgroup IA, but obturation was done using AH Plus (Dentsply Maillefer, Ballaigues, Switzerland) root canal sealer. BODY.GROUP II: Another thirty single- or multi-rooted teeth were treated in one visit. It was further divided into two subgroups. BODY.SUBGROUP IIA: In this subgroup, cleaning and shaping was done in the same manner as that of subgroup IA. However, after cleaning and shaping, canals were dried with paper points and obturated at the same appointment with gutta-percha cones with Apexit Plus sealer in similar method and materials used for subgroup IA. An immediate postobturation intraoral periapical radiograph was taken after permanent restoration. BODY.SUBGROUP IIB: In this group, cleaning and shaping was done in the same manner as that of subgroup IIA, but obturation was done using AH Plus sealer. A preoperative, immediate postoperative, and recall intraoral periapical radiographs were taken using the standardized paralleling technique with the help of paralleling kit. Patients were recalled at intervals of 1, 3, and 6 months to evaluate the treated teeth both clinically and radiographically. Clinical assessment included the presence of clinical signs and symptoms (spontaneous pain, presence of sinus tract, swelling, mobility, periodontal probing depths greater than baseline measurements, or sensitivity to percussion or palpation) at each follow-up visit. Radiographic evaluation was done using the PAI scoring system given by Orstavik in 1986.[7] PAI score description of radiographic findings: Normal periapical structures Small changes in bone structures Changes in bone structure with mineral loss Periodontitis with well-defined radiolucent area Severe periodontitis with exacerbating features. BODY.RESULTS: In the present study, sixty teeth were examined at 1-, 3-, and 6-month follow-up, thirty in Group I, further subdivided into subgroup IA and IB with 15 teeth each and thirty in Group II, further subdivided into subgroup IIA and IIB with 15 teeth each. Results were evaluated and tabulated. Clinical signs and symptoms at 1-, 3-, and 6-month follow-up examination were recorded and compared with preoperative records but not subjected to statistical analysis. Since clinical symptoms were rare during the follow-up period. Thus, the outcome was classified mainly on the radiographic evaluation using PAI scoring system. When scores obtained by radiographic evaluation were submitted to Kruskal–Wallis test, there was no statistically significant difference in periapical healing between teeth in subgroups IA, IB, IIA, and IIB at the baseline, 1-, 3-, or 6-month evaluation with P = 0.342, 0.839, 0.218, and 0.780, respectively. Results when evaluated by one-way ANOVA test revealed no significant difference between periapical healing of different subgroups from preoperative periapical status to immediate postoperative, 1-, 3-, and 6-month follow-up with P = 0.104, 0.368, 0.672, and 0.317, respectively [Table 1 and Graph 1]. Table 1Comparison of mean periapical index scores between Group I and Group II Graph 1Mean of change in periapical index score from pre- to postoperative follow. up conditions in all four subgroups indicating periapical healing Even though results when rendered to Wilcoxon signed-rank test showed no statistically significant difference between four subgroups, the decrease in mean PAI scores showed improvement in all the subgroups, with highly significant P ≤ 0.001 [Table 2]. Table 2Comparison of periapical healing on different intervals within the same groupsc BODY.DISCUSSION: The main objective for root canal treatment is to prevent or heal endodontic diseases such as apical periodontitis and to minimize patient discomfort. The basic biologic rationale for achieving ultimate success with root canal treatment consists primarily of eliminating microorganisms from the entire root canal system and creating an environment that is most favorable for healing.[8] Mechanical instrumentation and irrigation with antibacterial solutions have been considered essential for the elimination of bacteria during endodontic treatment, whereas the need for intracanal dressings has been questioned.[3] Only teeth with definite periapical lesions were included in the study making the comparison between treatment protocols viable. The expectation that teeth treated in two visits with an interappointment dressing would result in improved healing when compared with one-visit root canal therapy was not supported by this study. No statistically significant differences were found between the two treatment groups in the healing of periapical pathology. The findings of this study were consistent with the majority of well-controlled clinical studies.[59101112] When comparison was done between initial and final PAI score in each group, statistically significant improvement in PAI score was found in every group, which corroborates the study of Gesi et al. that with the proper use of aseptic operating procedures, proper instrumentation, and filling, an interappointment dressing with calcium hydroxide does not seem to influence outcome.[13] Successful endodontic therapy depends on many factors. One such important step in any endodontic treatment is obturation. The obturation requires the use of materials and techniques capable of densely filling the entire root canal system and providing a fluid tight seal from the apical segment of the canal to the cavosurface margin to prevent reinfection. A hermetic seal cannot be achieved with gutta-percha alone; hence, a root canal sealer is essential.[14] In this study, root canals were filled with AH Plus (Dentsply Maillefer, Ballaigues, Switzerland) and Apexit Plus sealer (Ivoclar-Vivadent, Schaan, Liechtenstein). However, no statistically significant relationship was found between the type of sealer used (Apexit Plus and AH Plus) and the healing of periradicular tissue, whether used during multiple or single-visit root canal treatment. These results were comparable with the findings of earlier studies done by Leonardo et al., Waltimo et al., and Tanomaru-Filho et al., who concluded that sealers had a statistically insignificant association to the rapid healing of apical periodontitis.[151617] Single-visit treatment has many potential advantages. It is less expensive, very well accepted by patients, has shown to result in a lower flare-up rates and similar periapical healing as that in case of multivisit root canal treatment. The findings of our study are also in accordance with the studies of Weiger et al., Sathorn et al., and Su et al. which showed that single-visit root canal treatment appeared to be slightly more effective than multiple visits. However, the difference in healing rate between these two treatment regimens was not statistically significant.[4618] These results are in contradiction with earlier studies by Sjögren et al. and Katebzadeh et al. which emphasized that the objective of completely eliminating bacteria from the root canal system before obturation cannot be reliably achieved in a one-visit treatment because it is not possible to eradicate all infections from the root canal without the support of an interappointment antimicrobial dressing and that the Ca(OH)2 treatment was statistically superior to one-step treatment.[819] The above results may be attributed to the difference in the sampling of patients, designs of the studies, and treatment procedures. The findings of our study give support to the assertion that no statistically significant differences occurred between single- and multivisit endodontic procedures when treating teeth with periapical rarefactions regardless of the technique used. The healing potential is within anticipated and acceptable levels when single visit is compared with multivisit cases. Based on clinical and radiographic outcomes of this study, it can be summarized that no additional benefit is provided by the use of an interappointment antibacterial dressing such as calcium hydroxide. Probably, elimination of bacteria is not strictly necessary and maximum reduction of bacteria, and effective canal filling may be sufficient in terms of healing, rather than complete eradication. BODY.CONCLUSION: Our study concludes that multiple and single-visit root canal treatment demonstrated almost equal success in endodontic treatment of teeth with periapical pathology. However, long-term follow-up and big sample size are required to further corroborate the findings of this study. BODY.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Effect of cross‐match on packed cell volume after transfusion of packed red blood cells in transfusion‐naïve anemic catsSYLVANE et al. ABSTRACT.BACKGROUND: Novel feline RBC antigens might contribute to decreased efficacy of RBC transfusion and increased incidence of acute transfusion reactions (ATR). ABSTRACT.OBJECTIVES: To examine the effect of major cross‐match in transfusion‐naïve anemic cats on the incidence of acute immunologic transfusion reaction and transfusion efficacy for up to 24 hours after transfusion. ABSTRACT.ANIMALS: Forty‐eight client owned transfusion‐naïve anemic cats. ABSTRACT.METHODS: Prospective, randomized, controlled study. All transfusion‐naïve cats receiving packed red blood cells (pRBC) transfusions from January 2016 to August 2017 were eligible for inclusion. Cats in the study group received cross‐match and blood type compatible pRBCs and cats in the control group received noncross‐matched blood type compatible pRBCs. Incidence of ATR and change in PCV after transfusion was recorded. ABSTRACT.RESULTS: No significant difference in incidence of transfusion reactions between cross‐matched and noncross‐matched groups (CM+ 4/24; 17%, CM– 7/24; 29%, P = .16). No significant difference between groups in mean change in PCV after transfusion scaled to dose of pRBCs administered at any time point after transfusion (immediate: CM+ 0.62 ± 0.59, CM– 0.75 ± 0.48, P = .41; 1 hour: CM+ 0.60 ± 0.66, CM– 0.74 ± 0.53, P = .43; 12 hours: CM+ 0.70 ± 0.55, CM– 0.66 ± 0.60, P = .81; 24 hours: CM+ 0.64 ± 0.71, CM– 0.55 ± 0.48, P = .70). ABSTRACT.CONCLUSIONS AND CLINICAL IMPORTANCE: Our results do not support use of the major cross‐match test to increase efficacy of, and to decrease adverse events associated with, RBC transfusion in AB blood typed transfusion‐naïve cats. BODY.ABBREVIATIONS: CM+cross‐matched, type specific transfusion, study group CM–noncross‐matched, type specific transfusion, control group FNHTRfebrile non‐hemolytic transfusion reaction Hbhemoglobin HCThematocrit HTRhemolytic transfusion reaction PCVpacked cell volume [pHb]plasma hemoglobin concentration pRBCpacked red blood cell RBCred blood cell 1BODY.INTRODUCTION: Blood transfusion to cats has become a fundamental component of advanced veterinary care over the past 25 years.1, 2, 3, 4 RBC transfusion is indicated to improve tissue oxygen delivery in cats with clinical anemia resulting from hemorrhage, hemolysis, or reduced RBC production. With the growing availability of feline blood components, there has been increasing interest in compatibility testing before transfusion.5, 6, 7, 8, 9, 10 The most well‐recognized feline blood group is the AB system, consisting of the A, B, and AB blood types. It is well established that feline blood contains naturally occurring alloantibodies against absent AB blood group antigens, and therefore it is imperative that all cats receive type‐specific blood.11, 12, 13 More comprehensive compatibility testing before transfusion includes the major and minor cross‐match which detect recipient antibodies to donor RBCs and donor antibodies to recipient RBCs, respectively. Currently, the cross‐match test is only recommended as a standard of care before transfusion in previously transfused cats, however, this practice has been challenged in recent years.1, 2, 13, 14 A novel RBC antigen, Mik, was discovered in a group of domestic short haired cats in 2007.6 Four Mik‐negative type A cats had an incompatible cross‐match with 30 Mik‐positive type A cats.6 Furthermore, one such Mik‐negative recipient had an acute, hemolytic transfusion reaction (HTR) after an AB type‐matched RBC transfusion.6 These results imply the presence of naturally occurring alloantibodies in feline blood which are not identified by conventional AB blood typing. Cross‐match before RBC transfusion is necessary for their detection. In 2014, a significantly greater increase in the PCV after transfusion in cats that received type‐specific cross‐match compatible blood as compared with cats that received type‐specific noncross‐matched blood was retrospectively documented.7 These results suggest there might exist a subclinical level of hemolysis in transfused RBCs because of undetected alloantibodies in uncross‐matched blood transfusions. However, markers of HTR such as changes in clinical variables during the transfusion period, plasma hemoglobin concentration [pHb], and serum bilirubin after transfusion are not reported in this study.7 Furthermore, because of its retrospective nature, the reported population was biased as the majority of cats in the noncross‐matched group were transfusion‐naïve, whereas the majority of cats in the cross‐matched group had received previous RBC transfusions or had unknown transfusion history.7 A prospective, randomized study is required to further investigate these findings. The purpose of this study was to examine the effect of cross‐match before transfusion in transfusion naïve anemic cats on the incidence of acute immunologic transfusion reaction for up to 24 hours after transfusion. Transfusion efficacy, as measured by PCV increase from baseline per mL/kg of packed red blood cells (pRBCs) transfused, was also evaluated. We hypothesized that transfusion of cross‐match and blood type compatible pRBCs would decrease the incidence of acute transfusion reactions (ATR) and result in an increased PCV after transfusion when compared to noncross‐matched cats given blood type compatible pRBCs. 2BODY.MATERIALS AND METHODS: The study protocol was approved by the Institutional Animal Care and Use Committee at The Animal Medical Center (protocol number AMC_11–24‐15). All transfusion‐naïve cats receiving pRBC transfusions from January 2016 to August 2017 were eligible for inclusion in this prospective, randomized controlled study. All owners signed an informed consent agreement regarding this study and financial incentives offered for entry. Exclusion criteria included pediatric cats (<4 months of age), unstable cats in whom delay in transfusion for cross‐matching purposes was deemed life‐threatening, and cats with unknown transfusion history. After enrollment, all cats were blood typed for the AB blood group. Each cat was randomized to have a major cross‐match performed (CM+/study group) or to not undergo any further testing before transfusion (CM–/control group). Cats in the study group received cross‐match and blood type compatible pRBCs and cats in the control group received noncross‐matched blood type compatible pRBCs. All cats received 1 unit of pRBCs at a rate determined by the primary clinician not to exceed 30 mL/h and to be completed in all cats within 4 hours. Cats received blood within 2–3 hours of initial PCV measurement. The start of transfusion for the CM– cats was purposely delayed by 2 hours to mimic the delay in transfusion time in the CM+ group because of laboratory reporting times. Information recorded for each cat included age, sex, breed, weight, reason for transfusion, volume of blood transfused, age of pRBCs, source of pRBCs, dose of pRBCs (mL/kg), PCV before transfusion, duration of time between PCV measurement before transfusion and pRBC administration, duration of time of transfusion, incidence of ATR, and PCV after transfusion measured immediately, 1, 12, and 24 hours after transfusion. Increase in PCV after transfusion was calculated and scaled to dose of pRBCs administered (%/mL/kg). If a cat was discharged from the hospital, died, or was euthanized before collecting the PCV measurements at 12 or 24 hours after transfusion then these values were not included in statistical analysis. The reason for transfusion was categorized into 3 discrete categories: blood loss, decreased RBC production, or increased RBC destruction. Each cat was assigned to one or more of these categories based on clinical assessment. Plasma hemoglobin concentration of each unit of pRBC was measured (HemoCue Hb 201, HemoCue America, Brea, California) before transfusion to ensure that transfused units did not already contain free Hb. Each cat also had a [pHb] measured (HemoCue Hb 201, HemoCue America, Brea, California) within 3 hours before transfusion as a baseline reading with subsequent measurements taken immediately after transfusion, 1, 12, and 24 hours after transfusion to screen for subclinical hemolysis. Febrile nonhemolytic transfusion reaction (FNHTR) was defined as an increase of the body temperature before transfusion by 1°C during the transfusion without evidence of intravascular hemolysis.15 Acute HTR was defined as an unexpected drop in the PCV or less than expected PCV after transfusion in association with elevated [pHb] after transfusion as well as clinical and laboratory abnormalities consistent with hemolysis. Expected increase in PCV after transfusion was defined as 1%/mL/kg of pRBCs.2, 4, 13, 16 2.1BODY.PRBC SOURCES AND TRANSFUSION: Packed red blood cells were obtained from one of three commercial blood banks (Ohio State University Blood Bank, Columbus, Ohio; Hemosolutions, Colorado Springs, Colorado; Animal Blood Resources International, Dixon, California). The blood banks used protocols to collect and process feline pRBCs similar to previously described methods.17 The blood banks reported that donor cats were not tested for the presence of Mik antigen. All transfused pRBC were administered before the unit's expiration date determined by the commercial blood bank. The pRBCs were administered as a constant rate infusion through a dedicated IV cannula or dedicated port of a multilumen cannula via syringe pump (Medfusion 3500, Smiths Medical ASD, St Paul, Minnesota) and microaggregate 18 μm filter, which is standard of care at our facility to control rate of blood administration more precisely (Hemo‐Nate filter, Utah Medical Products, Midvale, Utah). The protocol for pRBC transfusion was as follows: vital parameters (temperature, heart rate, respiratory rate, blood pressure) were recorded before initiation of the pRBC transfusion, every 15 minutes for the first 90 minutes of the transfusion, and then every 30 minutes until the end of the transfusion. The transfusions started at ¼ of the desired final rate and were then increased by 25% every 15 minutes until the final rate was achieved. 2.2BODY.BLOOD TYPE IDENTIFICATION: Blood type identification was performed in standardized fashion by trained laboratory or veterinary personnel using either the tube agglutination procedure or the card agglutination technique (RapidVet‐H Feline Blood typing card, DMS Laboratories, Flemington, New Jersey) as described previously.18 2.3BODY.CROSS‐MATCH PROCEDURE: Trained laboratory personnel performed all major cross‐match procedures in standardized fashion as reported previously5, 19 to detect for both macroscopic and microscopic evidence of agglutination and hemolysis. 2.4BODY.STATISTICAL ANALYSIS: Baseline descriptive statistics are presented as mean and standard deviation for normally distributed variables while non‐normally distributed variables are presented as median and range. Analyses of baseline variables between the CM– and CM+ groups was performed using ANOVA or the Wilcoxon as appropriate for the data distribution. The normality of the error residuals were analyzed by Kolmogorov‐Smirnoff test for descriptive and multivariate models. Analysis for proportions of categorical variables was evaluated with a Chi‐Square analysis or Fisher's Exact test where appropriate. A simple linear regression model was used to determine which of the following parameters were independently associated with the change in PCV after transfusion for each time point: age, reason for transfusion, dose of pRBCs (mL/kg), cross‐match status, and PCV before transfusion. All analyses were deemed significant at P < .05 and carried out using a commercially available statistical program (SAS Statistical Software, Version 9.2, SAS Institute, Cary, North Carolina). 3BODY.RESULTS: 3.1BODY.DEMOGRAPHICS: A total of 48 cats met the inclusion criteria and were enrolled in the study. Twenty‐four cats were randomized to the CM+ (24/48) and 24 to the CM– (24/48). The median age of the study population was 11 years (range 1–19 years), and the median weight was 3.95 kg (range 2.3–7.3 kg). There were 27 male cats (intact 2/27, castrated 25/27) and 21 female cats (intact 1/21, spayed 20/21). Breeds included domestic shorthair (40/48), Himalayan (2/48), Abyssinian (2/48), Siamese (1/48), Sphinx (1/48), and Maine Coon (2/48). There were 43 cats with blood type A (43/48; 90%), 4 cats with blood type B (4/48; 8%), and 1 cat with blood type AB (1/48; 2%). The reason for transfusion was categorized as decreased production (34/48; 71%), destruction (2/48; 4%), and blood loss (24/48; 50%). Twelve cats had more than one reason for transfusion and were therefore assigned to more than one group. Eleven of these cats (5 CM+ and 6 CM–) were categorized as decreased production and blood loss. One cat in the CM+ group was assigned to all three groups. Mean time from initial PCV to the start of pRBC transfusion was 2.23 hours (SD: 0.53). There was no significant difference between groups with regard to age, weight, breed, sex, blood type, PCV before transfusion, reason for transfusion, age of transfused pRBCs, source of pRBCs, and duration of time between PCV before transfusion and the start of the pRBC transfusion (Table 1). All cats received blood over 4 hours with the exception of 2 cats categorized as nonregenerative (one each from the CM+ and CM– groups). The mean volume of pRBCs administered was not significantly different between CM+ (29.56 ± 1.04) and CM– (29.54 ± 1.07) groups (P = .99). The mean dose of pRBCs administered was not significantly different between CM+ (7.42 ± 2.34 mL/kg) and CM– (8.38 ± 2.74 mL/kg) transfusions (P = .20) Table 1Comparison of demographic data between the cross‐match (study) group and the noncross‐match (control) group CategoryCM+CM– P valueSex .38 Female 12 (50) 9 (37.5) Male 12 (50) 15 (62.5) Age (years) 9.8 ± 5.0 10.7 ± 5.3 .54 Weight (kg) 3.9 ± 1.2 4.5 ± 1.44 .14 Breed .23 DSH 21 (87.5) 19 (79.2) Non‐DSH 3 (12.5) 5 (20.8) Blood type .38 A 22 (91.7) 21 (87.5) B 2 (8.3) 2 (8.3) AB 0 (0) 1 (4.2) Pre‐transfusion PCV (%) 16.3 ± 3.1 17.3 ± 4.2 .33 Time to transfusion (hours) 2.2 ± 0.5 2.2 ± 0.3 .58 Age of pRBCs (days) 12.9 ± 1.7 13 ± 1.8 .98 Source of pRBCs .76 Animal Blood Resourcesd 21 (87.5) 19 (79.2) Hemosolutionsc 2 (8.3) 3 (12.5) Ohio Stateb 2 (8.3) 2 (8.3) Reason for transfusion Destruction 1 (4.2) 1 (4.2) .25 Blood loss 12 (50) 12 (50) .51 ↓ Production 17 (70.8) 17 (70.8) .23 Abbreviations: CM+, cross‐match (study) group; CM–, noncross‐match (control) group; PCV, packed cell volume; DSH, domestic short hair; ↓, decreased; SD, standard deviation. Values are presented as mean ± SD for continuous variables and as number (%) for categorical variables. bOhio State University Blood Bank, Columbus, OH. cHemosolutions, Colorado Springs, CO. dAnimal Blood Resources International, Dixon, CA. 3.2BODY.CROSS‐MATCH COMPATIBILITY AND TRANSFUSION REACTION: In the CM+ group, each cat was cross‐matched to at least 2 units of pRBC except for one of the blood type B cats because of availability of only one unit of type B blood at that time. That cat was cross‐matched to the single unit of type B blood and was compatible. There were a total of 52 crossmatches performed, 10 of which were incompatible (19%). Five cats in the CM+ group were incompatible with a single unit (21%), one cat was incompatible with 2 out of 4 units tested, and one cat was incompatible with 3 out of 4 units tested. There were 4 transfusion reactions in the cross‐match group (4/24; 17%). Three (3/24; 13%) were FNHTR and 1 (1/24; 4%) was a suspected HTR. There were 7 transfusion reactions in the noncross‐match group (7/24; 29%) all of which were FNHTR. There was no significant difference in the incidence of any type of transfusion reaction between the CM+ and CM– groups (P = .16) or in the incidence of FNHTR between CM+ and CM– (P = .11). Plasma hemoglobin concentration was mildly elevated (range, 0.1–0.2 mg/dL) in 4/48 (8%) of the transfused pRBC units before transfusion. Plasma hemoglobin concentration before transfusion was 0 mg/dL for all cats. The [pHb] remained 0 mg/dL at all time points after transfusion except in the one cat (CM+ group) suspected to have had a HTR (pHb 0.4mg/dL and 0.7 mg/dL at 12 and 24 hours after transfusion, respectively). The suspected HTR was also characterized by an acute drop in the 24 hour PCV after transfusion as well as a new onset Heinz body anemia. 3.3BODY.PCV AFTER TRANSFUSION: There was no significant difference in mean PCV after transfusion scaled to dose of pRBCs administered between the CM+ and CM– transfusions for any time point (Table 2). Table 2Change in PCV after transfusion scaled to dose of pRBCs (%/mL/kg) TimeCM+CM– P value0 0.62 ± 0.59 0.75 ± 0.48 .41 1 0.60 ± 0.66 0.74 ± 0.53 .43 12 0.70 ± 0.55 0.66 ± 0.60 .81 24 0.64 ± 0.71 0.55 ± 0.48 .70 Abbreviations: pRBCS, packed red blood cells; CM+, cross‐match (study) group; CM–, noncross‐match (control) group; PCV, packed cell volume; mL/kg, milliliters per kilogram; SD, standard deviation. Values are presented as mean ± SD. Time is in hours after transfusion. Regression models were constructed to evaluate for independent predictors of PCV after transfusion at each time point and two significant linear regression models were established. The factors evaluated included age, cross‐match status, dose of pRBCs administered (mL/kg), reason for transfusion (decreased production, blood loss, destruction), and PCV before transfusion. Of these variables, only PCV before transfusion was a statistically significant predictor of PCV after transfusion at the immediate (F 1,47 = 4.76, adjusted r 2 = 0.094; coefficient −0.04, P = .034), and 1 hour (F 1,45 = 5.44, adjusted r 2 = 0.0741; coefficient −0.05, P = .024) time points after transfusion. Lower PCV before transfusion therefore leads to a higher PCV after transfusion in this model. Data was not collected at the 12 and 24 hour times after transfusion for some cats because of death, euthanasia, need for second RBC transfusion, or discharge from the hospital. In the CM+ group this included 1 cat at 12 hours and 10 cats at 24 hours after transfusion, and in the CM– group this included 5 cats at 12 hours and 10 cats at 24 hours after transfusion. 4BODY.DISCUSSION: This study aimed to address the limitations of previous retrospective studies on transfusion compatibility testing by prospective investigation. In our study, transfusion‐naïve cats that received blood type and cross‐match compatible pRBCs compared with those that received only blood type compatible pRBCs did not show significantly greater increases in PCV after transfusion when scaled by dose of pRBCs administered at any time point. Additionally, there was no significant difference in incidence of transfusion reactions between the CM+ and CM– groups. These results suggest that, if present, unidentified feline RBC antigens might not always be clinically significant. Compatibility testing before transfusion in cats has recently shifted focus from solely the AB blood type system to more recently identified RBC antigens such as the Mik antigen.6 Subclinical hemolysis from undetected antigens has been postulated to decrease the efficacy of feline blood transfusion or result in life threatening HTR.1, 2, 7, 13, 14 The major cross‐match procedure should detect these incompatibilities between donor and recipient blood and result in greater efficacy of transfusion as suggested by a recent study.7 However, that study design was retrospective and therefore had various limitations such as the inability to control for transfusion‐naïve cats, delay in transfusion for the cross‐matched cats, unknown pRBC delivery technique, lack of data on transfusion reactions, and substantial heterogeneity between groups.7 A substantial part of the rationale for the suspicion of subclinical hemolysis secondary to unidentified RBC antigens in cats has been the finding of a less than expected PCV after transfusion, which has been recently evaluated as a marker of the efficacy of RBC transfusion.1, 2, 7 An expected rise of 1% in the hematocrit (HCT) for each 1 mL/kg of pRBCs or 2 mL/kg of whole blood administered has been suggested.2, 4, 13, 19 Retrospective evaluation of the effect of cross‐match procedure on PCV after transfusion found an increase in PCV of only 0.78%/mL/kg pRBC in a group of uncrossmatched cats.7 Our study had a similar result of an immediate increase in PCV of 0.62%/mL/kg and 0.75%/mL/kg for the CM– and CM+ groups, respectively. The finding that CM+ and CM– groups in our study did not significantly differ with respect to PCV after transfusion at any time point suggests that these lower than anticipated values are not secondary to incompatibilities between donor and recipient blood. Other explanations for lower than expected PCV after transfusion include inaccuracy of formulae used to calculate PCV increase,16 repeated blood sampling,20 ongoing loss or destruction of RBCs, or dilution via administration of asanguinous fluids. Additionally, PCV after transfusion is dependent on the HCT of the transfused unit of pRBCs. Standard practice for blood banking in human transfusion medicine in the United States is to achieve a HCT of 55%‐65% for each unit of pRBCs.21 This accomplishes standardization such that 1 unit of pRBCs in a human will increase the HCT by ∼1%/mL/kg.22, 23 To our knowledge, the HCT of pRBC units from veterinary commercial blood banks has never been evaluated so based on our data and the data of others, extrapolation of these formulas to predict PCV after transfusion in cats could be inaccurate.7 Future studies incorporating HCT of pRBC units on predicting efficacy of transfusion are warranted to more specifically address the efficacy of feline pRBC transfusion. Additionally, a more accurate measure of efficacy of transfusion is via chemical labeling and flow cytometry detection of transfused RBCs, which accurately measures the half‐life of transfused RBCs in cats.24 Use of this technique in a future pretransfusion study is warranted. The current recommendation in human medicine is that the cross‐match procedure is only necessary if clinically relevant antibodies have already been identified on antibody screening test or if antibody screening test is not available.15, 25 Typically, >95% of humans have a negative antibody screening test and do not need to undergo further testing other than initial blood group identification.15, 25 In fact, a number of studies have shown a proportion of humans with negative antibody screening test to have an incompatible cross‐match result.15, 25 Furthermore, transfusion of these incompatible units did not result in clinical or serological evidence of hemolysis.15, 25 Therefore, the cross‐match might detect clinically insignificant antibodies in humans after conventional testing. In our study, 19% of all major cross‐matches performed in transfusion‐naïve cats in the CM+ group revealed an incompatibility. The inclusion of transfusion‐naïve cats in this study controlled for previous exposure to foreign RBC antigens so all incompatibilities identified here were primary in nature. The results of a major cross‐match are not commonly reported in transfusion‐naïve cats so the significance of this finding is unclear. However, similar to findings in human transfusion medicine, the results of our study suggest that cross‐match identified incompatibilities in transfusion naïve cats might not be clinically relevant. This conclusion is supported by the lack of a significant difference between CM+ and CM– groups with regards to PCV after transfusion and incidence of transfusion reactions. Febrile nonhemolytic transfusion reaction was suspected in 10 cats in the overall population, which is higher than previously reported. However, those numbers are derived from retrospective studies, which could have underestimated the occurrence of FNHTR.1, 2 The incidence of FNHTR should not be expected to differ between cross‐matched or noncross‐matched cats receiving blood products because both blood typing and the major cross‐match do not detect incompatibilities between donor and recipient leukocytes.5 Our results supported this concept as there was a similar incidence of FNHTR between CM– and CM+ groups. Our study screened for subclinical hemolysis to detect for any acute HTR. Plasma hemoglobin of the transfused units of pRBC as well as each cat was measured at all time points before and after transfusion. Two units of pRBCs in each group had a mild increase in the pHb before transfusion. This is suspected to have had negligible clinical implication because none of the cats that received those units had evidence of hemolysis; however, 1 of these cats was only monitored for 12 hours after transfusion because of euthanasia, which could have precluded detection of hemolysis. The other three cats were monitored for the full 24 hours. Furthermore, these units were not likely to contribute to decreased efficiency of transfusion because 3 of the cats that received these units had a higher PCV (%/mL/kg) after transfusion than the overall population (data not shown). One cat in the CM+ group had a suspected acute HTR. The HTR was characterized by a normal pHb before and after transfusion followed by a progressive increase in the 12 and 24 hours pHb after transfusion. This cat additionally had a drop in PCV from 34% to 22% at 24 hours after transfusion, as well as a new onset Heinz body anemia. Other causes of hemolysis in this cat were unlikely and included microbial contamination of the transfused unit, repeated blood sampling, underlying systemic disease or administration of drugs resulting in Heinz body hemolytic anemia, or hemolysis secondary to use of a mechanical delivery system.20, 24, 26, 27 This suggests that the transfusion of a major cross‐match compatible unit of pRBCs might not completely eliminate the risk of HTR. In fact, it has been shown that low titers of antibodies can be present below the threshold of major cross‐match detection.5, 6, 25, 28, 29 This therefore implies that the major cross‐match test might not be sensitive enough to detect all pRBC antibody incompatibilities between donor and recipient cats, which precludes its value as a pretransfusion test. It is also possible that hemolysis can occur from antibodies in donor plasma, which would only be detected by performing a minor cross‐match and would not be detected by the major cross‐match. The utility of the minor cross‐match should be evaluated in future prospective studies. Surprisingly, PCV before transfusion was a significant independent predictor of change in PCV after transfusion in this study, such that PCV before transfusion was inversely related to PCV after transfusion. This finding has been previously documented,7 however in the prior study, the pretransfusion PCV of the cross‐matched cats was significantly lower than that of the noncross‐matched cats. This suggests that the effect of PCV before transfusion rather than the effect of cross‐match could have contributed to the significant difference seen in PCV after transfusion between the groups.7 The implications of this phenomenon are unclear as, to the authors' knowledge, there are no studies investigating this topic in the human or veterinary literature. Many of the cats with the most severe anemia in our study likely suffered from chronic disease with secondary decreased production of RBCs and were not actively bleeding because an acute severe anemia would have required immediate pRBC transfusion resulting in ineligibility from this study. It is possible that chronically anemic cats could have developed protective mechanisms to reduce the clearance of endogenous or transfused RBCs, resulting in greater efficacy of transfusion. It is also possible that judicious administration of asanguinous fluids to cats that were more severely anemic could have resulted in a higher PCV after transfusion in those cats compared to the less anemic cats that received unrestricted fluids. Because we did not control for dose of fluids administrated IV in this study this could have been a confounding effect. However, none of the cats were administered IV fluids during the pRBC transfusion, so there should be no dilution effect from administration of IV fluids at the immediate PCV time point after transfusion. This finding should be further investigated. There are several limitations to this study. The HCT of transfused units of pRBCs was not recorded in this study and to the author's knowledge has never been assessed in the veterinary literature. If variation of commercial feline pRBC HCT exists, this would impact the results of this study as well as all studies investigating efficacy of pRBC transfusion in cats or use of formulas to predict PCV after transfusion. Future studies are warranted to further explore this topic. We were unable to collect data points at the 12 and 24 hours time after transfusion for some cats because of death, euthanasia, need for second RBC transfusion, or discharge from the hospital. It is possible that insufficient data at these time points could have masked a significant effect (type II error). However, this is considered unlikely because no effect was seen at the immediate or 1 hour after transfusion time periods and efficacy of a blood transfusion should not increase over time after the transfusion is finished. Biochemical profile, complete blood count, and urinalysis were not obtained after each transfusion to assess for variables consistent with hemolysis (ie, RBC morphology, total bilirubin, urine free Hb). Although [pHb] is an indicator of hemolysis, clinicopathologic data would have been helpful in supporting a diagnosis of AHTR or could have been used to investigate for other causes of hemolysis. Additionally, aerobic and anaerobic blood cultures of the transfused units were not performed to rule out microbial contamination as a source of transfusion reaction. Previous retrospective analyses have found no association of etiology of anemia on transfusion efficacy, similar to the results in our study.1, 4, 7 However, the classification scheme used in our study could have oversimplified the complex, multifactorial disease processes of these cats, resulting in failure to identify a significant difference between individuals. Additionally, some cats were classified into multiple categories for etiology of anemia, which could have precluded the ability to individually assess the impact of each type of anemia on PCV after transfusion. Hemolytic anemia was also uncommon in our study so the impact of this category of anemia on transfusion efficacy should be further assessed. Furthermore, critically ill cats were excluded from this study based on urgent need for transfusion so the results of this study might not apply to all feline transfusions. Future studies with a homogenous anemic population of cats are warranted. In conclusion, results of this prospective, randomized study do not support the major cross‐match test before transfusion to increase efficacy of and to decrease adverse events associated with RBC transfusion in AB blood typed transfusion naïve cats. Until the discovery of clinically relevant feline RBC antigens in addition to the AB and Mik blood group systems, or the development of an accurate antibody screening test for transfusion‐naïve cats, the major cross‐match procedure could still be warranted. This is particularly true when AB blood typing is not available. While our data do not support the major cross‐match test before transfusion, others recommend cross‐match when the Mik status of the donor and recipient cats are unknown.6 Further investigation evaluating PCV after transfusion with labeled RBCs or with assessment of the PCV of transfused units of pRBCs is warranted to further investigate determinants of PCV after transfusion. BODY.CONFLICT OF INTEREST DECLARATION: The authors declare that they have no conflict of interest with the contents of this article. BODY.OFF‐LABEL ANTIMICROBIAL DECLARATION: Authors declare no off‐label use of antimicrobials. BODY.INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION: The study protocol was approved by the IACUC at The Animal Medical Center (protocol number: AMC_11–24‐15).

TITLE: Visual Acuity Improvement in Continuous vs Divided Occlusion in Anisometropic Amblyopia ABSTRACT.PURPOSE:: To compare visual acuity improvement between continuous and split part-time occlusion for the treatment of moderate and severe anisometropic amblyopia. ABSTRACT.METHODS:: Randomised clinical trials in 6 – 13 y.o children with moderate and severe anisometropic amblyopia. Each patient was consecutively selected with continuous or split part-time occlusion. Best corrected visual acuity’s improvement was followed up to six weeks and statistical data were analyzed using chi square and unpaired t-test. ABSTRACT.RESULTS:: Best corrected visual acuity’s improvement was comparable between continuous and split part-time occlusion (0.20±0.27 vs 0.21±0.25; p = 0.79). ABSTRACT.CONCLUSION:: Split part-time occlusion may be considered as an alternative treatment for moderate and severe anisometropic amblyopia treatment. 1BODY.INTRODUCTION: Occlusion therapy is still the treatment of choice for anisometropic refractive amblyopia [1, 2]. Amblyopia may cause permanent universal vision loss in children. The prevalence of amblyopia in school-aged children in the world is 2% [1, 3-5]. Compliance is an important aspect in undergoing occlusion therapy. Several studies were conducted to increase treatment compliance for occlusion therapy, by dividing the occlusion half time continuously or divided [6-8]. This study aimed to compare the increase of visual acuity on moderate and severe anisometropic amblyopia between continuous occlusion half time and divided occlusion half time in order to assess its effectiveness as an alternative therapy. 2BODY.METHODS: The study was a controlled randomized clinical trial on patients with anisometropic refractive amblyopia aged 6 – 13 years from pediatric ophthalmology clinic in tertiary eye hospital, Bandung, Indonesia. The inclusion criteria were moderate and severe anisometropic amblyopia (with visual acuity in eye with amblyopia > 0.3 logMar, visual acuity difference between both eyes ≥ 0.2 unit logMar), had worn adjusted correction glasses for at least 4 weeks, had not received prior therapy for amblyopia. The exclusion criterion was patient being uncooperative during ophthalmological examination. The study was conducted upon approval from Ethics Committee of Medical Faculty Padjadjaran University. The patients were divided into 2 groups using random blocks permutation, on the first group continuous half time occlusion was performed and on the second divided half time occlusion was performed. Occlusion was performed using patch treatment as shown in Fig. (1). Continuous occlusion was performed continuously for 4 hours a day and divided occlusion was performed for 2 hours twice a day with an interval of 2 hours. The observation was performed for 6 weeks, assessing the visual acuity after usage of correction glasses for 4 weeks, visual acuity was performed on 2nd, 4th, and 6th week, while undergoing occlusion therapy. The visual acuity was evaluated using logMar chart from 4 meters distances. Compliance of treatment was observed by filling in the activity calendar accompanied with patients’ parents while undergoing occlusion therapy. Treatment compliance was divided into 3 categories: good (>85% occlusion time), moderate (70-85% occlusion time), and bad (<70% occlusion time). Statistical analyses used in this study were Chi Square test and unpaired t-test. The significance of the study p< 0.05. 3BODY.RESULTS: There were 30 subjects in the continuous occlusion group and 31 subjects in the divided occlusion group. Characteristics of the subjects are shown in Table 1. Occlusion was performed on the subjects for 6 weeks with 2 different occlusion techniques: continuous half time occlusion and divided half time occlusion. The resulting visual acuity and mean of visual acuity increase is shown in Table 2. No significant differences were found in increase of visual acuity and visual acuity difference between continuous half time occlusion group and divided half time occlusion group. (0.35 ± 0.26 vs 0.38 ± 0.22 (p = 0.72) ; 0.20 ± 0.27 vs 0.21 ± 0.25 (p= 0.79). The end result of visual acuity and mean of increase of visual acuity on moderate and severe amblyopia groups are shown in Table 3. Average of visual acuity in moderate amblyopia group after 6 weeks of occlusion treatment in group A was 0.22±0.1 logMar and in group B was 0.20±0.1 logMar. In severe amblyopia, the mean visual acuity after 6 weeks of occlusion treatment in group A was 0.53±0.31 logMar and in group B was 0.47±0.21 logMar. There was no significant difference between the visual acuity of moderate and severe amblyopia in both groups (p = 0.64; p = 0.52). The average increase of visual acuity in moderate amblyopia after 6 weeks of occlusion therapy in group A was 0.25±0.05 logmar and in group B was 0.27±0.08 logMar. The average increase of visual acuity in severe amblyopia in group A was 0.30±0.14 logMar and in group B was 0.37±0.15 logMar. There was no significant difference in increase of visual acuity between moderate and severe amblyopia in both groups. (p = 0.42; p = 0.19). 4BODY.DISCUSSION: Kane et al., had compared treatment compliance of continuous occlusion and divided occlusion therapy and had shown the identical increase of visual acuity in both groups (p = 0.82). [9] This study has shown that increase of visual acuity was not significantly different between both treatments (0.35±0.26 logMar vs 0.38±0.22 logMar; p = 0.72). This is different with the study conducted by Bhoompally et al, whose result was the visual acuity in the group that had received continuous occlusion therapy was better than the group that had received divided occlusion therapy (0.51±0.26 logMar vs 0.59±0.04 logMar; p = 0.27). [6] Different results were also acquired compared to the same study, in the group that had received continuous occlusion therapy had a higher increase of visual acuity compared to the group that had received divided occlusion therapy. (0.47±0.04 logMar vs 0.37±0.05 logMar; p = 0.15) [6] This study had shown that the mean of visual acuity increase on the continuous occlusion group was not significantly different compared to the divided occlusion group. (0.21±0.25 logMar vs 0.20±0.27 logMar; p = 0.79). This study had shown that, in moderate amblyopia, the mean of end visual acuity and the increase of visual acuity was higher in the divided occlusion group compared to the continuous occlusion group after 6 weeks of treatment. Meanwhile, in severe amblyopia, there was a difference, although statistically insignificant, in mean of end visual acuity and the increase of visual acuity between both groups. The end visual acuity and mean increase of visual acuity in severe amblyopia was higher in divided occlusion group compared to continuous occlusion group. This may be caused due to higher interocular visual acuity difference in the divided occlusion group compared to the continuous occlusion group (the visual acuity in amblyopic eye is worse compared to continuous occlusion group), making the occlusion therapy effects on visual acuity becoming more noticeable in this group. Several alternatives to improve comfort during occlusion therapy is to minimalize occlusion time by dividing the occlusion time and finding an alternative method for occlusion. One of them is by dividing occlusion time into several parts to increase treatment compliance. One of the most critical aspect in amblyopia treatment successistreatmentcompliance. According to a study conducted by Al-Zuhaibi et al. suggested that with bad treatment compliance affect success chance in treating amblyopia, with increase of visual acuity was strongly associated with treatment compliance in occlusion therapy (p = 0.08) [10] A study conducted by ATS 2B had found that the end visual acuity and increase of visual acuity in patients with moderate amblyopia in occlusion half time for 2 hours was as good as 6 hours of occlusion half time [7]. Identical results were acquired in severe amblyopia, according to a study conducted by ATS 2A, that had found the end visual acuity and increase of visual acuity in patients with severe amblyopia were better in half time occlusion for 6 hours compared to full occlusion.7 This study had indicated that shorter duration of occlusion increases therapy compliance. Singh et al, found that half time occlusion on mild amblyopia for 2, 4, and 6 hours were identically effective with full occlusion, while in severe amblyopia it has been shown that full occlusion and 6 hours occlusion were more effective compared to 2 hours occlusion [11]. Evaluated from compliance aspect, Jessica Kane et al. found the compliance in continuous occlusion group to be better compared to divided occlusion group (p = 0.023) [9]. According to a study conducted by Bhoompally et al. however, the compliance in divided occlusion (82%) was better compared to continuous occlusion (75%) [6]. The result differed with this study, where the compliance in continuous occlusion group (92%) was better compared to divided occlusion group (88%) although the difference was not statistically significant (p = 0.24). The difference of compliance levels may be caused by the duration of observation on both of the studies. The study conducted by Bhoompally observed the patient for 6 months compared to 6 weeks (which was performed in this study). Occlusion therapy for a long period of time may cause discomfort due to usage of occluder, thus reducing the treatment compliance. Additionally, usage of half time occlusion therapy may fit better to the patients’ schedule. In this study, the compliance was found to be good and moderate with an increase of visual acuity occurred in subjects with moderate and severe amblyopia. The limitations of the study were the small sample size and relatively short duration of observation. The difference in adaptation period of corrective glasses (between 4 -16 weeks) may be a confounding factor in the increase of visual acuity in occlusion therapy. BODY.CONCLUSION: In this study, the half time occlusion therapy resulted in an increase of visual acuity identical with full time occlusion therapy in anisometropic moderate and severe amblyopia. There were no difference in compliance between the group with continuous half time occlusion and the group with divided half time occlusion. Half time occlusion therapy may be used as an alternative for anisometropic moderate and severe amblyopia therapy.

TITLE: Phase II Prospective Randomized Trial of Weight Loss Prior to Radical Prostatectomy (PCAN-17-0128) ABSTRACT.BACKGROUND: Obesity is associated with poorly differentiated and advanced prostate cancer and increased mortality. In preclinical models, caloric restriction delays prostate cancer progression and prolongs survival. We sought to determine if weight loss (WL) in men with prostate cancer prior to radical prostatectomy affects tumor apoptosis and proliferation, and if WL effects other metabolic biomarkers. ABSTRACT.METHODS: In this Phase II prospective trial, overweight and obese men scheduled for radical prostatectomy were randomized to a 5–8 week WL program consisting of standard structured energy-restricted meal plans (1200–1500 Kcal/day) and physical activity or to a control group. The primary endpoint was apoptotic index in the radical prostatectomy malignant epithelium. Secondary endpoints were proliferation (Ki67) in the radical prostatectomy tissue, body weight, body mass index (BMI), waist to hip ratio, body composition, and serum PSA, insulin, triglyceride, cholesterol, testosterone, estradiol, leptin, adiponectin, interleukin 6, interleukin 8, insulin-like growth factor 1, and IGF binding protein 1. ABSTRACT.RESULTS: Twenty-three patients were randomized to the WL intervention and twenty-one patients to the control group. Subjects in the intervention group had significantly more weight loss (WL:−3.7 ± 0.5 kg; Control:−1.6 ± 0.5 kg; p=0.007) than the control group and total fat mass was significantly reduced (WL:−2.1 ± 0.4; Control: 0.1 ± 0.3; p=0.015). There was no significant difference in apoptotic or proliferation index between the groups. Among the other biomarkers, triglyceride and insulin levels were significantly decreased in the WL compared to the control group. ABSTRACT.CONCLUSIONS: In summary, this short-term WL program prior to radical prostatectomy resulted in significantly more WL in the intervention vs. the control group and was accompanied by significant reductions in body fat mass, circulating triglycerides, and insulin. However, no significant changes were observed in malignant epithelium apoptosis or proliferation. Future studies should consider a longer term or more intensive weight loss intervention. BODY.INTRODUCTION: Obesity is associated with poorly differentiated and advanced prostate cancer, increased risk of biochemical failure following radical prostatectomy and radiation therapy, and increased prostate cancer mortality (1–8). Multifactorial mechanisms may explain the link between obesity and the increased risk of advanced prostate cancer. Insulin metabolism, IGF-1, IGF binding protein, altered serum levels of sex hormones, pro-inflammatory cytokines, and adipokines may be involved (9–12). Visceral obesity may play an important role in the link between obesity and development of prostate cancer. One study among men in China showed that men in the highest quartile of waist-to-hip ratio had an almost 3-fold increased risk of developing prostate cancer (13). Another study described that central body fat mass was associated with increased high-grade prostate cancer (14). Likewise, increased periprostatic fat mass was associated with higher Gleason grade (15–17). Whether weight loss (WL) has the potential to delay prostate cancer progression is of great interest. In preclinical studies, energy restriction is well-known to decrease prostate cancer progression and prolong survival in mouse models (18–21). The IGF-AKT pathway, cytokines and adipokines, and microvessel density/vascular endothelial growth factor (VEGF) gene expression play a role in the decrease of prostate cancer progression in mouse models (18, 19, 21). However, evidence from human studies is inconclusive (22). To examine if WL has the potential to slow the progression of prostate cancer, we designed a prospective randomized pre-prostatectomy trial to determine whether WL from a hypocaloric diet and increased physical activity results in anti-proliferative and pro-apoptotic effects on prostate cancer tissue histopathology. Other examined endpoints included weight change, body composition and fat depots, and concentrations of potential mechanistic markers such as serum lipids, cholesterol, insulin, IGF-1, IGFBP-3, leptin, adiponectin, IL-6, IL-8, testosterone, and estradiol. BODY.MATERIALS AND METHODS: BODY.PATIENT ELIGIBILITY AND RECRUITMENT: Participants were recruited from the urology clinics at the Veterans Administration Greater Los Angeles Healthcare System, UCLA, and Santa Monica UCLA from 2009 to 2013. Inclusion criteria included BMI of > 25 kg/m2, physical ability to undergo a physical activity intervention, and able to come to the VA Clinical Research Center for 7 study visits if randomized to the WL group. Subjects were ineligible if they had Gleason score >4+5=9, a history of receiving androgen deprivation therapy, antiandrogen therapy, finasteride, radiotherapy, or cryotherapy. Patients were also excluded if they had a history of diabetes and were receiving insulin, if they were taking weight loss medication or enrolled in a weight loss program, taking lycopene supplements, or had significant co-morbidities (i.e. cardiac, pulmonary, liver disease, and ongoing alcohol/drug abuse) or a cardiac pacemaker. The study was approved by the Institutional Review Boards at the VA and UCLA and registered with ClinicalTrial.gov (NCT#00475982). BODY.STUDY DESIGN: This was a randomized two-arm open label intervention study. All subjects signed informed consent documents prior to study entry. Subjects were randomized using a permuted block design to either a weight loss program or control group.us After randomization, baseline weight, height, waist, hip circumference, and body composition by dual-energy X-ray absorptiometry (DEXA) (GE Lunar DEXA, Chicago, IL; maximum weight capacity 350 lbs) were determined, and fasting blood was collected. The control group proceeded with the scheduled radical prostatectomy. Within 3 days prior to radical prostatectomy, the control group had one additional visit to the clinical research center (CRC) for measurement of weight, height, waist, hip circumference, and fasting blood collection. Performance of a second DEXA scan in the control group prior to radical prostatectomy was added as a protocol modification after the beginning the trial, and therefore the first nine participants in the control group did not have an end of study DEXA scan. The control group was offered a free weight loss program after prostatectomy. Subjects randomized to the WL group received one of two standard structured energy-restricted meal plans (1200 and 1500 Kcal/day) using commercially available meal replacements and portion-controlled foods. Patients were taught how to count portions and how to consume the recommended calories from commercial ready-made frozen dinners. Prescribed plans were based upon lean body mass as determined by DEXA. The goal of the meal plan was to provide a total calorie intake incorporating 500–800 calorie deficits per day. Subjects had weekly visits with the CRC dietitian for the first 4 weeks, and every 2-week visits for the following 4 weeks and received instruction on recipes, grocery shopping strategies, education on healthy food preparation, and preparation of meal replacements. Prior to each visit, subjects in the WL group completed a 3-day food record to promote compliance and as a tool to assist the dietician with counseling. Subject’s wives or life partners were encouraged to attend and participate in the sessions. The goal for recommended diets was to contain 20–25% energy from fat, 15–20% from protein, and 50–65% from carbohydrates largely from fruits and vegetables with some whole grains. Fiber recommendations were a total of 25 grams/day from fruits, vegetables, legumes, and high fiber cereals. Patients in the WL group were counseled on performing 1 hour of exercise/day including aerobic, resistance, and flexibility activities such as walking and stretching. As an exercise incentive, patients were provided pedometers (Omron HJ-112 Digital Premium Pedometer, Bannockburn, IL) and exercise logs to complete and results were reviewed with the dietitian at each visit. As shown by Bravata et al., the use of pedometers is associated with significant increase in physical activity (23). Following the 5 to 8-week weight loss intervention and within 3 days prior to radical prostatectomy, weight, height, and waist to hip ratio were measured, fasting serum for biomarker analysis collected, and a DEXA scan was performed. BODY.OUTCOMES: The primary endpoint was apoptotic index in the radical prostatectomy malignant epithelium. Secondary endpoints were proliferation (Ki67) in the radical prostatectomy malignant epithelium, body weight, BMI, waist to hip ratio, body composition, and serum PSA, insulin, triglyceride, cholesterol, testosterone, estradiol, leptin, adiponectin, IL-6, IL-8, IGF-1, and IGFBP-1 levels. BODY.SERUM ANALYSES: Serum PSA, triglyceride, total cholesterol, LDL- and HDL-cholesterol, insulin, testosterone, and estradiol were measured by the UCLA clinical laboratory using standard laboratory methods. Serum leptin and adiponectin concentrations were measured using ELISA kits according to manufacturer instructions (Invitrogen, Carlsbad, CA) with coefficient of variation (CV) for leptin: Intra-assay CV 3.0–3.8% and inter-assay CV 3.9–4.6% (147–884 pg/mL), and adiponectin intra-assay CV 3.8–3.0% and inter-assay CV 5.2–2.8% (1.9–24.8 pg/mL). Serum IL-6 and IL-8 were quantified using ELISA kits from BD Biosciences (San Jose, CA) according to instructions with the following CV for IL-6: Intra-assay CV 4.1–10.8% (43.6–146.9 pg/mL) and inter-assay CV 10.9–7.9 % (39.1–147.8 pg/mL) and IL-8: Intra-assay CV 4.2–5.5% (27.4–92.1 pg/mL) and 3.4–3.2% (27.2–99.9 pg/mL). Plasma IGF-1 and IGFBP-1 concentrations were determined using a validated ‘in-house’ ELISA assay at the University of Southern California Aging Biomarker Service Core (Los Angeles, CA) with CV <10% (24). BODY.IMMUNOHISTOCHEMISTRY: Serial sections for immunohistochemical analyses were cut from archived paraffin embedded blocks with the largest cancer volume and with the Gleason grade corresponding to the grade on the final pathology report. Slides were stained for Ki67 (monoclonal mouse anti-human Ki67 antigen [Dako Omnis/Agilent, Santa Clara, CA]) and TUNEL (ApopTag® Plus Peroxidase In Situ Apoptosis Kit [Millipore, Temecula, CA]) (25) to assess proliferation and apoptosis, respectively. The areas of adenocarcinoma were circled by the study pathologist (J.S.) Slides were digitized on a ScanScope AT (Aperio Technologies, Inc., Vista, CA) and morphimetric analysis performed with Definiens’ Tissue Studio (Definiens Inc., Parsippany, NJ) to determine the percentage of Ki67 or TUNEL positive cells in a non-biased method. Briefly, using the pre-defined nuclear detection module and classification tool, positive and negative nuclei within each region of interest were identified. Thresholds were set to classify hematoxylin stain for negative nuclei and DAB stain for positive nuclei. The data were exported to Excel for further statistical analysis. The number of Ki67 or TUNEL-stained nuclei per total cells in the adenocarcinoma region was used to calculate the percent of positive stained cells (26). Scanning and analyses were performed through the Translational Pathology Core Laboratory, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA. BODY.STATISTICAL ANALYSIS: A sample size of 65 subjects [no weight loss = 35, weight loss = 30] was estimated to provide 80% power to detect an effect size of 0.74 using a two group t-test with a 0.050 two-sided significance when comparing the apoptotic index (primary outcome variable) in the radical prostatectomy malignant epithelium between the WL and no WL groups. To put this effect size in context, a similar study looking at a nutritional intervention by Kim et al. observed an effect size of 1.81 on apoptotic index using a nutritional intervention with similar study characteristics (27). To account for a post-randomization attrition rate of up to 15% in the WL group, we planned to randomize 35 subjects to the WL group. After 34 subjects fully completed the trial, an interim analysis was performed to evaluate the feasibility of finding a statistically significant difference between the groups for the primary outcome. A conditional power analysis demonstrated that, with enrollment of 65 participants, there was only a 0.9% chance of finding a significant difference in the apoptotic index between the treatment and control group. At this point, study enrollment was closed and secondary endpoint analyses were performed. Baseline patient characteristics were compared between groups using mean ± SD and frequencies. Secondary outcomes were measured both at baseline and prior to surgery and change from baseline to surgery was calculated. Within each group, changes from pre to post were calculated using the paired t-test. Next these changes were compared between control and WL intervention groups using the two sample t-test. For outcomes that had skewed distribution, a log transformation was implored prior to conducting the t-test. P values less than 0.05 were considered significant. Statistical analyses were carried out using IBM SPSS V24 (Armonk, NY). The data are presented as mean ± SD or SEM where appropriate. BODY.RESULTS: BODY.BASELINE CHARACTERISTICS: Twenty-three patients were randomized to the WL intervention and twenty-one patients to the control group. Seven patients in the WL group and three from the control group withdrew from the study with 34 patients completing the trial (Figure 1). The baseline characteristics of the 34 patients that completed the trial are shown in Table 1. All participants were overweight or obese. In the WL group, 44% were overweight, 44% obese, and 12% morbidly obese at baseline, while, in the control group, 31% were overweight, 44% obese, and 25% morbidly obese. Sixty seven percent of patients in the control group and 69% in the WL group were taking statins. The mean PSA in the WL group was 6.8 ± 2.5 compared to control group 7.5 ± 8.0 (Table 1). There was no significant difference in age, race, ethnicity, statin use, Gleason Score, and disease stage between the two groups (Table 1). BODY.BODY WEIGHT AND COMPOSITION: The mean time from study enrollment until radical prostatectomy was 62 ± 19 days in the control group and 51 ± 16 days in the WL group. Although patients had significant WL in both groups, subjects on the WL intervention had more weight loss (−3.7 ± 1.9 kg) compared to the control group (−1.6 ± 2.3 kg) (Table 2). Patients in the WL group also had a greater decline in BMI (−1.2 ± 0.6) as compared to the control group. (−0.5 ± 0.7) (Table 2). The WL intervention group had a greater decrease in fat mass (WL group −2.1 ± 1.8 vs. control group 2.2 ± 6.2) and decrease in percent gynoid fat (WL group 1.4 ± 1.9 vs. control group 0.2 ± 1.3, Table 2). There was a trend for a greater decrease in percent body fat in the WL group (p = 0.06) vs. the control group. No significant change was observed in lean body mass, percent trunk fat, and percent android fat between the groups. BODY.PROSTATE CANCER TISSUE APOPTOSIS AND PROLIFERATION: There was no significant difference in apoptotic index in radical prostatectomy tumor tissue (Primary Endpoint) as measured by TUNEL between the groups (Figure 2A). Likewise, there was no significant difference in the proliferation index as measured by Ki67 staining between the groups (Figure 2B). BODY.SERUM LIPID, ADIPOKINE, HORMONE, CYTOKINE, AND IGF/IGFBP-1: During the intervention period, there was a significant 33% decrease in serum triglyceride from 208 ± 194 to 140 ± 79 mg/dL in the WL group, while triglycerides increased in the control group from 131 ± 51 to 138 ± 71. Likewise, serum insulin levels decreased in the WL group from 17 ± 10 to 13.8 ± 7 and increased in the control group from 12 ± 7 to 14 ± 9 μIU/mL. There was no significant difference in serum total cholesterol or HDL-cholesterol between the WL and control groups (Table 3). There was a significant between group difference in LDL-cholesterol levels with a decrease in the control group as compared to the WL group. There were no significant between group changes in adipokines, testosterone, estradiol, IL-6, IL-8, IGF-1, and IGFBP-1 levels. BODY.DISCUSSION: Obesity is a well-established factor associated with increased risk and mortality of a number of human malignancies (28). Given the well-known impact of obesity on prostate cancer aggressiveness and mortality and the significant effect of caloric restriction on prostate cancer progression in preclinical models, there is strong evidence to support the conduct of clinical trials evaluating WL as a therapy for men with prostate cancer. Although the short-term WL intervention in our trial did not impact apoptosis or proliferation in radical prostatectomy malignant epithelium, biomarker results from our trial and others suggest the potential for clinical benefit of WL for men with prostate cancer. For example, a significant reduction in percent body fat was observed over the course of the trial. Previous studies showed that central body fat mass was associated with increased high-grade prostate cancer (14). Likewise, increased periprostatic fat mass was also associated with higher Gleason grade (15–17). Iyengar et al. recently reviewed the pro-inflammatory and pro-carcinogenic effects of hyperadiposity on the tumor microenvironment, and discussed the link between chronic low-grade inflammation and hyperinsulinemia (29). Noteworthy, in the current study circulating insulin levels were also reduced in the WL group (−18% compared to +18% in the control group), and insulin is a known growth factor for prostate cancer. A number of short-term clinical studies examined whether WL favorably modifies hormonal factors associated with prostate cancer progression. A small (n=11) short-term intensive WL intervention with a very low fat diet (<10% kcal from fat) in overweight and obese men was found to modify serum factors (IGF-1 decreased by 20%, IGFBP-1 increased by 53%, and insulin decreased by 25%) and decreased serum-stimulated growth of LNCaP cells was observed in an ex-vivo bioassay (30). It is noteworthy that patients lost more weight in this trial (4.2% WL over an 11-day period) as compared to our trial (1.5%WL in the control group and 3.7% WL in the treatment group), and in our trial IGF-1 and IGFBP-1 levels did not change. Given that the IGF axis may play a key role in the effects of weight loss on prostate cancer, it may be that the degree of weight loss in our trial was not adequate to affect tissue apoptosis. In another trial, Lin et al. reported that a 6-week low-fat, low-glycemic load diet resulted in significant WL (5.5%) and gene expression changes in 26 genes comparing biopsy tissue collected before and after diet intervention (31). In another small randomized trial, a 6-week caloric restricted diet in men with prostate cancer resulted in significant WL (−1.7%) and an increase in serum IGFBP-3 (+2.8%) compared to controls (WL +1%, IGFBP-3 −6.9%) (32). No significant changes in serum insulin, IGF-1 and adiponectin were observed (31). Heymach et al. conducted a controlled prospective randomized 4-arm pre-prostatectomy trial incorporating a low-fat diet and flax seed. They reported that WL significantly correlated with reduction in plasma VEGF levels, TNF-related apoptosis-inducing ligand (TRAIL) levels, and five other pro-inflammatory cytokines (33). Fabian et al demonstrated a 10% weight loss along with a significant decrease in Ki67 staining in breast cancer tissue (34). Another preprostatectomy weight loss study is ongoing (35). These investigators reported 9% weight loss in the intervention group and 6.2% weight loss in the control group, but have not yet reported on changes in tumor biomarkers (36). A number of mechanisms have been proposed linking obesity to advanced prostate cancer. As shown in animal studies, alterations in insulin metabolism, IGF-1, IGF binding protein, altered serum levels of sex hormones and pro-inflammatory cytokines, and adipokines might be involved (37, 38). The IGF axis is a frequently investigated target in prostate cancer since it plays an important role in cell proliferation, cell differentiation, apoptosis, and glucose and lipid metabolism (39). In a previous study by our group, long-term consumption of a low-fat/high-fiber diet, including soy for 6 months in prostate cancer patients following radical prostatectomy resulted in decreased serum IGF-1 compared with the consumption of the USDA recommended diet (40). In the present trial, we did not find any change in IGF-1 or IGFBP-1. In the current trial, we found a decrease in serum triglycerides, total cholesterol, and fat mass, which was expected with WL. A recent meta-analysis found that there was no relationship between serum triglyceride and prostate cancer risk (41). Although a decrease in fat mass may potentially result in a decrease of inflammatory cells in adipose tissue, we did not find any decrease in serum IL-6 or IL-8 levels. Noteworthy, in our present trial, there was significant WL in the control group (−1.6 ± 2.3 kg). However, between group comparisons demonstrated significantly more WL in the intervention group (−3.7 ± 1.9 kg) as compared to the control group. Through the standard consenting process, research subjects are aware of general aspects of the control and intervention groups and often desire randomization to the intervention group. Our trial and others demonstrated that patients randomized in the control group often self-implement behavioral changes and, in this case, achieved WL (42). In our trial, the incremental difference in weight loss between the intervention and control group was relatively low at 2.1 kg. Weight loss in the control group also occurred in other studies (36). Consideration should be given to implementing active interventions in the control group to prevent WL. Future trials should also consider more intensive weight loss interventions to achieve a greater difference in weight loss between the treatment and control groups. In this regard, data from our trial may be useful for power calculations for future randomized WL intervention trials in men with prostate cancer. A number of study designs may be appropriate for future WL intervention trials in men with prostate cancer. For example, men on active surveillance undergoing targeted same sight biopsies may be an ideal study population to undergo a long-term intervention. Another population to consider would be men on androgen deprivation for prostate cancer. Androgen deprivation is known to increase body fat, making this potentially an ideal population for future studies (43). In a prospective randomized trial incorporating diet and exercise, O’Neill et al. reported significant WL, reduction in total body fat, and improved functional capacity in men on androgen deprivation therapy, though they did not report on biomarkers related to prostate cancer progression (44). 27, 45). In addition, our trial was offered to all overweight and obese patients undergoing radical prostatectomy at the VA and UCLA. Future research should focus on selecting out specific patients more likely to respond to WL interventions based on inflammatory markers and thus incorporate “precision medicine” markers prior to enrollment. BODY.CONCLUSION: In summary, our short-term WL program prior to radical prostatectomy resulted in significantly more WL in the intervention vs. the control group and was accompanied by significant reduction in body fat mass, circulating triglycerides, and insulin levels. However, there were no significant changes in malignant epithelium apoptosis or proliferation levels. Based on the known association of obesity and lethal prostate cancer, future longer-term or more intensive weight loss intervention trials are warranted to further examine if WL has therapeutic benefits in men with prostate cancer.

TITLE: Use of 0.5% bupivacaine with buprenorphine in minor oral surgical procedures ABSTRACT.BACKGROUND:: Minor oral surgical procedures are the most commonly performed procedures by oral and maxillofacial surgeons. Performance of painless surgical procedure is highly appreciated by the patients and is possible through the use of local anesthesia, conscious sedation or general anesthesia. Postoperative pain can also be controlled by the use of opioids, as opioid receptors exist in the peripheral nervous system and offers the possibility of providing postoperative analgesia in the surgical patient. The present study compares the efficacy of 0.5% bupivacaine versus 0.5% bupivacaine with 0.3 mg buprenorphine in minor oral surgical procedures. ABSTRACT.PATIENTS AND METHODS:: The present study was conducted in 50 patients who required minor oral surgical procedures under local anesthesia. Two types of local anesthetic solutions were used- 0.5% bupivacaine with 1:200000 epinephrine in group I and a mixture of 39 ml of 0.5% bupivacaine with epinephrine 1:200000 and 1 ml of 300 μg buprenorphine (3 μg/kg)in group II. Intraoperative and postoperative evaluation was carried out for both the anesthetic solutions. ABSTRACT.RESULTS:: The mean duration of postoperative analgesia in bupivacaine group (508.92 ± 63.30 minutes) was quite less than the buprenorphine combination group (1840.84 ± 819.51 minutes). The mean dose of postoperative analgesic medication in bupivacaine group (1.64 ± 0.99 tablets) was higher than buprenorphine combination group (0.80 ± 1.08 tablets). There was no significant difference between the two groups regarding the onset of action of the anesthetic effect and duration of anesthesia. ABSTRACT.CONCLUSION:: Buprenorphine can be used in combination with bupivacaine for patients undergoing minor oral surgical procedures to provide postoperative analgesia for a longer duration. BODY.INTRODUCTION: Local anesthetic agents are the mainstay of perioperative pain control for most office-based oral surgical procedures. Amide types of local anesthetics (bupivacaine, etidocaine, lidocaine, mepivacaine, prilocaine, and articaine) with a moderate-to-long duration of action are commonly used for these surgical procedures.[1] Bupivacaine hydrochloride, introduced in 1963, is a long-acting amide type of local anesthetic. It is a powerful anesthetic with an intermediate onset of action (2–5 min), allowing a slow return to normal sensation (180–600 min).[2] It provides additional analgesia time, known as residual analgesia, and minimizes the duration of postoperative pain, facilitating postoperative care, and maintenance of proper oral hygiene.[3] Infection if present alters the ability of local anesthetic to achieve adequate pain control during surgery as the low pH of the inflamed tissue leads to quick dissociation of local anesthetic to cation form, which is not able to penetrate the phospholipid membrane of the neuronal cells. Locally injected opioids may act synergistically with local anesthetics in inflamed tissues and increase the perioperative analgesic effect.[4] Buprenorphine is a semi-synthetic, oripavine alkaloid derived from thebaine. It is a long-acting, lipid-soluble, mixed agonist-antagonist opioid analgesic which was first synthesized in 1966.[5] The low abuse liability of the drug in humans soon turned it into a widely used therapeutic agent in patients with opioid dependence. The principal clinical application of buprenorphine is as an analgesic for moderate-to-severe pain in perioperative setting.[6] The analgesic effect of buprenorphine appears to depend on the integrity of descending fibers from the rostral ventromedial medulla. Residual analgesic effects of opioids after inactivation of descending fibers may be caused by peripheral effects in the presence of inflammation.[7] Buprenorphine is shown to be fully efficacious with an antinociceptive potency 20-70 times higher than morphine. It binds to mu, kappa, and delta opioid receptors and dissociates slowly from these receptors. Buprenorphine acts as a partial mu opioid agonist and a kappa opioid antagonist.[8] The parenteral formulation of buprenorphine has an onset time of 5-15 min, and duration of action is about 8 h after administration. It is metabolized by the gut and liver. The various advantages associated with the use of buprenorphine are that it has a longer duration of analgesic action, low addiction propensity, and a high therapeutic index. The adverse effects associated with it include sedation, nausea, itching, constipation, addiction in higher doses, confusion, hallucinations, dry mouth, blurred vision, and respiratory depression with the overdose of drug.[67] The purpose of the study was to compare the efficacy of 0.5% bupivacaine versus 0.5% bupivacaine with buprenorphine in providing prolonged postoperative analgesia during various minor oral surgical procedures. BODY.PATIENTS AND METHODS: Fifty healthy adult patients who reported to the department of oral and maxillofacial surgery requiring minor oral surgical procedures were included in this study. Various minor surgical procedures included incision and drainage of abscess, removal of impacted third molars, apicoectomy, neurectomy, surgical extraction of teeth, cyst enucleation, and fracture reduction and fixation under local anesthesia. Diagnosis was made on the basis of history, clinical examination, and radiological examination. Patients were informed about the surgical procedure, postsurgical recommendations, and possible complications. Informed consent was obtained from each patient. Exclusion criteria included patients with a history of uncontrolled medical illness, sensitivity to local anesthesia, tolerance or addiction to analgesic drugs, pregnancy, bleeding disorders, chronic obstructive pulmonary disease, neurologic, psychiatric illness, or positive drug abuse history. The study protocol and informed consent form was approved by the Institutional Ethical Committee. BODY.TREATMENT GROUPS: Patients were assigned to one of the two equal groups by randomization method using table of random numbers. Patients in Group I (25 patients) received various intraoral nerve blocks as indicated using 0.5% bupivacaine with 1:200,000 epinephrine, whereas patients in Group II (25 patients) received the same blocks using the mixture of 39 ml of 0.5% bupivacaine with 1:200,000 epinephrine and 1 ml of 300 microgram buprenorphine (3 μg/kg). In addition, local infiltration was given in both the groups wherever needed to achieve hemostasis of the site. Analgesics were prescribed postoperatively only when the patient began to complaint of pain. The prescribed analgesic was tablet ketorol 10 mg (ketorolac 10 mg - Dr. Reddy's Laboratories Limited). Patients were asked to note down the number of tablets required, if any, to relieve pain. BODY.ASSESSMENT: It included evaluation of following parameters in both the groups intraoperatively and postoperatively: Total volume of anesthetic solution used during the surgery (in ml) Onset of action of anesthetic agent: The onset of anesthesia was determined by evaluating the subjective and objective symptoms of anesthesia of the respective nerve block used Duration of surgery after anesthetic administration (in minutes): The duration of surgery corresponded to the period between the first incision and the last suture Duration of anesthesia (in minutes): The duration of anesthesia was determined as the time from onset of anesthesia to the time when symptoms of anesthesia began to wear off Duration of postoperative analgesia (in minutes): The duration of postoperative analgesia was taken as the time from the end of surgery to the time for the need of first analgesic medication. The total amount of analgesic medication ingested during the postoperative period, and the percentage of patients who required medication in each group was also evaluated Efficacy of postoperative analgesia: The efficacy of analgesia was recorded with the aid of a 100 mm-length visual analog scale (VAS) with the markings between: 1–25: Mild pain 26–50: Moderate pain 51–75: Intense pain 76–100: Unbearable pain Each patient scored pain intensity every hourly for the first 10 h and then again at 24, 36, and 48 h. Patients were observed for side effects such as sedation, pruritus, nausea, vomiting, and respiratory depression. BODY.STATISTICAL ANALYSIS: The data obtained were subjected to statistical analysis and expressed as mean ± standard deviation. Unpaired t-test was used to analyze the data for the mean volume of anesthetic solution, onset and duration of anesthesia, postoperative analgesia, and duration of surgery for both the groups. Data for the percentage of patients taking postoperative analgesics were analyzed using nonparametric Chi-square test. Mann–Whitney test was used for the evaluation of pain with VAS because the data were not normally distributed, P < 0.01 was considered statistically significant. BODY.RESULTS: Out of total 50 patients selected for the study, there were 29 (58%) male and 21 (42%) female [Figure 1]. The mean age of patients was 27.60 years in bupivacaine group and 27.50 years in buprenorphine combination with bupivacaine group. Figure 1Demographic data Patients were divided into two groups and the surgical procedures included surgical removal of impacted mandibular third molars, incision and drainage of space infections, enucleation of cysts, dentoalveolar fractures, isolated mandibular fractures, maxillary fractures, elective implant removal, and excision of tumors [Figure 2]. Figure 2Various minor oral surgical procedures done In both groups, the minimum volume of anesthetic solution used was 4 ml. However, the mean volume of bupivacaine solution including the amount needed for infiltration/reanesthesia was slightly higher (4.15 ± 0.52 ml) than the mean volume of buprenorphine combination with bupivacaine solution (4.12 ± 0.60 ml). The difference in the mean volume of both the solutions used was found to be statistically nonsignificant (P = 0.84). The mean subjective onset of action in bupivacaine group (3.00 ± 1.08 min) was slightly longer than buprenorphine combination group (2.92 ± 1.03 min) whereas, on pinprick test, reverse was the case with the mean of 7.40 ± 1.93 min in buprenorphine combination group and 7.28 ± 1.59 min in bupivacaine group [Table 1]. Statistically, no significant difference was observed between both groups regarding subjective (P = 0.79) and objective onset of action (P = 0.81). Table 1Evaluation of onset of action Similarly, there was no significant difference in the duration of surgical procedure (P = 0.76) and duration of anesthesia with respect to initial recovery from anesthesia and complete recovery of sensation in both groups (P = 0.32, P = 0.63, respectively). However, the mean duration of postoperative analgesia in bupivacaine group (508.92 ± 63.30 min) was observed to be quite less than buprenorphine combination group (1840.84 ± 819.51 min), and value was found to be highly significant statistically (P < 0.001) [Table 2]. Table 2Evaluation of duration of anesthesia, postoperative analgesia, and duration of surgery On comparing the total amount of analgesic medication ingested during the postoperative period, the mean dose of analgesic medication in bupivacaine group (1.64 ± 0.99 tablets) was observed to be higher than buprenorphine combination group (0.80 ± 1.08 tablets). The difference between both the anesthetic solutions regarding number of analgesic tablets was found to be statistically significant (P = 0.006) [Table 3]. Table 3Evaluation of analgesic medication The percentage of patients who had taken analgesic medication in the early postoperative period (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h) and in the later postoperative period (12, 24, 36 and 48 h) was compared in both the groups, and it revealed that the patients in buprenorphine combination group had significantly less pain during both periods [Figure 3]. At the end of 10 h, all patients (100%) in buprenorphine combination group were pain free, as compared to only 4% of patients who were absolutely pain free in bupivacaine group. In this group, 12% of patients had mild pain but did not take analgesic medication. Only 84% of patients took analgesic medication till the 10th h evaluation. However, at 12, 24, and 36 h, 4%, 12%, and 16% of patients, respectively, in buprenorphine combination group had taken analgesic medication as compared to 100% of patients in bupivacaine group at all-time intervals. At the 48th h, 20% of patients were still pain free in buprenorphine combination group as compared to no pain-free patient in bupivacaine group. Figure 3Patients who had taken analgesic medication The difference between number of analgesic tablets taken in both groups was statistically highly significant at the 10th, 12th, 24th, 36th, and 48th h (P < 0.001) [Table 4]. Table 4Percentage of patients who had taken analgesic medication The efficacy of postoperative analgesia was recorded with the help of 100 mm-length VAS in both the groups [Figure 4]. Till the 5th postoperative hour, the P value was found to be statistically insignificant as all the patients in buprenorphine combination group had no pain and only three patients in bupivacaine group had very mild pain. Difference in median postoperative pain score between both anesthetic solutions was found to be statistically significant at the 6th and 10th h (P = 0.001). However, it was observed to be statistically highly significant from 7th h to 9th h (P < 0.001). As all the patients in the bupivacaine group had taken analgesic medication by the 12th h, they were excluded from further statistical analysis [Table 5]. Figure 4Evaluation of pain using visual analog scale Table 5Evaluation of pain using visual analog scale None of the patients in either group reported opioid-related side effects such as nausea, vomiting, pruritus, or any evidence of respiratory depression during intraoperative and postoperative assessment. BODY.DISCUSSION: Pain may be described as an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Buprenorphine has been used for the treatment of acute and chronic pain as a supplement to anesthesia for behavior and psychiatric disorders and as a maintenance medication for heroin dependence.[67] The analgesic effect of buprenorphine appears to depend on the integrity of descending fibers from the rostral ventromedial medulla. Prolonged duration of analgesia is due to the fact that buprenorphine dissociates very slowly from opioid receptors. The postoperative analgesic effects of buprenorphine added to local anesthetic have been discussed by various authors.[78910] Being a partial mu opioid agonist, buprenorphine has a wider safety profile as compared to full mu agonists. Further, the slow dissociation of buprenorphine from the receptor may result in fewer signs and symptoms of opioid withdrawal upon termination of buprenorphine therapy than those which occur with full mu opioid agonists such as morphine, heroin, and methadone. Antagonist effects at the kappa receptors are associated with limited spinal analgesia, dysphoria, and psychomimetic effects.[6] Buprenorphine can be used by various routes in human beings such as epidural, intravenous, intramuscular, sublingual, transdermal, delivery routes[678] and for intraoral nerve blocks.[8] The pH of the tissue and pKa of drug are the most important factors which affect the time of onset of anesthesia. The pKa defines the pH at which the ionized and nonionized forms of a drug are in complete equilibrium, that is, half of the drug is ionized. Only the nonionized form of the local anesthetic can diffuse across lipid nerve sheath and cell membrane. pKa also reflects the proportion of local anesthetic that is in a diffusible nonionized state and therefore contributes greatly to the rate of onset of anesthesia. The pKa values of commonly used local anesthetics are greater than the normal tissue pH (approximately 7.4) which means the drugs exist predominantly in the ionized form after injection. At normal tissue pH, the proportion of nonionized form of bupivacaine is 20%; this contributes in part to slightly slower onset of anesthesia with bupivacaine, particularly for nerve block anesthesia.[21112] In the present study, the mean value for time of onset as referred to lip and tongue numbness and also on pinprick test in bupivacaine group was 3.00 ± 1.08 and 7.28 ± 1.59 min, respectively, as compared to 2.92 ± 1.03 min and 7.40 ± 1.93 min, respectively, in buprenorphine combination group. Contrary to our results, Brkovic et al.[13] found the mean time of onset as referred to lower lip numbness as 8.7 ± 2.2 min and 7.4 ± 1.4 min on pinprick test while using 0.5% bupivacaine (2 ml) for the lower third molar surgery. Swarnkar et al.[14] reported the onset time of sensory block (with intravenous regional anesthesia) when 0.3 mg buprenorphine was added to the local anesthetic as 5.0 ± 1.0 min and the onset time of sensory block as 4.0 ± 0.4 min when 0.3 mg buprenorphine was given intramuscularly. Sarkar et al.[15] reported the onset time of sensory block as 3.28 min (supraclavicular block) when 1 ml (0.3 mg) buprenorphine was added to the mixture of local anesthetics. Our results are in agreement with those of Trullenque-Eriksson and Guisado-Moya[3] who found the mean onset of time for inferior alveolar nerve block as 3.68 ± 3.11 min and for buccal nerve as 1.95 ± 1.25 min while using 0.5% bupivacaine with 1:200,000 epinephrine for surgical extraction of mandibular third molars. Duration of the effect of an anesthetic is proportional to its degree of protein binding. Those agents who have a high affinity for the protein component of a nerve are less liable to diffuse from the injection site and be absorbed into the systemic circulation. Bupivacaine's long duration of action is largely due to this characteristic. Bupivacaine has one of the greatest protein binding values out of all the amide local anesthetics. The reported protein binding value for bupivacaine is 95%. However, the duration of the effect of the local anesthetic is also dependent on the injection site and the concentration of vasoconstrictor present in the anesthetic solution.[21112] In the present study, there was no statistically significant difference in the mean duration of anesthetic effect of 0.5% bupivacaine with epinephrine 1:200,000 (526.84 ± 101.13 min) and buprenorphine combination group (540.80 ± 103.78 min). Our results are in agreement with Brkovic et al.[13] who reported that duration of anesthesia was slightly longer with 0.5% bupivacaine (688 ± 85 min and 550 ± 48 min) as compared to 0.75% ropivacaine group (582 ± 67 min and 450 ± 73 min) with reference to lower lip numbness and pinprick test, respectively. In contrast to the present study, Sarkar et al.[15] reported the total duration of sensory block and motor block when 1 ml (0.3 mg) buprenorphine was added to the mixture of local anesthetics for supraclavicular block as 261.84 ± 53.30 min and 328.32 ± 47.94 min, respectively, which was less than the present study. Trullenque-Eriksson and Guisado-Moya[3] reported the duration of soft-tissue anesthesia being 8.20 ± 4.54 h in case of 0.5% bupivacaine with 1:200000 epinephrine group. Our results are also supported by Sancho-Puchades et al.[16] who compared 4% articaine and 0.5% bupivacaine both with epinephrine 1:200000 and reported that mean duration of soft-tissue anesthesia with respect to the final lip recovery (621.2 ± 148.4 min) was longer than final tongue recovery (512.1 ± 127.3 min). Singam et al.[17] reported the total duration of sensory block and motor block when 2 ml (0.3 mg) buprenorphine was added to the local anesthetic and then used for supraclavicular block as 647.83 ± 55.70 min and 306.33 ± 20.12 min, respectively, which is quite close to the findings of the present study. Postoperative pain control has been the subject of continuous research in the field of oral and maxillofacial surgery since pain can interfere with patient's quality of life. Surgical trauma elicits a variety of tissue responses producing and releasing biochemical mediators involved in the pain process.[18] Several authors have studied the various methods to control pain, such as the use of long-acting local anesthetics to decrease analgesic intake, the preoperative prescription of steroidal anti-inflammatory drugs for decreasing edema, pain and the postoperative use of soft-tissue lasers for better healing, and less postoperative pain and inflammation.[19] In the present study, duration of postoperative analgesia in bupivacaine group was 508.92 ± 63.30 min and in buprenorphine combination group being 1840.84 ± 819.51 min. Opioids exert their analgesic effect by acting exclusively in the central nervous system. Various mechanisms are proposed for activation of opioid receptors on peripheral neurons. Opioids increase potassium current and decrease calcium current in the cell bodies of sensory neurons. This inhibits the neuronal firing and transmitter release as well as the calcium-dependent release of excitatory pro-inflammatory compounds (e.g., substance P) which contributes to their analgesic and anti-inflammatory actions Opioid antinociceptive effect is particularly prominent in inflamed tissue Inflammation disrupts the perineurium (normally an impermeable membrane) and facilitates the passage of corticotropin-releasing hormones, interleukin 1B, and other cytokines. These substances apparently stimulate the release of opioid peptides from immune cells which activate opioid receptors on the sensory nerve endings leading to antinociception Inflammation also enhances the peripherally directed axonal transport of opioid receptors (dorsal root ganglia → peripheral) which leads to receptor upregulation (increase in their number in peripheral nerve terminals). Furthermore, the previously inactive opioid receptors become active in an inflamed tissue enhancing the analgesic potential of Opioids. Prolonged duration of analgesia is due to the fact that buprenorphine dissociates very slowly from opioid receptors.[8202122] Our results are supported by Nespeca,[23] Bouloux and Punnia Moorthy,[24] and Modi et al.[8] who found duration of postoperative analgesia in 0.5% bupivacaine with 1:200000 epinephrine groups as 449 ± 19.25 min, 480 min, and 500.4 ± 6.6 min, respectively, in their studies. Viel et al.[9] reported duration of postoperative analgesia when 0.3 mg buprenorphine was added to 0.5% bupivacaine in brachial plexus block as 2103 ± 117 min. Our results are supported by Modi et al.[8] who found duration of postoperative analgesia in Group II patients (0.3 mg of buprenorphine combined with 0.5% bupivacaine with epinephrine 1:200,000) as 1690.8 ± 61.2 min. The authors stated that the addition of buprenorphine to the local anesthetic mixture prolonged the duration of postoperative analgesia up to three times provided by the local anesthetics alone. 2 is an abundant eicosanoid released after surgical trauma and has been associated with inflammation and pain. The synthesis of prostaglandin is suppressed by a number of anti-inflammatory agents including the nonsteroidal anti-inflammatory drugs.[2526] In the present study, the mean dose of postoperative analgesic medication tablet ketorol DT 10 mg (ketorolac 10 mg Dr. Reddy's) in bupivacaine group ([1.64 ± 0.99 tablets] 16.4 ± 9.9 mg) was higher than buprenorphine combination group ([0.80 ± 1.08 tablets] 8 ± 10.8 mg). Trieger and Gillen[25] reported that patients receiving 0.5% bupivacaine with epinephrine 1:200000 required only 2.3 doses of codeine phosphate (30 mg) as postoperative analgesics. Our results are supported by Crout et al.[26] who found that only 1.4 ± 1.0 tablets of postoperative analgesic medication (325 mg acetaminophen with codeine 30 mg) were required in 0.5% bupivacaine with epinephrine 1:200000 group. Contrary to our results, Brkovic et al.[13] reported that there was no requirement of analgesic tablet in 0.5% bupivacaine group patients. Swarnkar et al.[14] found that 56 ± 9 mg of postoperative analgesic medication (diclofenac 1 mg/kg) was consumed when 0.3 mg of buprenorphine was added to local anesthetic for intravenous regional anesthesia. Analgesic dose consumption was higher (120 ± 24 mg) when 0.3 mg of buprenorphine was given intramuscularly as compared to intravenous route. Mehta et al.[21] reported that total dose of postoperative analgesic medication (tramadol 50 mg) consumed was 12.5 ± 5.38 mg when 2 μg/kg of buprenorphine was added to local anesthetic for wound infiltration. In the present study, VAS was used for assessment of postoperative pain. At the end of 10th h, all patients (100%) in buprenorphine combination group were pain free, as compared to only 4% pain-free patients in bupivacaine group. At the 48th h, 20% of patients were still pain free in buprenorphine combination group as compared to none in the bupivacaine group. Viel et al.[9] compared the effectiveness of both buprenorphine and morphine when used in combination with local anesthetic (5% bupivacaine) in brachial plexus block for postoperative pain relief. The authors reported that 12 h postanesthesia, all patients (100%) had satisfactory or tolerable anesthesia in buprenorphine group as compared to 80% of patients in morphine group. At the 48th h, 5% of patients were still pain free in buprenorphine combination group as compared to no pain-free patient in morphine combination group. Candido et al.[10] also studied that the addition of buprenorphine to local anesthetic used for brachial plexus block provided long-lasting postoperative analgesia with complete analgesia persisting 30 h beyond the duration (6 h) provided by local anesthetic alone in 75% of patients. In their study, at the 48th h evaluation, 10% of patients were pain free in buprenorphine combination group as compared to no pain-free patient in local anesthetic group. Their findings are in agreement with the present study and with the findings of Modi et al.[8] in which also postoperatively at the 48th h evaluation, 20% of patients of buprenorphine combination group were pain free as compared to no pain-free patient in bupivacaine group. BODY.CONCLUSION: Buprenorphine in combination with 0.5% bupivacaine group in comparison to 0.5% bupivacaine group alone provided a longer duration of postoperative analgesia and markedly decreased the need for analgesic medication in postoperative period. Overall, buprenorphine is a highly effective analgesic for the treatment of moderate-to-severe pain. It has a unique pharmacological and physiochemical profile allowing for relatively safe use and flexibility with regard to dosage and dosage forms. Thus, buprenorphine can be used in combination with bupivacaine for patients undergoing minor oral surgical procedures to provide postoperative analgesia for a longer duration, but it should be used cautiously in individuals with a past or current history of substance abuse or dependence, as it produces opioid-like subjective and physiologic effects dependent on the dose and the route of administration. BODY.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONFLICTS OF INTEREST: The authors wish to state that this paper does not have any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work.

TITLE: Effect on survey response rate of hand written versus printed signature on a covering letter: randomised controlled trial [ISRCTN67566265] ABSTRACT.BACKGROUND: It is important that response rates to postal surveys are as high as possible to ensure that the results are representative and to maximise statistical power. Previous research has suggested that any personalisation of approach helps to improve the response rate. This experiment tested whether personalising questionnaires by hand signing the covering letter improved the response rate compared with a non-personalised group where the investigator's signature on the covering letter was scanned into the document and printed. ABSTRACT.METHODS: Randomised controlled trial. Questionnaires about surgical techniques of caesarean section were mailed to 3,799 Members and Fellows of the Royal College of Obstetricians and Gynaecologists resident in the UK. Individuals were randomly allocated to receive a covering letter with either a computer printed signature or a hand written signature. Two reminders were sent to non-respondents. The outcome measures were the proportion of questionnaires returned and their time to return. ABSTRACT.RESULTS: The response rate was 79.1% (1506/1905) in the hand-signed group and 78.4% (1484/1894) in the scanned and printed signature group. There was no detectable difference between the groups in response rate or time taken to respond. ABSTRACT.CONCLUSION: No advantage was detected to hand signing the covering letter accompanying a postal questionnaire to health professionals. BODY.BACKGROUND: Large surveys of clinical practice are often conducted by postal questionnaire, as this is a practical and economical method of obtaining information. However, if the response rate is low, the respondents may represent a biased sample of the population. It is therefore desirable that the response rate to a postal survey be as high as possible, to ensure that there is sufficient statistical power and the findings are representative. One factor that may influence response rate is whether the covering letter that is sent out with the questionnaire is hand signed or bears a photocopied or printed signature. The rationale is that individuals who receive a hand signed covering letter may feel that the letter is more personalised to them, and hence may be more likely to complete and return the questionnaire than those who receive a letter with a printed signature. A recent systematic review [1] included a meta-analysis of randomised controlled trials investigating the effects of personalisation of covering letters accompanying postal questionnaires. The results suggested that an approach in the form of personalised letters, questionnaires or envelopes improved final response rates by a modest amount (odds ratio 1.16, 95% confidence interval (1.07, 1.26)). However, various methods of personalisation such as hand written versus typed salutations and hand written versus typed postscripts were used by the studies in this analysis. Only five of the 48 studies in this analysis evaluated the effects of hand signing letters versus duplicated or printed signatures, and none of these involved a questionnaire sent to health professionals. These studies also had methodological problems such as low response rates and small sample sizes. None has suggested any major advantage to one group or the other [2-6]. The randomised controlled trial reported here aimed to determine whether a hand written or computer printed signature on the covering letter influenced the proportion of questionnaires returned or the time taken to return them, in a survey of clinical practice among obstetricians and gynaecologists in the UK. BODY.METHODS: BODY.SETTING AND PARTICIPANTS: This study was conducted as part of a national postal survey of surgical techniques used in caesarean section operations in the UK [7]. The survey questionnaire was sent to all Members and Fellows of the Royal College of Obstetrician and Gynaecologists (RCOG) resident in the UK. The three page questionnaire, comprising 27 questions, asked for information about obstetricians' usual technique for lower segment caesarean section operations. Addresses were obtained from a database supplied by the RCOG. This contained 3,969 names and addresses, of which 170 were ineligible to receive the survey questionnaire; 169 had an address not in the UK, and one was an investigator for this study (PB). 3,799 questionnaires were therefore sent out. BODY.INTERVENTIONS: The 3,799 survey recipients were randomly allocated (using a random number generator) to receive the covering letter accompanying the questionnaire signed by hand or bearing a computer printed signature. In the hand signed group, all letters were signed by the same investigator (PB) in blue ink. In the computer printed group, PB's signature was scanned into a computer file, which was imported into each letter and printed along with the letter in black ink. All letters had a personal salutation, and the letters were identical apart from the signature. Each recipient received a copy of the questionnaire, the covering letter and a return white envelope labelled with a FREEPOST address. The survey was sent out in February 1999. Two reminders were sent to non-respondents, six and ten weeks after the initial mailing. Reminder letters included computer printed or hand written signatures according to the original allocation. The sample size of 3,799 was sufficient to detect an absolute difference in the response rate between the groups of just over 4%, with 80% power, assuming a response rate of 70% in the control group. BODY.RESULTS: By October 1999, 2,990 questionnaires had been returned, giving an overall response rate of 78.7%. The response rate was 79.1% (1506/1905) for the hand signed group and 78.4% (1484/1894) for the computer printed group. These proportions were not significantly different (risk ratio 1.01, (95% confidence interval 0.98, 1.04); risk difference +0.7% (95% confidence interval -1.9%, +3.3%)). The overall median time taken to return the questionnaire was 16 days (interquartile range 9 to 49 days). Kaplan-Meier survival analysis was performed on time to response and no difference between the groups was detected (Log rank = 0.72, p = 0.39; Figure 2). The survival curves for the two groups were very close at all time points, and hence it is unlikely that there could be an advantage to either group in the number of reminder letters needed. BODY.DISCUSSION: This study was large enough to detect a difference between the intervention and control groups of 4% or larger. The results indicate that a hand-signed covering letter is unlikely to improve the response rate or time taken to respond to a postal survey when compared with a covering letter bearing a computer printed signature. However, smaller differences between the two groups cannot be excluded and there may be a small advantage to personalisation. These findings, in a group of health professionals, support those from previous randomised trials of this intervention using other groups of respondents [2-6]. Combining the results of the existing trials in a fixed effects meta-analysis gave a summary risk ratio of 1.01 (95% CI 0.98, 1.04; Figure 3). BODY.CONCLUSION: The findings suggest that there may be no or only a very small advantage to hand signing of covering letters, and future postal surveys could use covering letters with scanned and printed signatures without compromising the response rate. However, other methods of personalising covering letters (such as handwritten postscripts or salutations) may be more beneficial in terms of enhancing response rates to questionnaires. BODY.COMPETING INTERESTS: The author(s) declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: PB was involved in study design and delivered the intervention. SG was involved in study design, conducted the analysis and helped to draft the manuscript. KM was involved in study design, data management and helped to draft the manuscript. SA was involved in study design, data management and analysis. LT was involved in the analysis. All authors read and approved the final manuscript. Figure 1Flow chart of study participants. Figure 2Time to return of questionnaires. Figure 3Meta-analysis of randomised controlled trials comparing hand written with printed signature on a covering letter accompanying a questionnaire. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here:

TITLE: Desvenlafaxine Versus Placebo in a Fluoxetine-Referenced Study of Children and Adolescents with Major Depressive Disorder ABSTRACT.ABSTRACT: Objectives: To evaluate the short-term efficacy and safety of desvenlafaxine (25–50 mg/d) compared with placebo in children and adolescents with major depressive disorder (MDD). Methods: Outpatient children (7–11 years) and adolescents (12–17 years) who met DSM-IV-TR criteria for MDD and had screening and baseline Children's Depression Rating Scale–Revised (CDRS-R) total scores >40 were randomly assigned to 8-week treatment with placebo, desvenlafaxine (25, 35, or 50 mg/d based on baseline weight), or fluoxetine (20 mg/d). The primary efficacy endpoint was change from baseline in CDRS-R total score at week 8, analyzed using a mixed-effects model for repeated measures. Secondary efficacy endpoints included week 8 Clinical Global Impressions–Severity, Clinical Global Impressions–Improvement (CGI-I), and response (CGI-I ≤ 2). Safety assessments included adverse events, physical and vital sign measurements, laboratory evaluations, electrocardiogram, and the Columbia-Suicide Severity Rating Scale. Results: The safety population included 339 patients (children, n = 130; adolescents, n = 209). The primary endpoint, change from baseline in CDRS-R total score at week 8, did not statistically separate from placebo, for either desvenlafaxine (adjusted mean [standard error] change, −22.6 [1.17]) or fluoxetine (−24.8 [1.17]; placebo, −23.1 [1.18]). Week 8 CGI-I response rates were significantly greater for fluoxetine (78.2%; p = 0.017) than for placebo (62.6%); desvenlafaxine (68.7%) did not differ from placebo. Other secondary outcomes were consistent with those obtained with CDRS-R. Rates of treatment-emergent adverse events were comparable among treatment groups (desvenlafaxine, 60.0%; placebo, 70.5%; and fluoxetine, 64.3%). Conclusion: Desvenlafaxine did not demonstrate efficacy for treating MDD in children and adolescents in this trial. Because neither desvenlafaxine nor the reference medication, fluoxetine, demonstrated a statistically significant difference from placebo on the primary endpoint, this was considered a failed trial and no efficacy conclusions can be drawn. Desvenlafaxine 25–50 mg/d was generally safe and well tolerated in children and adolescents in this study. ABSTRACT.INTRODUCTION: Prevalence rates for major depressive disorder (MDD) have been estimated at up to 2.5% in children and 8.3% in adolescents (Birmaher et al. 1996). Untreated MDD in children and adolescents can affect family interactions, social development, and social functioning, as well as school performance (Birmaher et al. 1996, 2007; Siu 2016). For these reasons, screening for MDD in primary care settings is recommended for all pediatric patients aged 12–18 years (Siu 2016). The standard of care for treatment of moderate-to-severe depression in adolescents and children is psychological intervention, an antidepressant medication, or both (Birmaher et al. 2007; Cheung et al. 2007). Several antidepressant medications have been assessed for efficacy for treating MDD in pediatric populations, including selective serotonin reuptake inhibitors (SSRIs) and serotonin–norepinephrine reuptake inhibitors (SNRIs) (Emslie et al. 1997, 2002, 2007, 2009, 2014; Wagner et al. 2003, 2004; Atkinson et al. 2014). Fluoxetine demonstrated statistical superiority over placebo in studies that enrolled children and adolescents (Emslie et al. 1997, 2002) and is approved for the treatment of pediatric MDD in many countries, including the United States (Prozac [package insert] 2014). Escitalopram, which had significant efficacy compared with placebo in patients aged 12–17 years (Emslie et al. 2009), is approved for the treatment of MDD in adolescents (Lexapro [package insert] 2014). However, for patients who do not tolerate or respond fully to those medications, additional antidepressant therapy options are needed. The SNRI desvenlafaxine (administered as desvenlafaxine succinate) has demonstrated safety, tolerability, and efficacy in randomized, double-blind, placebo-controlled studies in adult patients with MDD (DeMartinis et al. 2007; Septien-Velez et al. 2007; Boyer et al. 2008; Liebowitz et al. 2008; Tourian et al. 2009; Dunlop et al. 2011; Clayton et al. 2013, 2015; Iwata et al. 2013) and is approved for the treatment of adults with MDD, with a recommended therapeutic dose of 50 mg/d (Pristiq [package insert] 2016). No significant efficacy compared with placebo has been observed for desvenlafaxine doses below 50 mg/d (Iwata et al. 2013; Liebowitz et al. 2013). The safety and tolerability of desvenlafaxine was examined in pediatric patients ages 7–17 with MDD in an 8-week, open-label, fixed-dose phase IIa study of doses ranging from 10 to 200 mg/d and a 6-month extension study, in which desvenlafaxine treatment appeared to be generally safe and well tolerated (Findling et al. 2014). Neither study, however, included a placebo arm. To effectively examine the efficacy and safety of desvenlafaxine for the treatment of pediatric patients with MDD, the sponsor (Pfizer Inc) planned four phase III studies, including two short-term and two 6-month extension studies. The pharmacokinetic profile of desvenlafaxine over a 10- to 200-mg dose range, determined based on samples collected during the phase IIa study (Findling et al. 2016), informed the dosing design of the phase III program. This study (NCT01372150) is the first of two similarly designed, short-term, double-blind, placebo-controlled studies of desvenlafaxine for the treatment of MDD in children and adolescents. Results from the second short-term study and extension studies will be reported separately. The objectives of this short-term, placebo-controlled, and fluoxetine-referenced study were to evaluate the efficacy, safety, and tolerability of desvenlafaxine in the treatment of children and adolescents with MDD. ABSTRACT.METHODS: Patients were randomized at 37 sites; 35 sites were in the United States (1–31 patients per site; median, 7) and 2 in Mexico (13 and 17 patients). Study sites included hospitals and academic institutions (5), clinical/private practices (3), and clinical trial research centers (29). Hospitals and academic institutions enrolled 41/340 patients (12%). Principal investigators at each site were child and adolescent psychiatrists (20) or general psychiatrists (17), who were board eligible or board certified (or completion of an equivalent training program for Mexican investigators), with experience in the diagnosis and treatment of pediatric depression and in conducting industry-sponsored studies. The study was conducted between November 2011 and March 2015 and in accordance with the International Council for Harmonisation Guideline for Good Clinical Practice (International Council for Harmonisation 1998) and the ethical principles that have their origin in the Declaration of Helsinki. The study protocol received institutional review board or independent ethics committee approval. Written informed consent and assent were obtained from legal guardians and participants before any study procedures were performed. Periodic safety review was conducted by an independent Data Monitoring Committee. ABSTRACT.STUDY DESIGN: This was a phase III, multicenter, randomized, double-blind, placebo-controlled, fluoxetine-referenced, 8-week, parallel group study. Patients who completed this acute-phase study were eligible to participate in a 6-month, open-label extension study of desvenlafaxine. Patients were randomly assigned (1:1:1) to placebo, desvenlafaxine, or fluoxetine, stratified by age group (child or adolescent) and country. Eight-week double-blind treatment was followed by a 1-week double-blind transition phase for patients who entered the extension study or a 1-week double-blind taper phase for those who did not. The selection of doses was based on two factors: first, the highest dose used in the study was 50 mg/d because no efficacy benefit has been demonstrated at doses higher than 50 mg/d in adults and tolerability decreases at doses higher than 50 mg/d (Thase et al. 2009). The recommended desvenlafaxine dose is 50 mg/d in adults with MDD (Pristiq [package insert] 2016). In studies of adults with MDD, no dose response for efficacy was observed for desvenlafaxine doses of 50 to 400 mg/d; however, a dose response for safety was demonstrated (Clayton et al. 2009; Thase et al. 2009). The 50 mg/d dose was generally safe and well tolerated in a phase IIa study in children and adolescents with MDD (Findling et al. 2016). Second, pharmacokinetic data from the phase IIa study demonstrated that body weight predicts desvenlafaxine exposure in a pediatric MDD population (Findling et al. 2016). Desvenlafaxine dose was therefore assigned based on the patient's body weight at the baseline (day 1) visit, with 50 mg/d as the highest dose, as follows: 20 to <35 kg: 25 mg/d; 35 to <70 kg: 35 mg/d; and ≥70 kg: 50 mg/d. Titration, taper, and transition dosing is shown in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/cap). ABSTRACT.STUDY PATIENTS: Study participants were male and female outpatients, aged 7 to <18 years who weighed at least 20 kg at the screening and baseline visits. All enrolled patients met Diagnostic and Statistical Manual of Mental Disorders, 4th edition (Text Revision) (DSM-IV-TR) (American Psychiatric Association 2000) criteria for MDD as the primary diagnosis, had depressive symptoms of at least moderate severity for at least 30 days, and would not require concomitant psychotherapy. The MDD diagnosis was confirmed by a psychiatrist at the study site and supported by the KIDDIE Schedule for Affective Disorders and Schizophrenia–Present and Lifetime Version (K-SADS-PL). Enrolled patients had a Children's Depression Rating Scale–Revised (CDRS-R) total score >40 and Clinical Global Impressions Scale–Severity (CGI-S) (Guy 1976) score of 4 or greater at screening and baseline. Patients and their parents/guardians provided informed consent and assent. Patients were excluded from the study if they: had a history or presence of MDD with psychotic features or any psychotic disorder, bipolar disorder (or first-degree relative with bipolar disorder) or manic episodes or comorbid primary psychiatric condition other than MDD, or a history of or current significant risk of suicide, or first-degree relative who had committed suicide. Detailed exclusions and prohibited treatments are listed in Supplementary Data S1. ABSTRACT.ASSESSMENTS: ABSTRACT.EFFICACY: Efficacy assessments were administered at weeks 1, 2, 3, 4, 6, 8, and/or at early termination in the double-blind phase. A week 9 assessment was administered after taper or after transition as the baseline assessment for the extension study for those who were continuing. The primary efficacy outcome was change from baseline in the CDRS-R (Poznanski et al. 1979) total score at week 8. The protocol-defined key secondary efficacy outcome was change from baseline in CGI-S score; other secondary efficacy outcomes were change from baseline in Clinical Global Impressions Scale–Improvement (CGI-I) (Guy 1976) score and CGI-I response (CGI-I score ≤2) at each visit. Site personnel administering the K-SADS-PL, CDRS-R, CGI-S, and CGI-I were qualified (with a minimum of 2 years' clinical experience with pediatric MDD), trained by the study sponsor, and approved as evaluators before conducting the assessments. Those administering the K-SADS-PL and CGI were further required to be either a doctoral level psychologist or a psychiatrist and have at least 2 years' experience using the scale. Raters for the CDRS-R were required to be either a doctoral or nondoctoral (e.g., MA, MS, MSW, RN, BS, BA) level clinician or a psychiatrist, to have at least 2 years' experience using the scale, and to be certified by the sponsor. Certification was a two-step process: raters had to (1) meet predefined interrater reliability criteria against the gold standard scores using video-taped assessments and (2) complete a one-on-one training on CDRS-R interview technique (applied training) through the rater training vendor, achieving acceptable technique and reliability in accordance with prespecified criteria using the Rater Applied Performance Scale (Kobak et al. 2005). The protocol recommended that, whenever possible, the same rater administered a given scale for the patient at each assessment. ABSTRACT.SAFETY: Adverse event (AE; MedDRA v17.1) assessments, vital sign measurements, and Columbia-Suicide Severity Rating Scale (C-SSRS) (Posner et al. 2011) assessments were performed at each study visit. A physical examination with Tanner assessment and laboratory evaluations were performed at screening and week 8, with liver function tests and serum lipids also evaluated at week 4; ECG was performed at screening, baseline, and week 8. Individuals completing the C-SSRS and Tanner assessments were qualified, trained by the study sponsor, and approved as evaluators before conducting those assessments. The protocol recommended that, whenever possible, the same rater administer the Tanner for the patient at each assessment. Serious AEs were defined as any untoward medical occurrence at any dose of study medication that resulted in death or events that posed immediate risk of death, required inpatient hospitalization or prolongation of hospitalization, resulted in persistent or significant disability/incapacity, or resulted in congenital anomaly/birth defect. Important medical events were also reported as serious AEs when it was determined that they may have jeopardized the patient or required intervention to prevent one of the other serious AE outcomes. Potentially clinically important (PCI) findings were identified based on changes in laboratory findings, vital signs, and ECG results defined according to criteria prespecified by the sponsor. Clinically important results were then identified by the medical monitor based on a review of patient data, relevant clinical information pertaining to a patient in case report forms, and clinical judgment. ABSTRACT.PHARMACOKINETICS: Pharmacokinetic samples were collected at weeks 4 and 8 to support an integrated population pharmacokinetic analysis and an exploratory concentration-effect analysis. The results of these analyses will be reported and published separately. ABSTRACT.STATISTICAL ANALYSIS: ABSTRACT.SAMPLE SIZE DETERMINATION: The sample size estimate was performed for the change from baseline in CDRS-R total score at week 8. A sample of 111 patients per group was considered sufficient to demonstrate a 5-point difference in the primary endpoint between the desvenlafaxine and placebo groups at a significance level of 5% and a power of 85%, assuming a pooled standard deviation (SD) of 12, and that no more than 5% of randomized subjects would fail to qualify for the primary analysis (<1% in the current study). Results of a planned interim analysis (Supplementary Data S2) indicated that no sample size increase was required. ABSTRACT.EFFICACY: Efficacy analyses were based on the intent-to-treat (ITT) population: all patients who were randomly assigned to treatment received at least one dose of study medication, and had a baseline and at least one postbaseline primary efficacy assessment. The analysis of the change from baseline in CDRS-R total score was performed using a mixed-effects model for repeated measures with terms for treatment, week, interaction of treatment and week, age group, country, gender, and baseline CDRS-R total score. Statistical superiority of desvenlafaxine over placebo was to be declared based on a p-value <0.05. Desvenlafaxine and fluoxetine were separately compared with placebo. Fluoxetine was included as an active reference to provide assay sensitivity only and was not compared directly with desvenlafaxine. Sensitivity analyses are described in Supplementary Data S2. The CGI-S score was analyzed using the same approach as the CDRS-R total score. The categorical CGI-I score was analyzed at each visit using the Cochran–Mantel–Haenszel (CMH) row-mean-score-difference test using ridit scores and controlling for age/country stratum. CGI-I response rates were analyzed at each visit using a logistic regression model with terms for treatment, age group, and country. ABSTRACT.SAFETY: Safety summaries were presented for the safety population, defined as all patients who were randomly assigned to treatment and received at least one dose of study medication. The incidence rates for prespecified AEs of clinical importance were compared between treatment groups using risk difference versus placebo and associated p-values without multiplicity adjustment. Confidence intervals for the risk difference were calculated using the method by Chan and Zhang (1999). ABSTRACT.RESULTS: ABSTRACT.STUDY POPULATION: A total of 340 patients were randomly assigned to treatment; 339 patients (children, n = 130; adolescents, n = 209) were included in the safety population (desvenlafaxine, n = 115; fluoxetine, n = 112; placebo, n = 112). The ITT population included 130 children and 207 adolescents. A total of 42 (12%) patients discontinued early (desvenlafaxine, 16 [14%]; fluoxetine, 13 [12%]; and placebo, 13 [12%]). Lost to follow-up was the most common reason for discontinuation from the desvenlafaxine and placebo groups; in the fluoxetine group, the most common reason for discontinuation was “no longer willing to participate” (Fig. 1). FIG. 1.Study flow. aOne patient was screened and, although not randomized, received a total of eight blinded placebo doses. The patient was discontinued after the week 1 visit once the error was identified and listed as a protocol violation. Demographic and baseline characteristics were comparable among treatment groups for both children and adolescents (Table 1). Mean (SD) CDRS-R Total Score at baseline was 56.5 (8.9), mean (SD) CGI-S score was 4.5 (0.6), and the duration of the most recent depressive episode ranged from 1 to 96 months (median, 7; mean [SD], 13.4 [15.1]). Overall, 30.4% (103) of patients in the safety population had a prior or active psychiatric condition other than MDD in their medical history (desvenlafaxine, 31.3%; fluoxetine, 25.9%; and placebo, 33.9%). The most common psychiatric conditions (reported by ≥2% of patients) included attention-deficit/hyperactivity disorder (desvenlafaxine, 12.2%; fluoxetine, 13.4%; and placebo, 5.4%), nonsuicidal self-injurious behavior (desvenlafaxine, 7.0%; fluoxetine, 8.0%; and placebo, 12.5%), insomnia (desvenlafaxine, 7.0%; fluoxetine, 6.3%; and placebo, 8.0%), generalized anxiety disorder (desvenlafaxine, 0.9%; fluoxetine, 6.3%; and placebo, 0), and oppositional defiant disorder (desvenlafaxine, 3.5%; fluoxetine, 3.6%; and placebo, 0). Table 1.Demographic and Baseline Characteristics, Safety Population  ChildrenAdolescents Placebo (n = 42)Fluoxetine (n = 45)Desvenlafaxine (n = 43)Total (n = 130)Placebo (n = 70)Fluoxetine (n = 67)Desvenlafaxine (n = 72)Total (n = 209)Age, mean (SD), years 9.4 (1.3) 9.6 (1.3) 9.3 (1.4) 9.4 (1.3) 14.6 (1.5) 14.7 (1.6) 15.0 (1.5) 14.8 (1.5) Sex, n (%)  Female 23 (55) 14 (31) 20 (47) 57 (44) 41 (59) 43 (64) 43 (60) 127 (61)  Male 19 (45) 31 (69) 23 (53) 73 (56) 29 (41) 24 (36) 29 (40) 82 (39) Race, n (%)  Asian 1 (2) 0 1 (2) 2 (2) 0 2 (3) 0 2 (1)  Black 15 (36) 15 (33) 14 (33) 44 (34) 10 (14) 19 (28) 18 (25) 47 (22)  White 25 (60) 26 (58) 23 (53) 74 (57) 56 (80) 41 (61) 49 (68) 146 (70)  Other 1 (2) 4 (9) 5 (12) 10 (8) 4 (6) 5 (7) 5 (7) 14 (7) Height, mean (SD), cm 140.5 (10.6) 141.3 (11.2) 144.3 (11.5) 142.0 (11.1) 164.5 (9.8) 165.8 (8.4) 167.3 (9.1) 165.9 (9.2) Weight, mean (SD), kg 39.4 (14.0) 44.0 (18.9) 46.0 (15.2) 43.2 (16.3) 68.0 (19.1) 73.3 (21.8) 71.7 (19.0) 71.0 (20.0) BMI, mean (SD), kg/m2 19.5 (4.7) 21.3 (6.4) 21.6 (5.0) 20.8 (5.5) 25.0 (6.0) 25.5 (5.8) 25.6 (6.3) Duration of most recent episode, median (range), months 11 (1–57) 6 (1–42) 8 (1–71) 7 (1–71) 8 (1–69) 7 (1–96) 7 (1–61) 7 (1–96) CDRS-R total score, mean (SD) 57.0 (8.6) 55.0 (8.7) 56.4 (10.9) 56.1 (9.4) 57.1 (9.1) 57.0 (8.1) 56.3 (8.8) 56.8 (8.7) BMI, body mass index; CDRS-R, Children's Depression Rating Scale–Revised; SD, standard deviation. ABSTRACT.EFFICACY: Statistical separation from placebo was not observed on the primary efficacy endpoint for either desvenlafaxine or for the fluoxetine reference group. Adjusted mean (standard error) change from baseline in CDRS-R total score at week 8 was −22.6 (1.17) and −23.1 (1.18) for the desvenlafaxine and placebo groups, respectively, and for fluoxetine was −24.8 (1.17) (Fig. 2). The change from baseline in CDRS-R over the 8-week treatment phase was similar for the three arms. Point differences were statistically significant between desvenlafaxine and placebo at week 2 and between fluoxetine and placebo at weeks 1 and 2; no significant differences were observed at any other time points. Results were similar for the child and adolescent groups in an exploratory analysis of change from baseline in CDRS-R total score (Supplementary Fig. S1). FIG. 2.Adjusted mean (SE) change from baseline in CDRS-R total score in children and adolescents; MMRM analysis, ITT population. *p < 0.01, fluoxetine versus placebo. †p < 0.05, fluoxetine versus placebo and desvenlafaxine versus placebo. CDRS-R, Children's Depression Rating Scale–Revised; CI, confidence interval; ITT, intent-to-treat; MMRM, mixed-effects model for repeated measures; SE, standard error. At week 8, the CGI-I response rate for fluoxetine (78.2%) was significantly greater compared with placebo (62.6%; p = 0.017). Week 8 CGI-I response rate for desvenlafaxine (68.7%) was also greater than placebo, although this comparison did not reach statistical significance (p = 0.343). Results for CGI-S and CGI-I scores were otherwise consistent with those for CDRS-R total score, with no statistically significant difference from placebo for either desvenlafaxine or fluoxetine at week 8 (Table 2). Table 2.Summary of Secondary Efficacy Outcomes at Week 8 CGI-SnAdjusted mean change (SE) from baselineaDifference in adjusted means (placebo-active)95% CIpPlacebo 99 −1.71 (0.12) — — — Fluoxetine 101 −1.88 (0.12) 0.18 −0.11 to 0.46 0.224 Desvenlafaxine 99 −1.70 (0.11) −0.01 −0.29 to 0.27 0.944 CGI-IbnVery much improved (%)Much improved (%)Minimally improved (%)No change (%)CMH testc p-valuePlacebo 99 27.3 35.4 32.3 4.0 — Fluoxetine 101 30.7 47.5 16.8 4.0 0.095 Desvenlafaxine 99 23.2 45.5 21.2 9.1 0.852 CGI-I responsedProportion responders%Adjusted odds ratioeWald 95% CIpPlacebo 62/99 62.6 — — — Fluoxetine 79/101 78.2 0.465 0.249 to 0.871 0.017 Desvenlafaxine 68/99 68.7 0.751 0.415 to 1.357 0.343 aEstimates of adjusted mean, SE, difference in means, 95% CI, and p-values were based on a mixed-effects model for repeated measures for the change from baseline in CGI-S with terms for treatment, week, interaction of treatment and week, age group, country, gender, and baseline CGI-S total score. bCGI-I scored as 1, very much improved; 2, much improved; 3, minimally improved; 4, no change; 5, minimally worse; 6, much worse; 7, very much worse. No patients scored 5–7 at week 8. cCMH test was controlled for age group and country. dCGI-I response was defined as CGI-I score of 1 (very much improved) or 2 (much improved). eEstimates of odds ratios, 95% CI, and p-values were based on a logistic regression model of the response rate, with terms for treatment group, age group, and country. CGI-I, Clinical Global Impressions–Improvement; CGI-S, Clinical Global Impressions–Severity; CI, confidence interval; CMH, Cochran–Mantel–Haenszel; SE, standard error. ABSTRACT.SAFETY: ABSTRACT.ADVERSE EVENTS: A total of 220/339 (64.9%) patients experienced AEs during the on-therapy period (desvenlafaxine, 69/115 [60.0%]; fluoxetine, 72/112 [64.3%]; and placebo, 79/112 [70.5%]). Most AEs were mild or moderate in severity. AEs considered by the investigator to be related to study medication were reported by 31.9% of patients (desvenlafaxine, 28.7%; fluoxetine, 32.1%; and placebo, 34.8%). Severe AEs (those that interfered significantly with patients' usual function) considered by the investigator to be unrelated to study medication were reported by 3.5% of patients (desvenlafaxine, 1.7%; fluoxetine, 5.4%; and placebo, 3.6%). In three patients, severe AEs were considered related to study medication: One desvenlafaxine-treated patient reported severe pruritus and severe macular rash and discontinued treatment. One placebo-treated patient reported severe agitation and severe anger, and a second reported severe insomnia; neither was discontinued due to those AEs. Five adolescents (0 children) discontinued due to AEs, two each from the desvenlafaxine (disinhibition; macular rash and pruritus) and placebo (vomiting; headache) groups and one from the fluoxetine group (suicidal ideation). ABSTRACT.TREATMENT-EMERGENT ADVERSE EVENTS: Treatment-emergent adverse events (TEAEs) reported by more than 10% of patients in any group were headache, upper abdominal pain, and nausea. Table 3 shows the most frequent (≥5% in any group) TEAEs by age group, and overall. There were no statistically significant differences between desvenlafaxine or fluoxetine and placebo in the incidence of any prespecified TEAEs of clinical interest for desvenlafaxine (tier-1 TEAEs) (Supplementary Table S2). Table 3.Number (%) of Patients Reporting Treatment-Emergent Adverse Events with Incidence ≥5% in Any Group, On-Therapy Period, Safety Population  ChildrenAdolescentsOverall Placebo (n = 42)Fluoxetine (n = 45)Desvenlafaxine (n = 43)Placebo (n = 70)Fluoxetine (n = 67)Desvenlafaxine (n = 72)Placebo (n = 112)Desvenlafaxine (n = 112)Desvenlafaxine (n = 115)Any TEAE 27 (64.3) 29 (64.4) 22 (51.2) 52 (74.3) 43 (64.2) 47 (65.3) 79 (70.5) 72 (64.3) 69 (60.0)  Headache 8 (19.0) 3 (6.7) 5 (11.6) 13 (18.6) 13 (19.4) 14 (19.4) 21 (18.8) 16 (14.3) 19 (16.5)  Abdominal pain upper 2 (4.8) 1 (2.2) 6 (14.0) 5 (7.1) 8 (11.9) 9 (12.5) 7 (6.3) 9 (8.0) 15 (13.0)  Nausea 3 (7.1) 4 (8.9) 2 (4.7) 7 (10.0) 9 (13.4) 6 (8.3) 10 (8.9) 13 (11.6) 8 (7.0)  Dizziness 3 (7.1) 1 (2.2) 2 (4.7) 3 (4.3) 2 (3.0) 5 (6.9) 6 (5.4) 3 (2.7) 7 (6.1)  Influenza 0 2 (4.4) 2 (4.7) 0 0 4 (5.6) 0 2 (1.8) 6 (5.2)  Nasopharyngitis 4 (9.5) 2 (4.4) 1 (2.3) 4 (5.7) 5 (7.5) 5 (6.9) 8 (7.1) 7 (6.3) 6 (5.2)  Upper respiratory tract infection 2 (4.8) 3 (6.7) 1 (2.3) 4 (5.7) 1 (1.5) 5 (6.9) 6 (5.4) 4 (3.6) 6 (5.2)  Vomiting 1 (2.4) 5 (11.1) 1 (2.3) 3 (4.3) 2 (3.0) 4 (5.6) 4 (3.6) 7 (6.3) 5 (4.3)  Diarrhea 1 (2.4) 0 4 (9.3) 2 (2.9) 3 (4.5) 1 (1.4) 3 (2.7) 3 (2.7) 5 (4.3)  Insomnia 1 (2.4) 1 (2.2) 0 2 (2.9) 4 (6.0) 4 (5.6) 3 (2.7) 5 (4.5) 4 (3.5)  Fatigue 1 (2.4) 3 (6.7) 0 1 (1.4) 3 (4.5) 2 (2.8) 2 (1.8) 6 (5.4) 2 (1.7)  Pharyngitis 2 (4.8) 3 (6.7) 1 (2.3) 0 1 (1.5) 1 (1.4) 2 (1.8) 4 (3.6) 2 (1.7)  Rash 0 3 (6.7) 0 0 1 (1.5) 1 (1.4) 0 4 (3.6) 1 (0.9)  Arthralgia 3 (7.1) 1 (2.2) 1 (2.3) 1 (1.4) 2 (3.0) 0 4 (3.6) 3 (2.7) 1 (0.9)  Fall 2 (4.8) 4 (8.9) 0 0 1 (1.5) 0 2 (1.8) 5 (4.5) 0  Constipation 3 (7.1) 0 0 1 (1.4) 0 0 4 (3.6) 0 0 TEAE, treatment-emergent adverse event. ABSTRACT.DEATHS AND SERIOUS AES: There were no deaths during the study. Five randomized patients experienced serious AEs: two assigned to fluoxetine (suicidal ideation [acute suicidality; discontinued] and suicide attempt [occurred poststudy], one patient each), three assigned to desvenlafaxine (suicidal ideation [discontinued due to lack of confidence in caregiver reliability to report patient status], disinhibition [discontinued], and postpartum hemorrhage with anemia and endometritis [occurred poststudy], one patient each), and no placebo-treated patients. The desvenlafaxine-treated patient with postpartum hemorrhage had been withdrawn from the study following a positive pregnancy test at the final treatment phase visit and delivered a healthy full-term infant. ABSTRACT.SUICIDALITY: A summary of C-SSRS results for treatment-emergent suicidal ideation or behavior is presented for the safety population in Table 4 (full C-SSRS results are presented in Supplementary Table S3). Treatment-emergent suicidal ideation or suicidal behavior, which included both new-onset and worsening suicidal ideation or behavior, was reported for 29 (8.6%) of 337 patients who had a C-SSRS assessment at baseline and at 1 or more postbaseline time points. A total of 29/337 (8.6%) patients had treatment-emergent suicidal ideation, and 1 patient (0.3%) also had treatment-emergent suicidal behavior. The suicidal behavior event was categorized as a preparatory act toward imminent suicidal behavior (preparatory acts or behavior; interrupted attempt) and was reported in a fluoxetine-treated adolescent (Supplementary Table S3). The patient was discontinued due to a serious AE of acute suicidality (described in “Deaths and Serious AEs” section). Table 4.Summary of Treatment-Emergent Suicidal Ideation and Behavior Reported on the Columbia-Suicide Severity Rating Scale at Any Postbaseline Assessment, Safety Population  Placebo (N = 112)Fluoxetine (N = 110)Desvenlafaxine (N = 115)Total (N = 337)Treatment-emergent SIBa 8/112 (7.1) 12/110 (10.9) 9/115 (7.8) 29/337 (8.6)  New-onset SIBb 7/104 (6.7) 10/97 (10.3) 8/102 (7.8) 25/303 (8.3)  Worsening SIBc 1/8 (12.5) 2/13 (15.4) 1/13 (7.7) 4/34 (11.8) Treatment-emergent SId 8/112 (7.1) 12/110 (10.9) 9/115 (7.8) 29/337 (8.6)  New-onset SIe 7/104 (6.7) 10/97 (10.3) 8/102 (7.8) 25/303 (8.3)   Wish to be dead 5 4 3 12   Nonspecific active suicidal thoughts 1 1 1 3   Active SI with any methods (no plan) without intent to act 1 5 4 10  Worsening SIf 1/8 (12.5) 2/13 (15.4) 1/13 (7.7) 4/34 (11.8)   Shift to nonspecific active suicidal thoughts 1 0 0 1   Shift to active SI with any methods (no plan) without intent to act 0 1 1 2   Shift to active SI with specific plan and intent 0 1 0 1 Treatment-emergent SBg 0/112 (0.0) 1/110 (0.9) 0/115 (0.0) 1/337 (0.3)  New-onset SBh 0/112 (0.0) 1/110 (0.9) 0/115 (0.0) 1/337 (0.3)   Interrupted attempt 0 1 0 1  Worsening SBi 0 0 0 0 There was one poststudy suicide attempt reported as a serious adverse event that was not captured on the C-SSRS; C-SSRS was not performed following that event. N represents the number of patients in this analysis, that is, patients who had a baseline and a postbaseline C-SSRS assessment. aTreatment-emergent SIB is defined as (1) new-onset SI or SB, (2) worsening SI or SB, or (3) postbaseline SB on patients reporting SI at baseline. bNew-onset SIB is defined as any SI or SB reported postbaseline on patients who reported no SI and no SB at baseline. cWorsening SIB is defined as (1) shift from SI at baseline to a more severe SI postbaseline, (2) shift from SI at baseline (and no SB at baseline) to any SB postbaseline, or (3) shift from SB at baseline to a more severe SB postbaseline. dTreatment-emergent SI is defined as new-onset SI or worsening SI. eNew-onset SI is defined as any SI reported postbaseline on patients who reported no SI at baseline. fWorsening SI is defined as shift to a more severe SI postbaseline on patients reporting SI at baseline. gTreatment-emergent SB is defined as new-onset SB or worsening SB. hNew-onset SB is defined as any SB reported postbaseline on patients who reported no SB at baseline. iWorsening SB is defined as shift to a more severe SB postbaseline on patients reporting SB at baseline. C-SSRS, Columbia-Suicide Severity Rating Scale; SB, suicidal behavior; SI, suicidal ideation; SIB, suicidal ideation or behavior. New-onset self-injurious behavior without suicidal intent was reported in two (1.8%) fluoxetine-treated adolescents and one (0.9%) desvenlafaxine-treated adolescent. The suicide attempt reported as a serious AE in the “Deaths and Serious AEs” section was not captured on the C-SSRS because it occurred poststudy. ABSTRACT.OTHER SAFETY MEASURES: The frequencies of patients with on-therapy potentially clinically important (PCI) vital sign values can be found in Supplementary Table S4. Upon review of patient data by the medical monitor, eight patients (all adolescents) were deemed to have clinically important vital sign results: postural hypotension in three desvenlafaxine-treated patients (35-, 35-, and 50-mg/d doses) and in four fluoxetine-treated patients, and increased supine systolic blood pressure in one desvenlafaxine-treated patient (122–126 mm Hg at weeks 3–6; baseline, 117 mm Hg). No patients assigned to placebo had clinically important vital sign findings, and the observed changes from baseline in body mass index (BMI) were negligible. Mean changes from baseline in BMI, blood pressure, and pulse measurements are reported by age group in Supplementary Table S5. Expected shifts associated with development assessed by Tanner staging were observed during the study. Clinically important ECG findings were reported for one patient assigned to fluoxetine who had Wolff–Parkinson–White syndrome at study entry, with no change over the course of the study. The frequencies of on-therapy PCI laboratory findings are presented in Supplementary Table S6. Findings in nine patients (four desvenlafaxine, five fluoxetine) were determined to be clinically important: three had elevated triglycerides (two desvenlafaxine, one fluoxetine), one high prolactin (desvenlafaxine), one low hematocrit and low hemoglobin (desvenlafaxine), three positive urine protein (fluoxetine), and one abnormal liver function test (fluoxetine). No clinically important laboratory values were observed in the placebo group. Mean changes from baseline for selected laboratory values are reported by age group and treatment in Supplementary Table S7. ABSTRACT.DISCUSSION: Neither desvenlafaxine nor fluoxetine demonstrated efficacy for treating MDD in children and adolescents in this study. No statistically significant difference between desvenlafaxine and placebo groups, or between fluoxetine and placebo groups, was observed for the primary efficacy endpoint (CDRS-R). At study week 8, patients in desvenlafaxine and placebo treatment groups achieved mean 22.6- and 23.1-point improvements from baseline in CDRS-R total score, respectively. However, because fluoxetine (24.8-point improvement) also did not statistically separate from placebo on the primary endpoint, the study meets the Division of Psychiatry Products of the U.S. Food and Drug Administration definition of a failed trial (Food and Drug Administration 2015), and the efficacy results for desvenlafaxine therefore were inconclusive. Desvenlafaxine also failed to demonstrate efficacy for treating pediatric MDD in a sister study, which was a negative trial (no active control arm) (Atkinson et al. 2017). Findings for CGI-S (key secondary endpoint) and CGI-I scores in the current study were generally consistent with the primary efficacy results. The fluoxetine reference arm was included in the current study to provide assay sensitivity because fluoxetine has demonstrated efficacy in children and adolescents with MDD in several placebo-controlled studies (Emslie et al. 1997, 2002). A significant difference between fluoxetine and placebo in improvement on the CDRS-R total score was therefore expected in the current study, although in some recent studies fluoxetine did not demonstrate efficacy in children and adolescents (Atkinson et al. 2014; Emslie et al. 2014). The CGI-I responder rate was the only efficacy measure in this study on which fluoxetine statistically separated from placebo. Because fluoxetine did not separate from placebo on the primary efficacy endpoint, no conclusion can be drawn from the results of this study regarding the efficacy of desvenlafaxine in the treatment of pediatric MDD. Desvenlafaxine and fluoxetine treatment in this study were associated with mean decreases in CDRS-R scores of 23 and 25 points, respectively, similar to antidepressant response observed in positive pediatric MDD trials (Emslie et al. 1997, 2002, 2009; Wagner et al. 2003, 2004). The placebo response in the current trial (−23 points), however, was in line with that observed in published negative or inconclusive pediatric depression trials (Emslie et al. 2006, 2007, 2014; Atkinson et al. 2014; DelBello et al. 2014). In a meta-analysis of 12 pediatric antidepressant trials, Bridge et al. (2009) found that placebo response was more predictive of the efficacy outcome of a trial than was study medication response. Factors associated with a larger placebo response in that analysis were greater number of study sites and less severe depression at baseline (Bridge et al. 2009). The number of study sites (37) and the baseline severity of depression (mean CDRS-R total score, 56.5) in the current study were within the ranges reported in positive pediatric antidepressant trials (study sites: 1–53; baseline CDRS-R total scores: 55.1–64.6) (Emslie et al. 1997, 2002, 2009; Wagner et al. 2003, 2004). Several approaches to minimizing placebo response were used in this study as follows: limited number of treatment arms, rater certification for the CDRS-R, use of the same rater for a given patient whenever possible, and instruction to clinician investigators to refrain from positive emotional response to improvements in symptoms during study assessments or communication of expectancies for improvement. Different approaches to addressing placebo response may vary in their effect on efficacy outcomes, however, and the specific methods used in the current study may not have been sufficient. Study design elements used in positive SSRI/SNRI studies but not in our study included the use of two treatment arms only (study medication and placebo) and 1:1 randomization (Emslie et al. 1997, 2002, 2009; Wagner et al. 2003, 2004), which has been associated with a lower placebo response (Weimer et al. 2015). All but one of those positive studies used a placebo run-in (Emslie et al. 1997, 2002, 2009; Wagner et al. 2003, 2004) and most included multiple diagnostic interviews by independent raters to confirm diagnosis before entry (Emslie et al. 1997, 2002, 2009; Wagner et al. 2004). Recent thinking about placebo response as ubiquitous in clinical medicine has led to additional recommendations, including systematic measuring and statistical control for nonpharmacological patient factors and therapeutic alliance, separation of recruitment from trial conductance to reduce selection bias by the clinician, and assessment of and controlling for the effectiveness of blinding (Enck et al. 2013; Rief et al. 2016). If implemented, such processes might have reduced the placebo response in this trial or allowed for analyzing its contribution separately from the drug/placebo effects. The increased care and attention from parents and site personnel that is associated with entering a trial and participating in repeated site visits are likely to have contributed to the reduction of symptoms in both groups. Whether this effect is stronger in the placebo than in the active drug group is not known, but recent early results suggest that this may be the case (Zilcha-Mano et al. 2015). Designing future studies to assess and analyze results to determine this difference could help identify pharmacologic effects separate from nonspecific treatment effects (Zilcha-Mano 2016). The current study provides valuable information regarding the safety and tolerability of desvenlafaxine treatment in a pediatric population. In the previous open-label short-term and extension studies of desvenlafaxine (Findling et al. 2014), desvenlafaxine (children, 10–100 mg/d; adolescents, 25–200 mg/d) was generally safe and well tolerated, with AEs similar to those reported in adult studies. Those studies did not include a placebo comparison, however (Findling et al. 2014), limiting conclusions regarding the balance of benefits and risks of desvenlafaxine treatment in pediatric patients (Emslie 2009). In this study, rates of TEAEs were comparable for desvenlafaxine and placebo groups among both children and adolescents, and no statistically significant differences were observed in the incidence of prespecified AEs of clinical interest, including events related to blood pressure or aggressive or suicidal behavior. Although some statistical differences between groups were noted in laboratory evaluations and physical and vital sign measurements, none was considered to be clinically meaningful. The increased number of fluoxetine- versus placebo-treated patients with orthostatic hypotension is consistent with previously reported effects (Pacher and Kecskemeti 2004; Jerrell and McIntyre 2009). New-onset suicidal ideation was reported for 8, 10, and 7 patients in the desvenlafaxine, fluoxetine, and placebo groups, respectively; suicidal behavior was reported for 1 fluoxetine-treated patient who reported suicidal ideation at baseline. No evidence of a signal for increased risk of suicidal thoughts or behaviors has been observed in adults receiving desvenlafaxine treatment for MDD (Tourian et al. 2010). However, for the antidepressant class (SSRIs/SNRIs), there appears to be a small increased risk of treatment-emergent suicidal ideation/suicide attempt associated with antidepressant treatment of MDD in children and adolescents (1% risk difference, antidepressant vs. placebo), based on a meta-analysis of 15 trials (Bridge et al. 2007). Bridge et al. (2007) concluded that the overall risk-to-benefit profile for antidepressants in the treatment of pediatric MDD is favorable based on the benefits of treatment; however, the risk/benefit balance may differ across patient populations. Limitations of the current study should be noted. First, the enrollment criteria selected for a study population that was generally medically healthy, excluding patients at risk for suicide or with co-occurring psychiatric conditions necessitating treatment with medication or other unstable medical conditions, thus limiting generalization of study conclusions to a broader pediatric MDD population. In addition, median CGI-S score at baseline indicated that the majority of patients in the study had moderately severe depression at baseline. Analysis of the effect of baseline severity in adult MDD trials suggests that larger treatment effect size might be observed in patients with more severe baseline depression (Fournier et al. 2010). ABSTRACT.CONCLUSIONS: This phase III, double-blind, placebo-controlled study failed to demonstrate efficacy of desvenlafaxine for treating MDD in children and adolescent patients. No significant difference between placebo- and desvenlafaxine-treated patients was observed for the primary efficacy variable, change from baseline in CDRS-R total scores at week 8. A fluoxetine treatment arm was included in the study because fluoxetine has previously demonstrated efficacy in children and adolescents with MDD. However, because fluoxetine also did not separate from placebo on the primary endpoint in this study, neither medication demonstrated efficacy in the current trial. Therefore, no conclusion can be drawn from these results regarding the efficacy of desvenlafaxine in the treatment of pediatric MDD. Desvenlafaxine was generally safe and well tolerated in children and adolescents in this study. No new safety signals were identified. Safety results were consistent with previous adult and pediatric desvenlafaxine MDD trials. ABSTRACT.CLINICAL SIGNIFICANCE: Untreated depression can affect family interactions, social development and functioning, and school performance in children and adolescents with MDD. However, few antidepressant medications have been approved for this patient population. The efficacy of desvenlafaxine was assessed in a placebo-controlled study that included an active medication (fluoxetine) reference arm. No statistically significant difference was observed between desvenlafaxine and placebo on the primary efficacy endpoint (CDRS-R). Because fluoxetine also did not separate from placebo, no conclusion can be drawn from these results regarding the efficacy of desvenlafaxine in the treatment of pediatric MDD. ABSTRACT.DISCLOSURES: K.L.W.: received compensation from Pfizer Inc for her role as one of the Principal Investigators who conducted this study; received compensation in the past from Pfizer Inc and other pharmaceutical companies for her role as a Principal Investigator in numerous clinical trials; and has been a Pharmacy and Therapeutics consultant for CVS/Caremark. W.M.: received compensation from Pfizer Inc for his role as one of the Principal Investigators who conducted this study; received compensation in the past from Pfizer Inc and other pharmaceutical companies for his role as a Principal Investigator in numerous clinical trials. R.A.: employee of Pfizer Inc. D.C.: employee of Pfizer Inc and has Pfizer Inc stock options. R.D.E.: employee of Pfizer Inc, owns Pfizer Inc stock, and has Pfizer Inc stock options. S.R.: employee of Pfizer Inc, owns Pfizer Inc stock, and has Pfizer Inc stock options. D.B.W.: employee of Pfizer Inc, owns Pfizer Inc stock, and has Pfizer Inc stock options. ABSTRACT.SUPPLEMENTARY MATERIAL: ABSTRACT.SUPPLEMENTAL DATA:

TITLE: Defining Plasmodium falciparum Treatment in South West Asia: A Randomized Trial Comparing Artesunate or Primaquine Combined with Chloroquine or SPTreatment of Plasmodium falciparum Malaria ABSTRACT.INTRODUCTION: Antimalarial resistance has led to a global policy of artemisinin-based combination therapy. Despite growing resistance chloroquine (CQ) remained until recently the official first-line treatment for falciparum malaria in Pakistan, with sulfadoxine-pyrimethamine (SP) second-line. Co-treatment with the gametocytocidal primaquine (PQ) is recommended for transmission control in South Asia. The relative effect of artesunate (AS) or primaquine, as partner drugs, on clinical outcomes and gametocyte carriage in this setting were unknown. ABSTRACT.METHODS: A single-blinded, randomized trial among Afghan refugees in Pakistan compared six treatment arms: CQ; CQ+(single-dose)PQ; CQ+(3 d)AS; SP; SP+(single-dose)PQ, and SP+(3 d)AS. The objectives were to compare treatment failure rates and effect on gametocyte carriage, of CQ or SP monotherapy against the respective combinations (PQ or AS). Outcomes included trophozoite and gametocyte clearance (read by light microscopy), and clinical and parasitological failure. ABSTRACT.FINDINGS: A total of 308 (87%) patients completed the trial. Failure rates by day 28 were: CQ 55/68 (81%); CQ+AS 19/67 (28%), SP 4/41 (9.8%), SP+AS 1/41 (2.4%). The addition of PQ to CQ or SP did not affect failure rates (CQ+PQ 49/67 (73%) failed; SP+PQ 5/33 (16%) failed). AS was superior to PQ at clearing gametocytes; gametocytes were seen on d7 in 85% of CQ, 40% of CQ+PQ, 21% of CQ+AS, 91% of SP, 76% of SP+PQ and 23% of SP+AS treated patients. PQ was more effective at clearing older gametocyte infections whereas AS was more effective at preventing emergence of mature gametocytes, except in cases that recrudesced. ABSTRACT.CONCLUSIONS: CQ is no longer appropriate by itself or in combination. These findings influenced the replacement of CQ with SP+AS for first-line treatment of uncomplicated falciparum malaria in the WHO Eastern Mediterranean Region. The threat of SP resistance remains as SP monotherapy is still common. Three day AS was superior to single-dose PQ for reducing gametocyte carriage. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov bold> BODY.INTRODUCTION: Antimalarial drug resistance is an ongoing threat to malaria control [1]. Despite widespread documented resistance, chloroquine remained widely used in Pakistan and Afghanistan for first line treatment of falciparum malaria at the time of this study (2000–2003) [2]–[4]. In many other parts of the world including Pakistan and Afghanistan the antifolate drug sulfadoxine-pyrimethamine (SP) is comparatively effective against falciparum malaria in contrast to the situation in South East Asia and many African settings. In Pakistan and Afghanistan transmission is seasonal, strong immunity seldom develops, infected individuals are symptomatic and it is thought that the majority seek treatment [2], [3], [5]. These conditions expose the majority of infections to antimalarial drugs and would be expected to exert strong selection for resistance [6]. SP has a long plasma half-life which may further contribute to selection. In such areas of low seasonal transmission the operational effectiveness of SP before resistance arises appears to be short [7]. Whilst current effectiveness remains comparatively good in the region low level in vivo resistance to SP has been demonstrated [5], [8], [9]. Wide-scale adoption of effective SP based combinations, rather than SP monotherapy, which was being considered as an option to replace chloroquine at the time of the study, could delay the selection of resistance and prolong the useful life of SP across the subcontinent. In areas of low or medium endemicity co-treatment of infections with a gametocytocidal drug may help to reduce transmission and may be particularly important when considering strategies for malaria elimination. In South Asia, the standard policy has been to co-treat with a single dose of primaquine to reduce gametocyte carriage. Primaquine is known to have poor efficacy as a direct treatment but has repeatedly been shown to be highly gametocytocidal [10]–[14]. Reduction in gametocyte carriage and infectivity to mosquitoes after artesunate treatment is widely documented [15], [16]. It appears to be due to a combination of rapid clearance of asexual stages, direct activity on immature gametocytes (either killing them or preventing their maturation) and possibly reduction in the infectivity of mature gametocytes [17], [18]. Use of ACTs has been shown to reduce transmission [19]–[23] and is recommended for epidemic response [24], as too is primaquine [25]. There is increasing interest in using primaquine with ACTs to accelerate gametocyte clearance [14]. SP treatment of clinical infections can lead to high gametocyte loads which persist for 10 days or more [15] with latent gametocytaemia detected by PCR for up to a year [26]. A direct comparison of artesunate and primaquine on treatment failure and gametocyte carriage has not been conducted before. Many Afghan refugee communities in Pakistan have a history of falciparum malaria and are prone to outbreaks [27]–[29]. A trial examining the comparative efficacy of chloroquine and SP combined with either artesunate or primaquine was therefore carried out in refugee villages in North-western Pakistan. BODY.MATERIALS AND METHODS: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. BODY.STUDY AREA AND POPULATION: The study recruited patients from five Afghan refugee villages within an 80 km radius of Peshawar, Khyber Province (formerly North West Frontier Province), Pakistan. The 5 villages were established in the early 1980s and each has a history of falciparum transmission. Three (Adizai, Naguman and Yakka Ghund) are situated on the banks of the Kabul river, and two (Mohammed Khoja and Kotki) are situated to the south of Peshawar in Kohat district. Malaria transmission is seasonal with vivax malaria occurring from March to November and falciparum from July to December. Participants were Afghan refugees who were permanently resident in the Pakistan villages. A minority might have acquired their infections in Afghanistan, but admission criteria required 4–6 weeks of follow-up which would have excluded the more regular travellers. BODY.STUDY SITES: The study was conducted through three sites with well-managed clinics run by Non-Governmental Organisations (NGO) and government agencies. Site 1 was Adizai village which also served the population of nearby Naguman. Site 2 was Yakka Ghund village. Site 3 was Kotki which also serviced Mohammed Khoja village. Health staff from the NGO HealthNet TPO were seconded to each clinic to manage the recruitment and follow-up of patients. The study took place over three malaria seasons from 2000 to 2003. In the first season only site 1 was used, in the second season site 2 was added, and in the third season site 3 was added. The stepped inclusion of sites resulted from unexpectedly low recruitment rates at the first study site, and is summarized in Table 1. An earlier in vivo survey across all refugee camps along the length of the 1000 km North-South axis of Khyber Province showed little heterogeneity in resistance frequency to chloroquine and SP [30]. (Note that SP is the only drug name abbreviated throughout the text. Abbreviations for chloroquine (CQ), primaquine (PQ) and artesunate (AS) are used when referring to specific study arms (e.g. CQ+PQ) but not at other points in the text.) 10.1371/journal.pone.0028957.t001Table 1BODY.SUMMARY OF TREATMENT ARMS TESTED AT EACH OF THE STUDY SITES OVER THE THREE TRANSMISSION SEASONS.: Study site Transmission season (July–January) Site 1 (Adizai) Site 2 (Yakka Ghund) Site 3 (Kotki) Season 1: 2000–2001 CQ, CQ+PQ, CQ+AS,SP, SP+PQ, SP+AS Season 2: 2001–2002 SP, SP+PQ, SP+AS CQ, CQ+PQ, CQ+AS Season 3: 2002–2003 CQ, CQ+PQ, CQ+AS SP, SP+PQ, SP+AS CQ, CQ+PQ, CQ+AS Study design and procedures. Individuals presenting with clinical symptoms and diagnosed microscopically with falciparum malaria were referred to trial staff for further assessment. Inclusion criteria were: over two years of age, not pregnant, P. falciparum mono-infection, more than 1 asexual parasite per 10 fields, no other serious disease, resident in the refugee village for the full period of follow-up, no verbal report of antimalarial use during the last 21 days, and no signs of severe malaria. Individuals meeting the inclusion criteria and giving informed consent were allocated using randomization tables stratified by age and sex to the following treatment arms: chloroquine (CQ1) (25 mg/kg) over 3 days; CQ (25 mg/kg) over 3 days plus primaquine (PQ) (0.5 mg/kg) on the last day of treatment; CQ (25 mg/kg) over 3 days plus artesunate (AS) (4 mg/kg per day) over 3 days; sulfadoxine (25 mg/kg) and pyrimethamine (1.25 mg/kg) (SP) on day 0; SP (25:1.25 mg/kg) plus PQ (0.5 mg/kg) on day 0; SP (25:1.25 mg/kg) on day 0 plus AS (4 mg/kg per day) over 3 days. The primaquine co-treatment regimens were based on recommendations for use of primaquine as a gametocytocide treatment for falciparum malaria [31]. On treatment days when any specific arm did not require a partner drug, a placebo was substituted. Patients were not allocated to all six treatment arms at each site (Table 1). During years two and three, at any one site, patients were allocated to either one of the three SP treatment arms or one of the three chloroquine treatment arms; only in year one were patients allocated to all six treatment arms in the original site. For each of the study sites patients were stratified by age and sex and assigned consecutive patient numbers at enrolment. Treatment groups were pre-assigned to patient numbers using simple randomization lists generated using Excel (Microsoft Corp., Seattle, USA). The assignment of treatment group to patient number was concealed until after enrolment. The random allocation sequence was generated by two of the investigators (KK, MR) neither of whom enrolled or assessed patients. The brands and manufacturers/suppliers were: SP (Fansidar ®, Roche), chloroquine (Nivaquine ®, Beacon), primaquine (IDA), artesunate (Plasmotrim ™, Mepha). Although chloroquine resistance was known to exist in the study area, chloroquine was the first line treatment policy of the Government of Pakistan and the UN refugee agency (UNHCR) at the time and was therefore included. The trial manager, who allocated the treatment arms and administered treatment, was not blind to the study drugs and allocations. Recruited patients, microscopists and health workers were partially blinded; full blinding was not achieved given (i) the appearance of the different drugs, and (ii) the different times of follow up for the SP arms compared to the chloroquine arms. Patients were given directly observed treatment (by the trial manager), monitored for 30 minutes and re-dosed if vomiting occurred. Health workers then recorded the other day 0 biomedical parameters. Patients were asked to return on each day of treatment (days 0, 1, 2) and on days 3, 7, 14, 21 and 28 post treatment. Patients in the three SP arms were also followed up on days 35 and 42 for detection of late recrudescence [32]. On each day of follow up thick and thin smears were taken, clinical symptoms recorded and blood spots collected on Whatman No. 1 filter paper for parasite typing and differentiation of recrudescent from new infections. Haematocrits and blood spots were taken either on the day of failure or on day 28. Patients not presenting at the clinic were followed up at home. Criteria for withdrawal were reported administration of additional anti-malarial drugs (protocol violation), emergence of any concomitant febrile illness that interfered with outcome classification (including a non-falciparum malaria, for which the withdrawn study patient would be given appropriate first line treatment), parasitaemia still present on day 7, or signs of severe malaria developing before completing the initial treatment course. Patients found to be parasitaemic on any day after day 3 were treated with SP (the official second line treatment) or with SP and mefloquine (Fansimef, Roche) for those whose initial treatment was SP based. SP resistant parasites were sensitive to the mefloquine component of Fansimef, which was used in lieu of mefloquine monotherapy which was unavailable in the study area. Those who developed severe malaria, severe anaemia or other complications were referred to Khyber Hospital, Peshawar for treatment. Laboratory tests. Thick and thin blood smears were stained with 2% Giemsa solution. All slides were read on the day of collection by a microscopist based at each site. Differential diagnosis of vivax and falciparum (trophozoites and gametocytes) was by examination of thick and thin smears according to standard microscopy methods [33]. Trophozoites and gametocytes were counted against 200 white blood cells (WBC) from the thick blood smear on the assumption of a WBC count of 8000/µl. A smear was declared negative if no parasites were seen after examining 100 fields. Slides from sites 2 and 3 were re-examined for accuracy by the site 1 microscopist. Comparison was made between the parasite counts made by the three microscopists. The mean variation in parasite count for trophozoites and gametocytes was less than 5%. Owing to electricity supply deficiency, the haematocrit microcentrifuge was only used at sites 1 and 3. Outcome measures. Patients completed the trial if treatment was administered fully and all follow-up appointments conducted, or if they failed treatment on any day of follow-up. The primary endpoint of the trial was any clinical or parasitological failure up to day 28, although a subset of patients in the SP treatment arms was also followed until day 42. Patient outcomes were classified under the WHO parasitological classification system of sensitive (S) or resistant (RI, RII, RIII) infections with those whose outcomes were classified as S (sensitive) being treatment successes and those with outcomes of RI, RII or RIII being classified as treatment failures [34]. This classification system was still commonly used at the time of the study and the ethical clearance for this study (received in 2000) was based on a protocol using this classification system. Patients were also classified against the newer WHO treatment outcome system [35], [36]. Presenting the outcomes under both of these systems allows better comparability with data past and present [37]. For the WHO classification system standard definitions were applied: adequate clinical and parasitological response (ACPR), early treatment failure (ETF) or late treatment failure (LTF) which incorporated standard definitions of late clinical and parasitological response [35]. Those cases with ETF or LTF were classified as treatment failures, and ACPR was classified as treatment success if they completed the follow-up period. Other parameters examined were time to fever resolution (with fever defined as axillary temperature ≥37.5°C), asexual parasite clearance, gametocyte clearance and gametocyte carriage on or after day 7. Molecular characterisation. PCR genotyping was conducted on a subset of samples to distinguish recrudescent from new infections using the protocol described by Brockman et al [38]. P. falciparum genes msp-1, msp-2 and glurp were genotyped according to polymorphisms present at variable loci on day 0 and day of failure. Approximately 50% of cases were available for genotyping. PCR corrected outcomes were applied as a secondary analysis using redefined outcomes based on the number and frequency of true recrudescent infections and reclassifying cases with new infections as treatment successes, excluding those with indeterminate outcomes or negative PCR results. PCR testing was performed at the Shoklo Malaria Research Unit, Thailand. We also conducted an analysis at the London School of Hygiene and Tropical Medicine (LSHTM), UK, for known resistance-associated mutations to chloroquine and antifolate drugs. Samples collected at enrolment were tested for known mutations in the pfcrt, pfmdr1, pfdhfr and pfdhps genes [39], [40]. PCR and sequence specific oligonucleotide probe assays were used to analyse nucleotide polymorphisms at pfcrt codon 76, pfmdr1 codons 86 and 184, pfdhfr codons 16, 51, 59 and 108 and pfdhps codons 436, 437, 581 and 613 [41]. BODY.SAMPLE SIZE AND DATA ANALYSIS: The sample size required to detect a difference in treatment failure or gametocyte carriage with 80% power and 95% confidence (two-sided significance level for type 1 error of 0.05) were calculated using the following predictions. In the chloroquine arms (i) the estimated frequency of failure in chloroquine monotherapy arm was 30% and in each of the combination arms (CQ+PQ or CQ+AS) it was 10% or less; (ii) the estimated proportion of gametocyte positive individuals after 7 days in the chloroquine arm was 50% and in each of the combination arms it was 25%. In the SP arms (i) the estimated frequency of failure in the SP arm was 10% and in each of the combination arms (SP+PQ or SP+AS) it was 1%; (ii) the estimated frequency of gametocyte positive patients after 7 days in the SP arm was 50% and in each combination arm it was 25%. The estimated samples sizes were 64 per arm in the chloroquine arms and 121 per arm in the SP arms. For gametocyte carriage the required sample sizes were 65 per arm. The target sample sizes for the study were 65 for the chloroquine arms and 121 for the SP arms. To allow for 15% loss to follow up the targets for recruitment were 76 for the chloroquine arms and 142 for the SP arms. The primary aims of the study were to evaluate: 1) the relative efficacy of the combination drugs in providing parasite clearance with no recrudescence compared to monotherapy, and 2) the effect on gametocyte clearance. The secondary aim was to determine the proportion of individuals in each arm with classifiable treatment failure. The primary outcome was the proportion of individuals in each treatment group classified as a treatment failure during the 28 days follow-up compared to the respective monotherapy. The odds of treatment failure between each of the study groups at day 28 were estimated after adjusting for potential confounders (age, sex, parasitaemia, PCV, study site) using logistic regression analysis or Mantel-Haenszel χ2 test. Treatment failure was also analysed using the definitions of ACPR and ETF or LTF, and parasitological classifications using the S-R scale in addition to the PCR corrected analysis [35]. Time to event data (time to treatment failure) were analysed with Kaplan Meier survival analysis. For assessing potential effects on gametocyte carriage, the primary outcome was the presence or absence of gametocytes on day 7 with a secondary analysis examining the presence or absence of gametocytes and the geometric mean gametocyte density on each day of follow-up. All data were entered in Microsoft Excel (1997) by one data clerk and the database checked patient by patient against paper records by a second data clerk. Analysis was performed using STATA 10.0 (STATA Corp, College Station, TX, USA). BODY.ETHICS STATEMENT: The study protocol was approved by the ethics committees of the Pakistan Medical Research Council and LSHTM, UK. All patients or their guardians gave written informed consent to participate. The trial was registered under clinicaltrials.gov [NCT00959517]. BODY.RESULTS: BODY.RECRUITMENT AND FOLLOW-UP: A total of 355 cases of microscopically confirmed falciparum malaria were enrolled into the study between July 2000 and December 2002. Table 2 shows enrolment characteristics and Figure 1 shows the trial profile. Patients were recruited to the chloroquine arms in line with recruitment targets. Insufficient numbers were recruited to the SP arms. This was due to considerably lower than expected malaria cases occurring in these locations during the study period (in contrast to the years leading up to the study). Additional sites were added over the three-year study period to increase recruitment but it was not possible to continue the trial for longer because of resource constraints. chloroquine arms met the recruitment targets because of the site allocations and unexpected pattern of case loads in the third year. 10.1371/journal.pone.0028957.g001Figure 1BODY.TRIAL PROFILE.: 10.1371/journal.pone.0028957.t002Table 2BODY.ENROLMENT CHARACTERISTICS OF THE TREATMENT GROUPS.: Variable CQ CQ+PQ CQ+AS SP SP+PQ SP+AS Number enrolled 76 76 74 45 40 44 Number evaluable at day 28 (%) 68 (89) 67 (88) 67 (91) 41 (91) 33 (82) 41 (93) Age [median (IQR) years] 12 (8–18) 12 (8–20) 12 (8–20) 17 (9–27) 14 (7–25) 18.5 (9.5–30) Percentage female 42 37 50 33 43 45 Weight [median (IQR) kg] 29 (20–47) 30 (20–53) 33 (20–45) 48 (25–58) 42 (22–55) 41 (21–57) Temperature [mean (SD) °C] 37.3 (1.0) 37.5 (1.2) 37.4 (1.2) 37.5 (1.0) 37.5 (1.2) 37.5 (1.5) Temperature ≥37.5°C on presentation [n (%)] 33 (43) 34 (45) 34 (46) 21 (47) 17 (43) 23 (52) PCV [mean (SD) % haematocrit]1 42.9 (9.7) 40.8 (3.9) 41.5 (4.3) 44.2 (7.7) 45.5 (6.9) 44.3 (5.1) PCV<30% [n (%)]1 0 0 0 1 (2.2) 0 0 Asexual parasite density [geometric mean (95% CI) per µl] 5161 (3536–7535) 5263 (3647–7595) 7366 (4972–10,915) 7600 (5185–11,140) 8091 (4236–15,454) 12,134 (7757–18,982) Gametocyte positive [n (%)] 12 (15.8) 15 (19.7) 9 (12.2) 14 (31.1) 7 (17.5) 7 (15.9) Gametocyte density [geometric mean (95% CI) per µl] 1.3 (0.4–2.6) 1.7 (0.7–3.4) 0.6 (0.2–1.2) 4.1 (1.4–10.1) 1.6 (0.3–4.1) 1.2 (0.2–2.9) Notes: (1) PCV was not recorded for all patients; a microcentrifuge was only available at one of the 3 clinics. For PCV percentages in the 6 treatment groups: CQ n = 10; CQ+PQ n = 19; CQ+AS n = 13; SP n = 19; SP+PQ n = 15, SP+AS n = 15. Characteristics of patients recruited into the 3 chloroquine arms were broadly similar, as were the three SP arms. The SP+AS arm showed slightly higher asexual parasite densities on enrolment than other groups (Table 2). Of 355 patients enrolled, 38 (10.7%) were either withdrawn or lost to follow up leaving 317 for possible inclusion (Figure 1). Of these 292 (82%) were evaluable for parasitological outcomes because 25 patients classified as RII parasitological failure did not meet the definition of clinical failure, i.e. they did not have fever on day 3. Likewise, 308 (87%) were evaluable for clinical outcomes because 9 patients who had classifiable Early Treatment Failure did not fit the definition for parasitological failures (they did not reach day 7 of the trial). Of the 266 patients followed up for 42 days, 221 (83%) were evaluable for clinical and parasitological outcomes. All treatments were well tolerated and no severe or serious adverse events were recorded during the study. Clinical Outcomes. The 28-day failure rate in the CQ and CQ+PQ arms was 81% and 73% respectively (Table 3, Figure 2A). The addition of artesunate improved the treatment response, with a treatment failure rate of 27%. The failure rates in the SP groups were 16% or less (Table 3) with the combination of SP+AS having the lowest failure rate (1/41 [2.4%]). Neither SP+AS nor SP+PQ arms were significantly different to the SP arm at the 28-day end point (Figure 2B). 10.1371/journal.pone.0028957.g002Figure 2BODY.KAPLAN MEIER SURVIVAL ANALYSIS SHOWING CUMULATIVE PROBABILITY OF FAILURE IN CQ TREATMENT GROUPS (A) AND SP TREATMENT GROUPS (B).: 10.1371/journal.pone.0028957.t003Table 3BODY.CLINICAL AND PARASITOLOGICAL FAILURE RATES AT 28 DAYS FOLLOW-UP AND FOR GAMETOCYTAEMIA AT DAY 7.: Day 28 clinical or parasitological failure Day 7 gametocytaemia No./Total (%) Odds Ratio (95% CI) P No./Total (%) Odds Ratio (95% CI) P CQ 55/68 (81) Reference - 52/67 (78) Reference - CQ+PQ 49/67 (73) 0.64 (0.29–1.4) 0.3 27/70 (39) 0.18 (0.09–0.4) <0.001 CQ+AS 19/67 (28) 0.09 (0.04–0.2) <0.001 12/72 (17) 0.06 (0.02–0.1) <0.001 SP 4/41 (10) Reference - 37/43 (86.1) Reference - SP+PQ 5/33 (16) 1.6 (0.41–6.7) 0.5 24/36 (66.7) 0.32 (0.10–1.0) 0.046 SP+AS 1/41 (2) 0.23 (0.02–2.2) 0.2 9/44 (20.5) 0.04 (0.01–0.1) <0.001 None of the potential confounding factors examined (age, sex, parasitaemia, study site, PCV) were associated with treatment outcome so adjustments were unnecessary in the final regression analysis. Crude odds ratios are presented. Clinical and parasitological outcomes are shown in Table 4. Only 32/133 (24%) of the individuals who failed had fever on the day of failure; the majority of ‘late treatment failures’ therefore fell into the category of ‘late parasitological failure’. Some recent history of fever may have gone unreported. 10.1371/journal.pone.0028957.t004Table 4BODY.CLINICAL AND PARASITOLOGICAL OUTCOMES AFTER 28 DAYS FOLLOW-UP: N (%).: CQ CQ+PQ CQ+AS SP SP+PQ SP+AS Clinical outcomes N = 56 N = 58 N = 67 N = 41 N = 30 N = 40 Adequate clinical response 13 (23) 18 (31) 48 (72) 37 (90) 28 (93) 40 (100) Early treatment failure 9 (16) 7 (12) 0 1 (2) 1 (3) 0 Late clinical failure 2 (4) 4 (7) 1 (2) 0 0 0 Late Parasitological Failure 32 (57) 35 (50) 18 (26) 3 (7) 1 (3) 0 Parasitological outcomes N = 63 N = 65 N = 67 N = 40 N = 32 N = 41 S 13 (21) 18 (28) 48 (72) 37 (93) 28 (88) 40 (98) RI 35 (56) 33 (51) 19 (28) 3 (7) 1 (3) 0 RII 13 (21) 11 (16.9) 0 0 3 (9) 1 (2) RIII 2 (3) 3 (5) 0 0 0 0 Trophozoite clearance time (days until negative smear) was lowest for the artesunate combination arms. Addition of primaquine to either chloroquine or SP did not appear to affect clearance times. Clearance times for each arm (median (interquartile range)) were: CQ, 3 days (2–7); CQ+PQ, 3 days (2–7); CQ+AS, 2 days (1–2); SP, 2 days (2–3); SP+PQ, 2 days (1.5–3); SP+AS, 1 day (1–2). Of the 133 failures, 79 (47%) matched pairs were available for PCR evaluation of MSP-1, MSP-2 and GLURP. Matched pairs were collected on day 0 and on the day of failure. Outcomes of the PCR are shown in Table 5, which excludes those with indeterminate results. Most re-infections took place at one site (Site 3, Yakka Ghund) where malaria transmission was higher than in the other villages. Treatment outcomes were reclassified for those failures where matched pairs were available and used to give adjusted failure rates for the sample (Table 5). Although the number of failures in the chloroquine arms was reduced by the correction, the failure rates remained high at >50% for chloroquine monotherapy. The PCR adjusted treatment failure rate of the CQ+AS combination (9%) was considerably lower that the in vivo rate (28%). 10.1371/journal.pone.0028957.t005Table 5BODY.OUTCOMES OF PCR ANALYSIS, AND PROJECTED ADJUSTED FAILURE RATES BY TREATMENT GROUP EXCLUDING INDETERMINATE RESULTS.: PCR Results [n (%)] Failure rates [n/N (%)] Reinfection Recrudescent Negative Total in vivo 1 PCR adjusted 2 CQ 6 (21) 20 (69) 3 (10) 29 55/68 (81) 42/68 (62) CQ+PQ 4 (21) 11 (58) 4 (21) 19 49/67 (73) 36/67 (54) CQ+AS 7 (64) 3 (27) 1 (9) 11 19/67 (28) 6/67 (9) SP 0 2 (100) 0 2 4/41 (10) 4/41 (10) SP+PQ 0 2 (100) 0 2 5/33 (16) 5/33 (16) SP+AS - - - 0 1/41 (2) 1/41 (2) Notes: (1) Taken from Table 3 for 28 day failures; (2) Adjusted by the ratio of PCR recrudescent to PCR re-infected cases to estimate number of true recrudescent cases among the observed in vivo failures. In the subset of patients followed-up for 42 days, recrudescence between day 28 and 42 was rare: only 8/317 (2.5%) of individuals classified as adequate clinical and parasitological response (ACPR) or sensitive (S) at day 28 went on to fail by day 42 (1 in the CQ arm, 2 in the CQ+PQ arm, 3 in the CQ+AS arm, 1 in the SP arm and 1 in the SP+PQ arm). Failure rates at day 42 were therefore similar to day 28. The failures between 28-day and 42-day rates were not corrected by PCR and hence could have resulted from new infections. Molecular outcomes. Molecular analysis of drug resistance-associated alleles was conducted on samples taken on day 0 (Table 6). The major marker of chloroquine resistance, pfcrt-76T, was fixed at 100%. Mutations in pfmdr1 are known to modulate resistance to quinoline and other drugs. The prevalence of chloroquine-resistance associated allele pfmdr1-86Y was 13.6% in this sample. Mutations associated with pyrimethamine resistance (on the pfdhfr gene) were seen frequently; 108 N was found in all but one of 74 typable samples (98.6%) and 59R occurred in 66/76 (86.8%) samples. However, 51I was only detected in 5/75 (6.7%) samples. Therefore pfdhfr ‘double mutants’ were common (at codons 108+59) but ‘triple mutants’ were rare. Interestingly, we observed one sample with the pfdhfr mutations 16V+108T. These are reportedly selected by the antifolate drug cycloguanil, rather than pyrimethamine [42]. No mutations on the dhps gene were seen. These PCR results broadly match what would be expected from the clinical outcomes. The pfdhr/pfdhps mutation profile suggests that the parasites would be killed by a full therapeutic dose of SP, but that some tolerance to lower doses existed. 10.1371/journal.pone.0028957.t006Table 6BODY.DRUG RESISTANCE ALLELES IN A SUB-SET COLLECTED AT ENROLMENT.: Locus Allele Number (%) Pfcrt 76 K 0 76 T 63 (100) Pfmdr1 86 N 76 (86) 86 Y 12 (14) 184 Y 22 (27) 184 F 60 (73) DHFR 16 A 76 (99) 16 V 1 (1) 50/51 CN1 70 (93) 50/51 C1 5 (7) 59 C 10 (13) 59 R 66 (67) 108 N 73 (99) 108 T 1 (1) DHPS 436/437 SA 43 (100) 581 A 41 (100) 613 A 43 (100) BODY.EFFECT ON GAMETOCYTES: The addition of artesunate or primaquine to chloroquine or SP reduced gametocyte carriage on day 7 (Table 3). Combining chloroquine or SP with artesunate succeeded in eliminating gametocytaemia from more individuals by day 7 compared to monotherapy or co-treatment with a single-dose of primaquine. Figure 3 shows the proportion of individuals with patent gametocytaemia over 28 days of follow-up. In the chloroquine and SP arms, gametocyte carriage persisted at day 28 in >30% of individuals in the CQ arm and >70% of individuals in the SP arm. The addition of primaquine reduced gametocyte carriage; the effect was more pronounced with chloroquine than with SP. The proportion of gametocytaemic individuals was lowest in the artesunate arms and the difference was evident within two days of the start of treatment. Peak gametocyte densities occurred 7 days after the start of treatment (Figure 4). Gametocyte density was higher after SP than after chloroquine treatment. Both primaquine and artesunate reduced density to low levels by day 7. 10.1371/journal.pone.0028957.g003Figure 3BODY.PERCENTAGE OF PATIENTS (+CI) CARRYING GAMETOCYTES ON SPECIFIED DAYS AFTER TREATMENT IN CQ (A) AND SP (B) TREATMENT ARMS.: 10.1371/journal.pone.0028957.g004Figure 4BODY.GEOMETRIC MEAN GAMETOCYTE DENSITY (+CI) AFTER TREATMENT IN CHLOROQUINE (A) AND SP TREATMENT ARMS (B).: Note the different scales on the Y-axis. Where the CI bars are absent, only 1 patient had gametocytes. Most patients did not have gametocytaemia on day 0 (291/355 (82.0%) had no gametocytes on day 0). However, the presence or absence of sexual stage parasites on day 0 was an important explanatory variable in the secondary analysis; patients who presented with gametocytes on day 0 were more likely to be gametocytaemic on day 7 than individuals who were not gametocytaemic on day 0 regardless of treatment group (Mantel-Haenszel OR: 3.5 [95%CI: 2.0–6.4], p<0.001). We therefore conducted further analysis comparing those who presented with gametocytes on day 0 and those who did not. The artesunate treatments were more effective at preventing the emergence of gametocytaemia between day 0 and 7 than in removing established gametocytaemia already present on day 0 (Table 7). For example, among the CQ+AS group who did havedid not have patent gametocytaemia on day 0, only 11% (7/63) were gametocytaemic on day 7 when treated with CQ+AS as compared to 47/56 (84%) among those treated with chloroquine monotherapy (χ2 p<0.001). Similar trends were evident in the SP arms treated with artesunate; prevalence of gametocytaemia on day 7 was significantly lower in the SP+AS group that was not gametocytaemic on day 0 than in the group that was (Fisher's exact test p<0.001). This indicates that artesunate is less active against older gametocytes than against those newly emerged or immature forms that are not yet emerged. Primaquine showed no such trend: the proportion of patients gametocytaemic on day 7 was not significantly different for patients who were or were not gametocytaemic on day 0. Primaquine therefore appears to be active against gametocytes of all ages. 10.1371/journal.pone.0028957.t007Table 7BODY.NUMBERS AND PERCENTAGES OF INDIVIDUALS WITH GAMETOCYTES ON DAY 7 POST TREATMENT.: No (%) parasitaemic on day 7 Amongst those who had gametocytes on day 0 Amongst those who did not have gametocytes on day 0 CQ 11/12 (91.7%) a, 1 47/56 (83.9%) a, 1 CQ+PQ 7/14 (50%) b, 1 21/56 (37.5%) b, 1 CQ+AS 8/9(88.9%) a, 1 7/63(11.1%) c, 2 SP 14/14 (100%) a, 1 25/29 (86.2%) a, 1 SP+PQ 5/7 (71.4%) b, 1 23/30 (76.7%) a, 1 SP+AS 5/7 (71.4%) b, 1 5/37 (13.5) b, 2 Chloroquine or SP treatments in the same column that share the same superscript letter are not significantly different. Treatments in the same row that share the same numeric superscript are not significantly different. Primaquine was more effective at controlling gametocytaemia when combined with CQ than with SP, regardless of whether individuals were gametocytaemic at the start of treatment (MH-OR = 0.22, P = 0.001) (Table 7). By contrast, artesunate seemed as effective when combined with chloroquine as it was when combined with SP (MH-OR = 1.04, P = 0.83). Though, as discussed, artesunate was more effective at preventing development of patent gametocytaemia than in clearing existing gametocytaemia. Artesunate was better than primaquine at preventing patent gametocytaemia among those that were not gametocytaemic at day 0 enrolment (CQ: OR = 0.30, P<0.001; SP: OR = 0.18, P<0.001). Using the presence of gametocytes on day 7 as the secondary endpoint, adjusted for the presence of gametocytes at day 0, both primaquine and artesunate with chloroquine or SP were more effective than their respective monotherapies at reducing the presence of gametocytes (Table 8). 10.1371/journal.pone.0028957.t008Table 8BODY.EFFECT OF TREATMENT ON GAMETOCYTE CARRIAGE AT DAY 7 AND DAY 28, FOR THOSE PATIENTS WHO WERE GAMETOCYTAEMIC ON ENROLMENT.: AOR for presence of gametocytes on day 7, (95% CI), p-value 1 AOR for presence of gametocytes on day 28, (95% CI), p-value 1 CQ Reference Reference CQ+PQ 0.15 (0.07–0.34), p<0.001 0.42 (0.08–2.2), p = 0.3 CQ+AS 0.05 (0.02–0.12), p<0.001 0.04 (0.004–0.39), p = 0.006 SP Reference Reference SP+PQ 0.37 (0.12–1.16), p = 0.09 0.14 (0.05–0.4), p<0.001 SP+AS 0.04 (0.01–0.13), p<0.001 0.02 (0.005–0.1), p<0.001 Notes: (1) AOR: Odds ratios using logistic regression analysis adjusted for presence of gametocytes on day 0 (CQ and SP arms analysed separately). Compared to monotherapy with chloroquine or SP, co-treatment with artesunate was negatively associated with having gametocytes on day 28 whether the accompanying drug was chloroquine or SP (Table 8). The presence of gametocytes on day 0 was not associated with the presence of gametocytes on day 28 (MH-OR, adjusted for treatment arm: 1.0, p = 1.0) suggesting that older gametocytes had cleared by day 28, regardless of treatment. BODY.DISCUSSION: The present study was designed to inform decision-making at several levels: to guide UNHCR on appropriate treatment in the Afghan refugee communities in Pakistan, to build evidence for national treatment policy in the Pakistan Directorate of Malaria Control and in the Afghanistan Ministry of Public Health, to guide the WHO Eastern Mediterranean Region Office (EMRO) on regional treatment recommendations, and to build evidence for international epidemic response guidelines. The findings of this study, when presented at an inter-country meeting of national malaria control managers coordinated by WHO/EMRO in 2004, contributed to a shift in policy from chloroquine monotherapy to ACT in South and West Asia [43], influencing the defining of SP+AS as the first line treatment of choice for the WHO Eastern Mediterranean Region. India is the latest neighbouring country to adopt SP+AS as first line treatment with the support of WHO [37]. The SP+AS combination is also effective against vivax malaria [44], although not as an approved treatment. SP monotherapy was effective in the study population, with treatment failure rates remaining in the 5–10% range, similar to that seen in other studies in the region [5], [8], [45]. Molecular studies in the region show that genetic markers for SP resistance are still at low levels in the parasite population [46], [47]. However, the South East Asian experience of rapid rise in SP resistance in settings of similar endemicity [7] points to SP resistance spreading rapidly if ACT policy is not implemented rigorously in both public and private sectors across the region [37]. The complete failure of chloroquine (which at the time of the study remained the officially sanctioned first line treatment) convinced policy makers of the need to redefine treatment practices in South Asia. The new data demonstrated higher levels of resistance than in previous studies dating back to the 1980s and 1990s [2], [3], [5], [48], [49] and emphasised the need for change to national treatment guidelines. The addition of artesunate to chloroquine did reduce treatment failure rates, but if this regimen was used as policy it would, in effect, constitute artesunate monotherapy in the majority of infections and accelerate the development of artesunate tolerance, as reported in South East Asia [50]. A constraint on the study design was that not all 6 arms could be included at each of the three study sites. Site is therefore a potential confounding covariate that cannot be fully controlled for in the data analysis. Differences in transmission could, for example, affect the risk of re-infection between sites but this was corrected for by the PCR analysis. At each site in any one year each triplet of treatment arms (all the SP arms or all the chloroquine arms) were examined at the same time, and it is comparisons within these triplets which are of greatest interest. The study did not complete its target sample size for the SP arms because of low recruitment rates. PCR corrected outcomes were not attainable for all failures, and higher numbers of patients were reinfected at one site where only the chloroquine arms were being tested. Low rates of transmission at the other sites make it unlikely that failing cases were due to reinfection. The secondary analysis with PCR-corrected outcomes did not change the overall conclusions on the suitability of the combinations for revised treatment policy. Treatment with either the clinically effective SP monotherapy or the failing chloroquine monotherapy resulted in persistence of gametocytaemia into the second and third weeks in the majority of individuals. This effect was more evident for SP than for chloroquine, SP being well known for high rates of gametocyte carriage post treatment [32]. This contrasts with situations of low-frequency chloroquine resistance where the proportion of gametocytaemic patients post treatment also tends to be lower [16], [51]. The effect of artemisinin derivatives on reducing gametocyte carriage is already documented [16], [52]. Artesunate also proved highly effective in reducing the prevalence of gametocytaemia in the present study. Regimens combining artesunate with chloroquine or SP saw marked reductions in gametocyte carriage and few cases had persisting gametocytaemia. As demonstrated with SP+AS in The Gambia [16] the SP+AS and CQ+AS combinations appear to have limited activity against mature circulating gametocytes [53]. Co-treatment with a single dose of primaquine was more effective against older gametocyte infections. Both primaquine and, more markedly, artesunate reduced the odds of persisting gametocytes from that seen post treatment with either chloroquine or SP monotherapy. Primaquine is more rapidly excreted than artesunate and this may account for the fact that the single dose of primaquine used in this study had a lower impact on gametocyte carriage than the artesunate regimens. The usefulness of primaquine as a gametocytocidal treatment may be improved by administering it after the ACT course [14], [54]. In our study, a key factor in the clearance of gametocytes was the presence or absence of gametocytes on day 0. Those who presented with gametocytes were more likely to have gametocytes on day 7, an effect which was independent of the treatment given. Although the majority of patients (80%) presented without gametocytaemia, artesunate appeared to be less active against these older parasites whereas primaquine appeared to be effective against all ages. The proportion presenting with gametocytes may vary according to background transmission levels. If gametocytes persist after treatment with ACTs, the effect on transmission in areas where a high proportion of cases present with gametocytes may prove suboptimal and justify the simultaneous use of primaquine. Primaquine is more rapidly excreted than artesunate and this may account for the fact that the single dose of primaquine used in this study had a lower impact on gametocyte carriage than the artesunate regimens. The usefulness of primaquine as a gametocytocidal treatment may be improved by administering it after the ACT [14], [54]. For treatment policy to have a major impact on transmission several criteria need to be met: i) transmission is low to moderate; ii) the majority of people use public health rather than private facilities, or effective private sector interventions ensure adherence to policy; iii) public health facilities correctly prescribe the approved regimen, and iv) patients take the full course. These conditions are largely met in the refugee villages of Pakistan. The region is characterized by low endemicity and low immunity, well supported health care facilities are available in the Afghan refugee villages, and diagnosis of malaria to species level is maintained to a high standard [28]. The health facilities are well utilized by the Afghan refugees and Pakistanis living nearby. The effect of this policy is evident in the refugee populations: since 2005 falciparum malaria has virtually disappeared in all but a handful of the refugee villages [International Rescue Committee, Final Report, unpublished]. This reduction is attributed to prompt and accurate diagnosis and wider access to ACTs as prevention interventions were minimal. The operational data from this region and elsewhere suggest that drugs showing faster gametocyte clearance in clinical trials do help to reduce transmission [20]–[23]. However, it is also important to note the results of recent research which suggest that drawing conclusions about the potential impact on transmission is not straightforward, for the following reasons: The effect of drugs on gametocyte carriage cannot be fully determined if only standard light microscopy (LM) is used. Research in the last decade shows that LM gives starkly different indications of gametocyte prevalence and densities compared to other techniques [55] and that sub-microscopic levels of gametocytes likely play an important role in transmission, perhaps particularly in low transmission settings [56]. Gametocytes that are readily identified in blood following treatment may or may not be viable. Gametocytes may not be infective to mosquitoes either because they are recently emerged (naturally, gametocytes are not infectious to mosquitoes in the first 1–2 d of emergence [57]) or because the effect of the drug regimens have rendered them ‘sterile’ [58]. It is unclear how the density of gametocytes in the blood links to potential for transmission success to blood feeding mosquitoes. Bousema & Drakeley [57] summarise the available data from membrane feeding studies showing that whilst there is an overall correlation, the variations within it are confusing; high density gametocytaemia can lead to minimal mosquito infection and very low densities lead to reasonable rates of infection. Even if solid data show that a particular drug does reduce the number of gametocytes which successfully infect, and lead to infectious, mosquitoes; the effect of this on malaria transmission cannot be stated with certainty. This effect would depend on what proportion of the gametocyte reservoir these patients would otherwise make up [56]. The comparative performance of artesunate and primaquine on gametocyte carriage in this study did not influence policy. The decision to use SP+AS was based on treatment efficacy and the need to protect against SP resistance through the use of an ACT combination. The fact that SP+AS led to reduced gametocyte carriage compared to the previous policy of chloroquine montherapy was reassuring, and raised the possibility of the regimen reducing transmission too. SP+AS is now the first line treatment policy in Pakistan, Iran, Afghanistan and India. The continuing collection of molecular data on SP resistance in this region will be vital, given that the treatment efficacy of artesunate could mask the emergence of SP resistance by acting alone to provide a degree of clinical cure in the presence of a failing companion drug [59]. This risk could be reduced by introduction of a co-blister of SP plus artesunate. BODY.SUPPORTING INFORMATION: Protocol S1 Trial Protocol. (DOC) Click here for additional data file. Checklist S1 CONSORT Checklist. (DOC) Click here for additional data file.

TITLE: Two Novel Cognitive Behavioral Therapy–Based Mobile Apps for Agoraphobia: Randomized Controlled Trial ABSTRACT.BACKGROUND: Despite the large body of literature demonstrating the effectiveness of cognitive behavioral treatments for agoraphobia, many patients remain untreated because of various barriers to treatment. Web-based and mobile-based interventions targeting agoraphobia may provide a solution to this problem, but there is a lack of research investigating the efficacy of such interventions. ABSTRACT.OBJECTIVE: The objective of our study was to evaluate for the first time the effectiveness of a self-guided mobile-based intervention primarily targeting agoraphobic symptoms, with respect to a generic mobile app targeting anxiety. ABSTRACT.METHODS: A Web-based randomized controlled trial (RCT) compared a novel mobile app designed to target agoraphobia (called Agoraphobia Free) with a mobile app designed to help with symptoms of anxiety in general (called Stress Free). Both interventions were based on established cognitive behavioral principles. We recruited participants (N=170) who self-identified as having agoraphobia and assessed them online at baseline, midpoint, and end point (posttreatment) over a period of 12 weeks. The primary outcome was symptom severity measured by the Panic and Agoraphobia Scale. ABSTRACT.RESULTS: Both groups had statistically significant improvements in symptom severity over time (difference –5.97, 95% CI –8.49 to –3.44, P<.001 for Agoraphobia Free and –6.35, 95% CI –8.82 to –3.87, P<.001 for Stress Free), but there were no significant between-group differences on the primary outcome (difference 0.38, 95% CI –1.96 to 3.20, P=.64). ABSTRACT.CONCLUSIONS: This is, to our knowledge, the first RCT to provide evidence that people who identify as having agoraphobia may equally benefit from a diagnosis-specific and a transdiagnostic mobile-based intervention. We also discuss clinical and research implications for the development and dissemination of mobile mental health apps. ABSTRACT.TRIAL REGISTRATION: International Standard Randomized Controlled Trial Number (ISRCTN): 98453199; http://www.isrctn.com /ISRCTN98453199 (Archived by WebCite at http://www.webcitation.org/6uR5vsdZw) BODY.INTRODUCTION: People with agoraphobia have considerable impairment in their daily lives as a result of persisting avoidance of places and situations. Lifetime prevalence of agoraphobia with and without panic disorder in the adult population has been reported to range from 0.8% to 2.6% in American and European community samples [1,2]. Agoraphobia has been ranked as one of the most chronically persistent disorders [3], with higher rates of work-related inactivity and disability than with other chronic conditions [4,5]. The standard psychological treatment for agoraphobia symptoms is cognitive behavioral therapy (CBT), which is also recommended by the UK National Institute of Health and Care Excellence [6]. People with agoraphobia have catastrophic ideas regarding the likelihood of threat in a situation or environment, such as fainting and being ignored or ridiculed by others [7,8], and as a consequence engage in “safety behaviors” to prevent the expected catastrophe and reduce anxiety. Such safety behaviors preserve and might even enhance the maladaptive beliefs and thus maintain anxiety [9,10]. In CBT, cognitive restructuring is used to challenge catastrophic cognitions and unrealistic predictions, and to generate alternative, more realistic expectations. Behavioral experiments may be conducted to help disconfirm such beliefs and drop safety behaviors [8]. Moreover, imaginal or in vivo exposure to the feared situations is used to reduce situational avoidance and phobic anxiety. Breathing and relaxation exercises are sometimes employed to help the patient cope with overwhelming stress. The effectiveness of CBT in the treatment of agoraphobia has been demonstrated in several studies. A systematic review and meta-analysis [11] examined the effectiveness of psychotherapy and pharmacotherapy in the treatment of panic disorder, with the majority of participants also having agoraphobia. They identified 23 randomized controlled trials (RCTs) and showed that, although combined psychotherapy and pharmacotherapy produced the best results in the short term, in the long term combined treatment was as effective as psychotherapy alone, and both treatment groups were superior to pharmacotherapy alone. Among the different types of psychotherapy, CBT had the strongest evidence. More recent studies have also provided support for CBT as an effective treatment in agoraphobia patients [12-15] in line with earlier studies [16]. Even though CBT has been established as the preferred and most effective treatment for agoraphobia and panic, many people remain untreated. From the population of people with anxiety disorders in developed countries, only 16.7% seek help from a mental health professional, and only 21.3% of those receive CBT [17]. Despite the fact that CBT has nowadays become more widely available, certain barriers to treatment may explain the low levels of self-seeking in people with agoraphobia. For example, fear of stigmatization, lack of psychoeducation, long waiting lists, transportation problems, and time constraints significantly limit access to CBT [18-20]. In addition, the very nature of agoraphobia, which may include fears of leaving the house and using public transport, may make it even more difficult for people to actively seek professional help. To overcome such barriers, effective interventions that are easily accessible and do not require therapist face-to-face contact should be available. Internet-based or computerized interventions can be considered as acceptable alternatives to standard treatments, as they can be clinically effective and minimize treatment barriers for users. Such interventions can be self- or therapist guided, presenting materials of cognitive behavioral principles and methods in a series of lessons, which are typically accompanied by homework tasks and supplementary information. Systematic reviews show that computerized CBT (CCBT) is as beneficial as therapist-led CBT in the treatment of anxiety disorders such as panic disorder, and is more effective than treatment as usual or waiting list conditions [21,22]. CCBT can also reduce therapist time [21] and have good acceptability, as shown by adherence and satisfaction levels [22]. A more recent review of Internet CBT, including 8 trials of panic disorder with and without agoraphobia, concluded that Internet CBT was as efficacious as face-to-face CBT and more efficacious than waiting list, attention, information, and online discussion control groups [23]. One promising mode of delivering computerized interventions are mobile phones, because of their relatively low cost and widespread use [24]. Since users carry their mobile devices with them in almost any situation, mobile phones, and particularly smartphones, might facilitate engagement with exposure exercises in the users’ natural environments. Although mobile apps have been tested for several conditions, such as unipolar depression [25], borderline personality disorder, and substance abuse [26], to date there has been no study testing an app that primarily targets agoraphobic symptoms. Because of the isolative nature of agoraphobia and the extreme avoidance behaviors, an app that could be easily downloaded over the Internet onto patients’ phones or tablets, requiring no traveling to sites, might be especially appealing and convenient for this population. This study was a Web-based RCT aiming to test the clinical effectiveness of a novel mobile app for agoraphobia in a community-based sample. The treatment app, Agoraphobia Free, developed by Health eLiving Partnership Ltd (HeLP) for the iOS and Android operating systems, provides an interactive game-based intervention using cognitive behavioral techniques that target agoraphobia and panic. The comparator was a stress-reduction app (Stress Free), which does not address agoraphobic symptoms or panic, but stress and anxiety in general. Both interventions were self-guided and were evaluated over a period of 12 weeks. Adults that self-identified with agoraphobia were randomly allocated to the 2 treatment arms and completed self-reported assessments at baseline, midpoint (6 weeks), and end point (12 weeks) of the trial. The primary objective was to examine whether an agoraphobia-specific intervention would be more effective than a generic, anxiety-related intervention. A secondary aim was also to assess the level of engagement with these interventions and the feasibility of conducting such a trial over the Web. BODY.METHODS: BODY.DESIGN: The study was a Web-based, assessor-blinded, parallel-group RCT with an active control group. Participants were individually randomly allocated (ratio of 1:1) to either the treatment group or the control group at baseline and were given an equal amount of time to complete each intervention (12 weeks). Data were gathered automatically through online collection of anonymized data, without any researcher intervention. The primary outcome was the degree of symptom severity, as measured by the self-reported version of the Panic and Agoraphobia Scale [27] at end point. BODY.PARTICIPANTS: BODY.INCLUSION CRITERIA: Participants needed to be adults (aged ≥18 years) and identify themselves as having agoraphobia. Participants also had to be willing and able to provide informed consent to participate in the trial. We used no diagnostic check, as the aim was to recruit a community sample that would reflect the nature of the population that would use the apps in a real-world setting, where no screening or check would be required. BODY.EXCLUSION CRITERIA: The exclusion criteria were as follows: (1) inability to give informed consent due to significant cognitive or intellectual impairment, (2) no adequate understanding of English as a first language, and (3) not having a mobile device than could run the app as designed. BODY.RECRUITMENT AND SETTING: A website was set up for the trial on which advertisements and all relevant information and updates were posted. The website was hosted by HeLP Ltd (currently known as Thrive Therapeutic Software). Advertisements of the trial were also posted on social media (eg, Facebook, Twitter), support groups and forums, websites of relevant organizations such as Anxiety UK, blogs, and university websites. We also individually contacted members of anxiety support groups via Facebook or forum messages. We created a mailing list of people subscribing their interest in the trial, whom we encouraged to stay in touch until the trial commenced. Moreover, contacts of the Chief Investigator that were working in health-related settings were encouraged to inform any relevant clients or representatives about the study. There were 2 rounds of recruitment in order to achieve a larger number of participants. We always contacted participants via email. Initially, those who expressed interest in participating followed a link to the online information sheet and consent form, which outlined the eligibility criteria and information about the trial. Through consenting to participate and answering a series of questions, participants confirmed that they met the criteria and understood the purpose of the study. Participants also provided their email addresses and names, though the latter was optional. Participant codes were then assigned to those who consented, and details about their mobile devices (smartphones or tablets) and demographics (age and sex) were obtained. Participants were instructed to use only their participant code to identify themselves. Participants received invitations for the apps, which were available to use for free. Emails also contained links to weekly surveys on app use and links to questionnaires, a description of the specific survey questionnaire, links to the calendar and the main website of the trial, information about the upcoming survey, and useful contact details. A Frequently Asked Questions section was set up on the main website. Data were collected online and could only be accessed by the researchers. BODY.INTERVENTIONS: BODY.AGORAPHOBIA FREE: The treatment app was Agoraphobia Free (version 0.8), developed by Thrive Therapeutic Software for the treatment of agoraphobia, and this was the first time that it was evaluated. The app was a game-based interactive intervention, with 3-dimensional characters and situations that simulate real-life environments. Specifically, the app presented a case example of a virtual character who had agoraphobia. The user was required to guide her, through the help of the virtual therapist, to complete the different therapeutic tasks. Those tasks were based on CBT principles, comprising psychoeducation, reflection, cognitive restructuring, interoceptive exposure, and systematic desensitization. The 3 overarching goals were to decrease the virtual character’s catastrophic cognitions, safety behaviors, and physiological arousal. In this way, users were able to progress through the character’s recovery and treatment, and build the formulation of her difficulties. At the same time, users were asked to apply the techniques they used in the case example to their own situation. Therefore, by using the character’s recovery journey as a template, users learned how to reduce their catastrophic thinking, their safety behaviors, and physiological arousal. At the beginning of the intervention, we asked participants to set a hierarchy of goals they would like to achieve (eg, going out of the house, going to the supermarket). At the end of each session, they were required to complete each one of those goals in order, using the techniques and strategies they had learned in the intervention. The sessions were designed so that the tasks became increasingly more challenging as participants progressed through the intervention. The sessions needed to be completed in the order they were presented in for the next ones to become unlocked. There were 10 sessions in total, and the participants were asked to complete 1 or 2 sessions per week at their own pace, and reminders for those were sent weekly. Multimedia Appendix 1 shows an outline of each session. BODY.STRESS FREE: The control app was Stress Free (version 1.3) developed by Thrive Therapeutic Software, which focused on teaching relaxation techniques and generic CBT skills though a virtual therapist. The app also included a few distraction techniques presented in the form of games that required attention to help individuals cope with acute anxiety. The intervention was presented initially as a linear training program using video and audio guides. The user first learned diaphragmatic breathing, then differential deep muscle relaxation, then self-hypnosis, and finally meditation. These relaxation techniques have been previously shown to be effective in reducing stress [28,29]. Participants rated their anxiety before and after relaxation sessions using a visual analog scale. After completing the training, they were familiarized with CBT concepts such as negative automatic thoughts and the process of challenging them. Finally, we gave participants a daily goal to complete, such as doing 3 sessions of diaphragmatic breathing or a 10-minute session of meditation. In between sessions, participants were prompted to record their anxiety on a CBT journal. The app only targeted stress and anxiety in general, so it was not specific to agoraphobia. In the original version, there was no maximum limit of sessions that users could go through until they mastered the techniques. However, for the purpose of the trial, we asked participants to complete 10 sessions in total, so as to match the number of sessions required from the treatment group. Table 1 compares the 2 apps by showing which components and exercises were present in each. Although some components were common to both apps, in Agoraphobia Free they were specifically tailored to agoraphobia. In Stress Free the exercises addressed stress and anxiety in general without referring to agoraphobia. The 2 apps were matched for the number of sessions required and time to complete the interventions (minimum: 6 weeks, maximum: 12 weeks). No training, supervision, or guidance was offered before or during the trial, and only a basic description of each app was provided. Any questions participants had regarding the app or any technical issues they encountered were resolved through email. Weekly reminders and short surveys were sent to promote engagement and monitor progress, and the completion of 1 to 2 sessions per week was recommended for both groups. We also informed participants at the beginning of the trial that, when they completed the intervention, they would receive a link to download the app they did not receive for free, as a reward for taking part and an additional incentive to complete their assigned intervention. Participants were assured that they would not be asked any questions about the second app, as it would not be part of the research. The 2 apps were available on Android and iOS. Multimedia Appendix 2 shows screenshots of the apps. BODY.OUTCOMES: The primary outcome was the severity of agoraphobic and panic symptoms, measured by the PAS [27]. The questionnaire was administered online in a self-report format at baseline, midpoint (6 weeks), and end point (12 weeks) of the trial. Reminders were emailed to those who did not reply to the questionnaires before the prespecified deadline. Participants rated the symptoms they experienced in the previous week on a 5-point scale. The questionnaire comprises 14 items, although only 13 of those are used to calculate severity scores. The items are grouped into 5 subscales: (1) Panic Attacks, assessing frequency, severity, and duration of panic attacks, (2) Agoraphobic Avoidance, assessing frequency of avoidance, and number and relevance of avoided situations, (3) Anticipatory Anxiety, assessing frequency and severity of anxiety, (4) Disability, assessing impairment in family life, social relationships, and employment, and (5) Worries about Health, assessing worries about damage to health and assumption of organic disease. The scale was originally validated in a sample of 235 panic patients and has shown good internal consistency (Cronbach alpha=.88), test-retest reliability, and good internal and external validity [27,30]. Cronbach alphas for this study’s sample indicated good internal consistency, with alpha=.84 for the overall scale. Coefficients for individual subscales ranged from .41 to .88. The scale has been shown to be sensitive to change due to treatment in 2 clinical trials [31,32]. The secondary outcomes were completion of the intervention and engagement with the apps. Completion of the interventions was assessed in the short online surveys that were sent weekly, by asking participants if they had used the app, how much time they used it for over the past week, and how many sessions they had completed. If participants claimed that they had not used the app, they were asked to give reasons. BODY.SAMPLE SIZE: We estimated the sample size on the basis of using the self-rated version of the PAS (SD 10.3) as the primary outcome measure. At least 68 participants in each arm were needed to detect a 5-point between-group difference, with a 2-sided significance level set at 5% and power at 80%. Given the high dropout rates in Web-based trials [33], the aim was to recruit at least 150 participants in total. Table 1The different features present (indicated by “X”) in each app. App features Stress Free app Agoraphobia Free app Relaxation technique training X X Automated activity goals X X CCBTa basic tutorial X X CCBT journal with prompts X X Maintenance sessions X X Self-soothing strategies X X Distraction techniques X X Structured CCBT program X Goal setting by user X Construction of exposure hierarchy X Development of a formulation X Relapse prevention session X aCCBT: computerized cognitive behavioral therapy. BODY.RANDOMIZATION: We used a random computer-generated sequence to randomly allocate participants to the 2 intervention groups. The random allocation sequence was retrieved from a website that generates truly random numbers [34], by a person outside the research team. We applied block randomization to ensure equal numbers of participants in each group (ratio of 1:1). Participants were automatically allocated to intervention groups by a formula on Excel version 14.6.6 (Microsoft Corporation) using the random number sequence, which was coordinated by another contact who was not a member of the research team. The random sequence and the allocation of participants to groups were concealed from research staff throughout the trial. The same person sent emails to participants containing the link to the assigned app after they had returned the baseline questionnaire. BODY.BLINDING: The trial was assessor blinded, as researchers were blinded to treatment allocation throughout the trial and during the statistical analysis. This was achieved by having a person outside the research team to manage treatment allocation and personal communications with the participants. Any questions or comments made in the surveys were forwarded from this contact to research staff, excluding any participant details or codes. This was to ensure that the researchers remained blinded to treatment allocation, as some comments contained information about the specific app the participants were using. Researchers did not have any access to data regarding treatment allocation, as those were stored on a secure database, separate and protected from other research files. During data collection and analysis, only numerical codes were used to indicate group allocation. Researchers did not know which groups those referred to until the end of the analysis. Blinding the participants was not possible, as the apps were clearly labelled as “Stress Free” and “Agoraphobia Free,” and masking those would require significant changes in the software, which were not feasible. Moreover, even if the apps were not differentially labelled, it is very likely that participants would have become aware of which treatment group they were assigned to because of the intervention content and the extent to which it addressed agoraphobia. BODY.ETHICS: Multimedia Appendix 3) [35]. Data were kept anonymized and protected according to the UK Data Protection Act [36]. We could obtain information about adverse events or effects of the interventions from feedback participants provided in the weekly surveys. BODY.ANALYSIS: We analyzed the data on Stata version 14 (StataCorp LLC). We checked baseline data for normality and obtained descriptive statistics to capture the demographic and clinical characteristics of the sample. The analysis performed was intention-to-treat, as requested in the CONSORT [37]. All participants who completed the baseline assessment were included in the analysis as they were randomly allocated. We used a linear mixed model to analyze the data, with a random effect of participant, and fixed effects of time (baseline, midpoint, end point), group (Agoraphobia Free and Stress Free), and the interaction between time and group. The estimated baseline PAS score was constrained to be identical in the 2 groups, thus adjusting for baseline and allowing the relationship between baseline and follow-up scores to differ at each time point. Another advantage of this statistical method is that the data from all participants contribute to the analysis, even if there is a substantial amount of missing data at follow-up [38]. We used an unstructured residual covariance matrix to allow for correlations within participants between the different time points. Statistical significance was taken at the 5% level (P<.05). We conducted a planned secondary completers’ analysis using the same data analytic strategy as the intention-to-treat analysis. This analysis included only those participants who were identified as intervention completers. BODY.RESULTS: BODY.PARTICIPANT FLOW: The first phase of recruitment started from September 2014 and ended in late February 2015, and the second phase started in March 2015 and concluded in April, 2015. In the first round, 153 participants consented to participate, and we recruited 17 additional participants in the second round. The procedure following recruitment was the same in both samples. Data collection ended in June 2015. Figure 1 shows how the total sample of 170 participants progressed through the trial. After 171 individuals consented to participate, 1 wanted to withdraw from the trial. The rest were randomly allocated into the 2 arms, but were not told which app they were assigned to at that point. Although the aim was to assign equal numbers of participants to each group, at each stage of randomization, the treatment group happened to have 1 more person than the other group, because the number of participants recruited each time was odd, and researchers were blinded to the allocation sequence. We asked participants to fill in a form with their demographic and device details to register for the intervention so that we could set up the corresponding app invitations. A total of 18 participants did not return the form and did not reply to emails, even after they had been sent several reminders; thus, we treated them as dropouts and excluded them from the trial. The baseline PAS questionnaire was then sent and had to be completed in order for participants to proceed and receive the intervention. A total of 10 individuals did not return the questionnaire and did not reply to any emails. Up to this point, participants were still unaware of the group they were assigned to; thus, their exclusion from the trial was very unlikely to introduce any bias. The remaining 142 who completed the PAS were sent their allocated intervention, but 6 participants could not download the app on their device, despite efforts to resolve the technical issues. Figure 1Flow of participants through the different stages of the trial. After baseline, 39 participants did not want to continue with the trial and dropped out, while 29 participants did not reply to emails and did not complete the questionnaires. We included all participants who provided baseline data in the analysis. BODY.BASELINE CHARACTERISTICS: Of the 142 participants who completed the baseline assessment, 2 did not give details of their age and sex (1 from each treatment arm). Of the remaining 140, 118 (84.3%) were female and had a mean age of 39.7 years (SD 11.3). Overall, the mean participant PAS total score was 30.3 (SD 8.7), and the scores were normally distributed (Shapiro-Wilk test: W=.985, P=.14). The mean PAS total score fells into the severe range and was much higher than that of the original sample used in the validation of the scale [27], which reported a mean score of 23.5, SD 10.3. Our sample also had a higher proportion of women than the original sample (of whom only 57% were women) and a slightly higher age (mean 36.09 years in the original sample). A significant proportion of our sample (114/142, 80.3%) had severe or extremely severe Agoraphobic Avoidance, while 85.2% (121/142) avoided more than 8 situations. None of the participants scored 0 on the Agoraphobic Avoidance subscale, which indicates that all of them experienced agoraphobic symptoms to some degree. The lowest score on that scale was 1.67, indicating mild Agoraphobic Avoidance. In contrast, 29 participants (20.4%) obtained a score of 0 on the Panic Attacks subscale, which suggests that some participants experienced agoraphobia in the absence of panic attacks. Overall, agoraphobic symptoms were more prominent than panic symptoms in this sample. Table 2 shows the demographic and clinical baseline characteristics of the participants by group. The 2 groups did not differ statistically on any of those characteristics at baseline (all P>.05). BODY.MISSING DATA: A total of 68 (47.9%) participants had missing outcomes. Overall, the differences between participants with missing data and those without were not statistically significant on any of the baseline variables examined. There were no significant differences in age (t138=0.85, P=.40) sex (χ21=0.6, P=.45), or clinical symptom severity (t140=1.32, P=.19). There were 37 participants (54%) with missing data in the Agoraphobia Free arm and 31 (46%) in the Stress Free arm. The relationship between missing data and treatment arm was not significant (χ21=0.5, P=.49). Therefore, participant attrition did not seem to be biased with regard to group or any other baseline factor. BODY.MAIN ANALYSIS: We produced a linear mixed model assessing the relative effects of each intervention on PAS scores at the 2 follow-up time points. Table 3 presents the estimated differences in PAS scores for the Agoraphobia Free group compared with the Stress Free group adjusted for baseline score at the 2 time points. At end point, symptom severity scores decreased in both groups, but there was no evidence that the changes were significantly greater among participants of the Agoraphobia Free group than among those in the Stress Free group. Similarly, at midpoint there were no significant differences in symptom severity changes between the 2 groups. Therefore, there were no significant differences between the 2 groups. Figure 2 presents the differences on the primary outcome for each group over time. We carried out the same linear mixed model analysis (n=142) using each PAS subscale as the dependent variable to examine whether there was a difference between the 2 groups in terms of symptom dimensions. We found no significant interactions between group and time for any of those outcomes (all P>.05). We conducted within-group contrasts to examine the degree of change in symptom severity over time. For both Agoraphobia Free (n=73) and Stress Free (n=69), there were statistically significant improvements in symptom severity from baseline to midpoint and end point. Table 4 presents the results. Table 2Baseline demographic and clinical characteristics of each treatment arm. Characteristics Agoraphobia Free app (n=73) Range of PASa scores Stress Free app (n=69) Range of PAS scores Age (years), mean (SD) 39.21 (10.45) N/Ab 40.23 (12.21) N/A Sex (female), n (%) 64 (88.9) N/A 54 (79.4) N/A PAS scores, mean (SD) Total 30.77 (8.72) 9-50 29.80 (8.72) 6-47 Panic Attacks 1.58 (1.05) 0-4 1.52 (0.88) 0-3.33 Agoraphobic Avoidance 3.30 (0.57) 1.67-4 3.24 (0.52) 2-4 Anticipatory Anxiety 2.70 (0.94) 0-4 2.59 (1.01) 0-4 Disability 2.39 (1.16) 0-4 2.47 (1.09) 0-4 Worries about Health 1.78 (1.17) 0-4 1.46 (1.11) 0-4 aPAS: Panic and Agoraphobia Scale. bN/A: not applicable. Table 3Intention-to-treat analysis at end point (12 weeks) and midpoint (6 weeks), n=142 . Time point of PASa score Agoraphobia Free mean (SD) Stress Free mean (SD) Effect estimate Difference 95% CI P value End point (primary outcome) 24.33 (16.81) 23.95 (16.51) 0.38 –1.96 to 3.20 .64 Midpoint 27.66 (13.37) 27.03 (13.27) 0.62 –3.13 to 3.89 .83 aPAS: Panic and Agoraphobia Scale. Figure 2Clinical symptom severity as indicated by the total score on the Panic and Agoraphobia Scale (PAS) in each group at trial baseline, midpoint, and end point. Table 4Within-group contrasts capturing the differences in Panic and Agoraphobia Scale total score between time points within each treatment arm (n=142). Treatment arms Baseline-midpoint contrast Baseline-end point contrast Difference 95% CI P value Difference 95% CI P value AFa –2.64 –4.48 to –0.79 .005 –5.97 –8.49 to –3.44 <.001 SFb –3.25 –5.09 to –1.43 <.001 –6.35 –8.82 to –3.87 <.001 aAF: Agoraphobia Free app. bSF: Stress Free app. BODY.COMPLETERS’ ANALYSIS: Data on completion were available from participants who consistently replied to the surveys and questionnaires (n=74). There were 56 participants who completed 80% or more of the assigned intervention; 25 of those received Agoraphobia Free and 31 received Stress Free. Of those who were deemed noncompleters, 11 were in the Agoraphobia Free arm and 7 in the Stress Free arm. There was no relationship between treatment arm and completion of intervention (χ21=1.5, P=.22). We examined differences in completers’ symptom severity between the 2 intervention groups. We produced a linear mixed model, with random effect of participant, and fixed effects of time (baseline, midpoint, end point), group (Agoraphobia Free and Stress Free), and the interaction between time and group. In line with the intention-to-treat analysis, there were no significant differences between the 2 groups at end point or midpoint, as Table 5 shows. The within-group changes at each follow-up time point compared with baseline were significant in both groups (all P<.001). Therefore, there were significant reductions in symptom severity in both groups over time, but those reductions were equivalent across the 2 groups. BODY.CLINICAL SIGNIFICANCE: Wichmann et al [39] recently showed that an overall postintervention (ie, face-to-face CBT) decrease in the total PAS score of about 4 to 5 points represents a clear clinical change in terms of quality of life and functioning. This is slightly smaller than the change we observed in our study (6 points) for end point PAS scores compared with baseline. The decrease in symptom severity was even more marked in the completers’ sample (7 points in the Stress Free group and 10 points in the Agoraphobia Free group). Therefore, it seems that both apps were overall successful in achieving clinically significant change. Of the 74 participants who provided data at end point, 46 (62%) had a reduction of 5 or more points on the PAS at end point. A total of 25 (66%) participants in the Stress Free group and 21 (58%) participants in the Agoraphobia Free group improved at least 5 points on the PAS at end point. BODY.SAFETY AND USE OF APPS: A total of 7 participants (Agoraphobia Free: n=3, Stress Free: n=4) commented that certain app components were mildly stressful (eg, the distraction games, background music). There were no reported adverse events experienced as a result of either intervention. Also, 7 participants commented that the Agoraphobia Free app was confusing to follow at certain points. Another participant explained that they could not use the treatment app as much as they wanted to because they had depression and did not feel motivated. Table 5Completers’ analysis at end point (12 weeks) and midpoint (6 weeks), n=56. Time point of PASa score Agoraphobia Free mean (SD) Stress Free mean (SD) Effect estimate Difference 95% CI P value End point 21.58 (13.10) 23.73 (12.12) –2.15 –6.21 to 1.91 .30 Midpoint 26.09 (11.15) 26.93 (10.40) –0.84 –4.05 to 2.37 .61 aPAS: Panic and Agoraphobia Scale. The mean baseline PAS score for both groups was 31.23. BODY.DISCUSSION: BODY.PRINCIPAL FINDINGS: The results from this RCT showed that participants who received Agoraphobia Free did not improve more than those who received the Stress Free app. Both groups showed reductions in symptom severity over time that were statistically significant, but those reductions seemed to be equivalent across the 2 groups. Both treatment apps were safe to use and yielded similar completion rates. Moreover, completers of either intervention showed marked improvements in symptom severity, with a 10-point drop in the PAS at end point compared with baseline in the Agoraphobia Free group. However, findings from this analysis should be considered with caution, as we performed the analysis on a specific subgroup of participants, and there is the possibility that factors other than the intervention influenced the outcome (eg, participant expectations). In addition, the power of this analysis was very low (below 50%) due to the small sample size. Throughout the trial, participant attrition was particularly high, as almost half of the participants recruited dropped out of the trial or stopped responding to emails. Dropout rates were similar to those reported in other Web-based trials of self-guided interventions [40,41]. Many participants dropped out after providing consent, which we had not expected. We had to exclude those participants from the analysis, as no baseline data were available. It is unlikely that this exclusion of participants could have led to bias, as participants at that point were unaware of the groups they were randomly allocated to, and thus attrition was random between the 2 groups. Similarly, participants who dropped out or stopped responding after receiving the intervention did not seem to differ from those who engaged in the trial, and attrition was equivalent across the 2 groups. The analytic strategy we chose is robust and uses all available data from each participant, producing less-biased results than other methods of analysis and data imputation [38]. Overall, the fact that participant attrition appeared random, in addition to the fact that we used data from all participants in the analysis, offer support and confidence in the validity of the study findings. BODY.LIMITATIONS: The trial had a few limitations. There was no follow-up after the completion of the intervention. Therefore, it was not possible to investigate whether the improvements in symptom severity were maintained over time or whether there were any differences between the 2 groups after a few months of using the apps. The information collected regarding the characteristics of the sample was also limited, as there were no data on comorbid disorders, other psychological or physiological treatments, or other demographic characteristics such as ethnicity and computer literacy. This information might have provided better insights into the sample and could have been related to intervention efficacy. There was also limited information on app completion, as the information available relied on participant report and we were not permitted to extract app use data for participants individually. Moreover, participants could not be blinded, which is a common limitation in eHealth trials. Another limitation of the study was the absence of a waitlist control group. The comparison of 2 active groups that shared very similar features tested the effects of agoraphobia-specific therapeutic elements over and above those of a generic anxiety-related treatment. There was no control, however, for the effect of time, and it is therefore not possible to conclude whether the improvements observed were not because of natural recovery processes or factors other than the intervention. Although a waitlist control group would be a necessary addition in a future trial in order to clearly establish treatment efficacy, the primary focus of this study was to demonstrate whether a disorder-specific mobile-based intervention is warranted in the treatment of agoraphobic symptoms, compared with a more generic approach addressing anxiety. Despite its limitations, the study also presented certain strengths. First, it is, to our knowledge, the first RCT to directly compare an agoraphobia-specific app with a generic anxiety-related app. Second, the sample recruited online was characterized by severe clinical symptoms and especially high levels of agoraphobia, which shows that recruitment based on self-identification is feasible and reliable. Such a sample may have also been hard to reach in a traditional multicenter trial with assessors conducting screening interviews, which would have been time consuming and would require a lot of resources. The trial was minimally intrusive for the participants, as it requested a limited amount of information, and the tools used were short and easy to complete. This helped achieve fair recruitment and response rates without any explicit individual guidance. Third, an important strength of the study was its external validity. The apps were offered as they would be offered in real-world settings, without requiring any screening of users or close supervision. There were very few restrictions on who could participate, therefore making our findings more easily generalizable to an Internet population who would simply download the app if they thought it would be relevant to them. Fourth, the results of the study provide an insight into what we would realistically expect from an unguided mobile intervention, such as a substantial dropout rate, small symptom change, and highest efficacy for those who consistently engage with and complete the intervention. BODY.COMPARISON WITH PRIOR WORK: The findings of this study relate to previous studies on the treatment of anxiety disorders. The findings add to the evidence base of computerized cognitive behavioral interventions examined in anxiety disorders [21-23]. Our findings are also consistent with previous studies showing that completers of Web-based interventions for panic (with or without agoraphobia) benefit the most [40,42]. Importantly, the lack of a significant difference between the 2 groups indicates that a generic anxiety-related app and a diagnosis-specific app are equally effective in treating agoraphobic symptoms. This finding is in line with evidence supporting the efficacy of transdiagnostic (ie, unified, nonspecific) cognitive behavioral treatments in anxiety disorders. Transdiagnostic treatments are based on the premise that “commonalities across disorders outweigh the differences” [43]. A recent systematic review and meta-analysis showed that transdiagnostic treatments can be as efficacious as diagnosis-specific treatments in reducing anxiety symptoms and more effective than waitlist or attention control conditions [44]. Transdiagnostic CBT programs tested in different anxiety disorders (including panic disorder) can also be successfully administered via the Internet [45], producing effects equivalent to those of disorder-specific Internet-based interventions [46,47]. Moreover, transdiagnostic CBT does not differ from diagnosis-specific CBT in terms of treatment credibility [48]. Our study adds to this body of evidence suggesting that, for people with agoraphobia, an agoraphobia-specific app does not produce any additional benefits in relation to a transdiagnostic anxiety-targeting app. BODY.CLINICAL IMPLICATIONS: The findings of this study have implications for clinical practice. This trial shows that mobile apps can be successfully administered to a particular population that is hard to reach otherwise, without requiring guidance by a clinician. This could potentially save time for clinicians, while it could also be more convenient for patients and help them overcome many barriers to treatment, such as traveling to sites or long waiting lists. Furthermore, a self-guided app could be easily introduced into a stepped care model as a minimal intervention, as it is less intense than a clinician-guided intervention or individual therapy [49]. Since there was no evidence of superiority of one type of intervention over the other in this study, a choice between a generic and a targeted approach could be ultimately based on clinical judgment or patient preference. However, as suggested by Norton and Barrera [48], a generic anxiety-targeting app might be more resource efficient and easier to implement and disseminate than a diagnosis-specific app. BODY.CONCLUSIONS: Overall, this RCT suggests that transdiagnostic anxiety-targeting mobile apps can be as effective as disorder-specific apps for people with agoraphobic symptoms. Results show that individuals who identify as having agoraphobia do not benefit more from an agoraphobia-specific than from a transdiagnostic app. Future research conducted by independent research teams should replicate the results of this study, further investigating the possibility that mobile-based transdiagnostic interventions for anxiety can be as effective as current gold standard disorder-specific interventions. A trial with a larger sample size and a waitlist control group is warranted to establish intervention efficacy and cost effectiveness in this population. Additionally, the collection of more-extensive demographic and clinical information can help examine under which circumstances a diagnosis-specific or a generic approach is more appropriate. For example, future research could investigate whether patients with more severe symptoms or with comorbid disorders (eg, depression) benefit more from one type of intervention than the other. While there is still much to learn about treatment approaches in anxiety disorders, many studies, including this one, show that e-mental health interventions can overcome barriers and be effective in reducing clinical symptoms.

TITLE: Does eating slowly influence appetite and energy intake when water intake is controlled? ABSTRACT.BACKGROUND: Slow eating has been associated with enhanced satiation, but also with increased water intake. Therefore, the role of water ingestion in regard to eating rate needs to be discerned. This study examined the influence of eating rate on appetite regulation and energy intake when water intake is controlled. ABSTRACT.METHODS: In a randomized design, slow and fast eating rates were compared on two occasions, in 30 women (22.7±1.2y; BMI=22.4±0.4kg/m2) who consumed an ad libitum mixed-macronutrient lunch with water (300 mL). Satiation was examined as the main outcome by measuring energy intake during meals. At designated times, subjects rated hunger, satiety, desire-to-eat, thirst, and meal palatability on visual analogue scales. Paired t-tests were used to compare hypothesis-driven outcomes. Appetite ratings were compared across time points and conditions by repeated measures analysis of variance (ANOVA) using a within-subject model. ABSTRACT.RESULTS: Energy intake and appetite ratings did not differ between conditions at meal completion. However, subjects rated less hunger and tended to rate lower desire-to-eat and greater satiety at 1 hour following the slow condition. ABSTRACT.CONCLUSIONS: Results tend to support a role of slow eating on decreased hunger and higher inter-meal satiety when water intake is controlled. However, the lack of significant differences in energy intake under these conditions indicates that water intake may account for the effects of eating rate on appetite regulation. BODY.BACKGROUND: Eating behaviors that promote excess energy intake may have contributed to the increases in the incidence of overweight and obesity [1]. For example, findings from population studies have shown that faster self-reported eating rates were associated with greater body mass index in Japanese individuals and in middle-aged women from New Zealand [2-4]. Altering eating behaviors to reduce the rate of eating has become a hallmark of many weight control programs [5,6], despite limited evidence supporting its effectiveness. In fact, a retrospective longitudinal study on this topic was recently published, and it showed that the fast-eating group of Japanese male workers had a higher average 8-year weight gain than the combined group of medium and slow eaters [7]. Similar findings have been earlier reported in fire fighters that reported eating faster at the station, whose weight gain over 7 years was 1.4 kg higher than all the others [8]. Slow eating has been hypothesized to help control energy intake since it allows satiation to register before too much food is consumed [1,9]. However, empirical evidence on this treatment approach is limited and has yielded inconsistent findings. Smaller bite sizes or pauses within meals have been associated with reduced energy intake in some studies [10,11], or conversely have led to no differences in overall meal intake [12,13]. These former studies have not directly manipulated eating rates and examined meal food intake between conditions using a within-subject design. Although an increased interest in eating rate has been noticed in recent years, studies of the effect of manipulating eating rates on energy intake have shown that slow eating reduced energy intake in some groups, but not consistently across studies [14-19]. For example, slower eating rate decreased food intake in men [16], but not in women [16], patients with bulimia nervosa [15], or highly restrained eaters [17]. Another study observed lower total energy intake and greater satiety after meal completion, with combined techniques of taking small bites, pausing between bites, and chewing thoroughly to alter eating rate, in healthy women [14]. However, eating slowly allowed time for the consumption of more water along with the meal, which led to higher total weight consumed. This may have increased stomach distension, and thus induced higher satiety [20]. Nonetheless, previous research has shown that drinking water with a meal, as opposed to the water incorporated into a food, does not necessarily affect energy intake [21]. Most previous studies of eating rate and energy intake have not reported fluid consumption, so the role of fluid intake in this relation needs to be clarified. Before slowing the pace of eating is recommended as a useful strategy to prevent overeating, the mechanisms underlying its effectiveness must be fully understood and nuances for practical strategies more clearly determined. To further validate the previous findings [14], the present study sought to compare the impact of slow and fast eating rates on energy intake and on the development of satiation in healthy women when water intake is controlled. BODY.METHODS: BODY.PARTICIPANTS: Thirty healthy, non-smoking, pre-menopausal females with body mass index (BMI) of 19-30kg/m2 were recruited with flyers and classroom announcements on the University of Rhode Island campus. Exclusion criteria included allergies to test foods, caffeine or alcohol dependency, diabetes mellitus, adrenal or thyroid disease, any chronic illness that might cause weight change, clinically-diagnosed eating disorders, medications that might alter appetite, or dieting. Participants’ characteristics are shown in Table 1. The study was approved by the Institutional Review Board of the University of Rhode Island. BMI was cross-checked by the investigators at the laboratory before starting the study, and written informed consent was obtained from participants. By design, the purpose of the study was not fully disclosed to participants, since meal duration and food intake were covertly recorded. Participants were financially compensated for completion of the study. Table 1Subject characteristics CharacteristicMeanSEMRangeAge (y) 22.7 1.2 18-45 Height (m) 1.7 0.0 1.5-1.9 Weight (kg) 62.4 1.7 44.9-91.9 Body Mass Index (kg/m2) 22.4 0.4 18.6-26.3 Waist circumference (cm) 79.7 1.8 67.5-100.0 % Body fat 26.2 1.3 17.5-38.5 H-P score 2 12.0 0.8 0-21 TFEQ Scores         Dietary restraint score 3 10.1 0.8 1-20   Disinhibition score 3 6.5 0.6 1-12   Hunger score 3 6.8 0.5 2-14 n = 30 females1. 1 Self-reported eating rate: “Slow”: n=5; “Medium”: n=16; “Fast”: n=9. 2 Scores from the Herman-Polivy Questionnaire (H-P). Anchor score: 0-35 [23]. 3 Scores from the Three-Factor Eating Questionnaire. Anchor score: restraint: 0-21; disinhibition: 0-16; hunger: 0-14 [24]. BODY.ANTHROPOMETRIC MEASUREMENTS: During visit 1, body weight was measured in minimal clothing on a digital scale accurate to 0.1kg (BodPod, Life Measurements Inc., Concord, CA), height was measured to the nearest 0.1centimeter (cm) on a wall-mounted stadiometer (Seca 240, SECA, Hamburg, Germany) accurate to 0.1cm, and BMI (in kg/m2) was calculated. Waist circumference was measured (in cm) at the level of the umbilicus with a flexible tyvek measuring tape. Body composition was assessed after a minimum 2-hour fast by air displacement plethysmography (BodPod, Life Measurements Inc., Concord, CA) using standardized techniques [22]. BODY.QUESTIONNAIRES: Participants completed a personal health history questionnaire. Eating rate was self-reported as “slow”, “medium” or “fast”. Two validated instruments, the 10-item Herman-Polivy (H-P) Questionnaire, and the 51-item Three-Factor Eating Questionnaire (TFEQ), were also administered to assess levels of chronic weight-focused behavior, as well as dietary restraint, disinhibition, and hunger, respectively [23,24]. Participants completed the three questionnaires at home and investigators carefully checked all the questions with the participant when she arrived to the laboratory for visit 1. BODY.STUDY PROTOCOL: The next two laboratory visits were the test visits. A randomized crossover design was used to compare the experimental conditions of slow versus fast eating, with tests separated by 3–7 days. These visits were conducted during the mid-follicular phase of each participant’s menstrual cycle, to control for possible effects of the menstrual cycle on appetite [25]. Each test visit was conducted with one individual participant at a time. For the purposes of comparison, the study design was virtually identical to our previous protocol [14] but with controlled water intake instead of ad libitum water. On the day prior to each test visit, participants were instructed to avoid strenuous physical activity, to refrain from alcohol and caffeine consumption, to maintain their diet as close to normal as possible (no extremes of nutrients and calories) and to match these conditions before both test days. On the morning of test days, participants’ breakfasts were consumed at home, and were specifically prescribed (described below) and matched between the slow and fast eating conditions. Between breakfast and the lunch test, physical activity was minimized, and they were allowed to drink water in moderation (enough to quench thirst) but not more than 600mL. A graduated bottle was provided to each participant with the purpose of recording the volume of water consumed during the morning before each lunch test. At lunch time, participants reported to the laboratory following a minimum 4-hour fast. Prior to each test meal, participants were asked if they followed the instructions regarding breakfast intake, physical activity, and their diet on the day before, and their visit was rescheduled if they failed to follow the instructions. Participants began each lunch meal at the same time. After voiding, they were offered generous pre-weighed portions (690 grams) of a mixed-macronutrient lunch (described below) and 300mL of water (refrigerator-chilled). These quantities were based on previous work in our lab [14]. They were instructed to drink the water in its entirety throughout the meal, and to consume as much of the food as they would like, to the point of comfortable satiation. Under the fast condition, participants used a large spoon (soup spoon) and were instructed to consume the meal as fast as possible with no pauses between bites. However, they were instructed not to eat so fast that it was uncomfortable for them. During the slow condition, a small spoon (teaspoon) was provided with these meals. Participants were instructed to take small bites, put down the spoon between each bite and chew each bite 20 to 30 times. During both conditions, the investigator carefully monitored the participant, prompting her to eat according to protocol. Exact clock time of meal initiation and completion were covertly recorded. The amount of the meal consumed was calculated by weighed differences (to the nearest 0.01gram) using a digital scale (Adventurer, OHAUS Corp., Pine Brook, NJ), and eating rate was calculated as kcal/min and g/min. BODY.ASSESSMENT OF APPETITE AND MEAL PALATABILITY: Appetite was assessed with validated 10-cm visual analogue scales (VAS) [26]. For both conditions, hunger (VAS-H), satiety (VAS-S), desire-to-eat (VAS-D), and thirst (VAS-T) were assessed before test meal intake, every 5 minutes (min) during the meal up to 30min, again upon meal completion, and then at 45 and 60min. Participants were not allowed to eat or drink anything between meal completion and the final appetite ratings at 60min after meal initiation. Meal palatability was also assessed by VAS at 1min into each meal and after meal completion. These scales were anchored by the statements “not at all” and “extremely”. BODY.TEST MEALS: On the morning of the two test days, participants consumed a standardized breakfast at home containing approximately 400kcals, consisting of 8oz of orange juice, 8oz of 1% or 2% milk, and 1 cup of ready-to-eat cereal (except Granola or Grape Nuts), with the option of consuming 8oz of decaffeinated tea or coffee. Participants consumed identical breakfasts on the morning before each test, and then fasted for a standard time (minimum 4hours) during which physical activity was minimized. The test meal contained 1000kcals and consisted of ditalini pasta, canned diced tomatoes with Italian seasoning, tomato paste, celery and minced garlic sautéed in olive oil, and parmesan romano cheese. Ditalini pasta was chosen specifically because its small size allowed for slow or fast eating rates with small or large utensils. The macronutrient distribution of the test meal was 48% of energy from carbohydrate, 39% from fat and 13% from protein. BODY.STATISTICAL ANALYSIS: The main study outcome was ad libitum energy consumption. Other outcomes included ratings of VAS-H, VAS-S, VAS-D, and VAS-T. Paired t-tests were used to compare energy intake, rate of energy consumption, and appetite ratings upon meal completion between fast and slow conditions. VAS-H, VAS-S, VAS-D, and VAS-T ratings were compared across time points and conditions by repeated measures analysis of variance (ANOVA) using within-subjects model. A square root transformation was applied to VAS appetite data prior to ANOVA because the data were not normally distributed. Violation of sphericity was corrected using the Box correction, and corrected degrees of freedom were reported. Significant results are reported, and the effect size is reported as partial eta squared (η2). The equation maximizing the time-by-condition interaction is reported (e.g. cubic model) for all significant ANOVA’s. Post hoc comparisons were performed using paired t-tests using Bonferroni correction [27]. Area under the curve (AUC) was calculated for ratings of appetite using the trapezoid method, and results were compared using paired t-tests. Meal palatability data were examined by paired t-tests. Data were also examined continuously by correlation analyses (Pearson’s tests) to determine the relationships of TFEQ and H-P scores with the main outcomes. Results are expressed as mean ± standard error of the mean (SEM), and were considered significant at p < 0.05. Data were analyzed using the software Statistica version 6.1 (StatSoft Inc., Tulsa, OK), and SPSS version 16.0 (SPSS Inc., Chicago, IL). BODY.RESULTS: Thirty healthy, non-smoking, pre-menopausal females were recruited and all completed the study (Table 1). The majority of participants were college students. Thirteen women scored higher than 10 in the TFEQ-restraint, which is common in this population. However, neither H-P nor TFEQ scores correlated with the ad libitum lunch variables (p > 0.05). There was no difference in the amount of water consumed during the morning before participants’ arrival at the laboratory for the lunch test (fast: 375.3 ± 30.9 mL; slow: 347.2 ± 33.9 mL; p > 0.05). As shown in Table 2, meal duration was approximately 18min longer under the slow condition. However, there was no significant difference in the weight of food consumed and therefore no difference in energy intake. Since water intake was controlled, all participants consumed 300 mL of water. The combination of small bites, pauses between bites, and thorough chewing during the slow condition resulted in a significantly decreased eating rate, expressed as kcal/min. Participants consumed 70.8 ± 2.6% of the available energy presented at the meal under the fast condition and 69.4 ± 3.3% under the slow condition (p > 0.05). No participant consumed the full amount of food offered under either condition. Table 2Meal duration, ad libitum meal intake, and eating rate in the two experimental conditions (fast and slow)  Experimental condition1Number of subjectsFastSlowFast > Slow2Slow > Fast3MeanSEMMeanSEMnnDuration of the meal (min) 8.4 0.6 26.1 1.8 * 0 30 Weight of food consumed (g) 488.2 17.9 478.6 22.5 17 13 Energy intake (kcal) 707.9 26.0 694.0 32.6 17 13 Energy density (kcal / total g) 0.9 0.0 0.9 0.0 17 13 Rate of energy consumption (kcal/min) 94.0 5.6 29.0 1.9 * 30 0 n = 30 females. 1 Statistical differences between conditions were determined by paired t-tests: * p < 0.05. 2 Number of subjects in which meal duration and intakes were higher in the fast condition than the slow. 3 Number of subjects in which meal duration and intakes were higher in the slow condition than the fast. VAS-H, VAS-S, VAS-D and VAS-T ratings upon completion of the ad libitum meal consumed under fast and slow conditions are shown in Figure 1. VAS-H, VAS-S, and VAS-D ratings did not differ between conditions immediately after finishing the meal (p > 0.14) while VAS-T ratings tended to be higher immediately upon completion of the meal consumed at a slow rate (p = 0.09). Figure 1Visual analogue scale appetite ratings (mean ± SEM) upon meal completion from thirty women who consumed an identical ad libitum meal and 300mL water under fast and slow eating conditions, in randomized order. The effect of eating rate on ratings of VAS-H, VAS-S, VAS-D and VAS-T over time is represented in Figure 2. The open and closed arrows represent average time of meal termination for the fast (8min) and slow (26min) conditions, respectively. There was a significant effect of eating rate on hunger (Linear model: F[1,29] = 32.72, p < 0.001, η2 = 0.530). There was also a significant effect of time on hunger (Linear model: F[1,29] = 241.270, p < 0.001, η2 = 0.893), and a significant time × eating rate interaction effect on hunger (Cubic model: F[1,29] = 68.21, p<0.001, η2 = 0.702). Post hoc testing revealed that in the slow condition, hunger was significantly higher at 5, 10, 15, and 20min (p < 0.001) and was significantly lower at 60min (p = 0.003) compared with the fast condition, as shown in Figure 2A. AUC for hunger in the fast condition (824 ± 87 mm·min) was significantly lower (t = 5.052, p < 0.001) than the slow condition (1431 ± 132 mm·min). There was a significant effect of eating rate on desire-to-eat (Linear model: F[1,29] = 39.23, p < 0.001, η2 = 0.575). There was also a significant effect of time on desire-to-eat (Linear model: F[1,29] = 188.94, p < 0.001, η2 = 0.867), as well as a significant time × eating rate interaction effect (Cubic model: F[1,29] = 77.73, p < 0.001, η2 = 0.728). Post hoc testing revealed that in the slow condition, desire-to-eat was significantly higher at 5, 10, 15, and 20min (p < 0.001) and tended to be lower at 60min (p = 0.008) compared with the fast condition, as shown in Figure 2B. AUC for desire-to-eat during the fast condition (834 ± 81 mm·min) was significantly lower (t = 5.337, p < 0.001) than the slow condition (1391 ± 126 mm·min). There was a significant effect of eating rate on satiety (Linear model: F[1,29] = 10.63, p = 0.003, η2 = 0.268) and a significant effect of time on satiety (Quadratic model: F[1,29] = 123.75, p < 0.001, η2 = 0.810), as well as a significant time × eating rate interaction effect (Cubic model: F[1,29] = 36.82, p < 0.001, η2 = 0.559), as shown in Figure 2C. Post hoc testing revealed that in the fast condition, satiety was significantly higher at 5, 10, and 15min (p < 0.001) and tended to be lower at 60min (p = 0.017) compared with the slow condition, as shown in Figure 2C. AUC for satiety in the fast condition (4643 ± 175 mm·min) was marginally higher (t = 2.018, p = 0.053) than the slow condition (4315 ± 159 mm·min). There was no significant effect of eating rate, time or time × eating rate interaction effect on thirst, as shown in Figure 2D Figure 2Visual analogue scale appetite ratings over time (mean ± SEM), from thirty women who consumed an identical ad libitum meal and 300mL of water under fast and slow eating conditions, in randomized order: A–Hunger; B–Desire-to-eat; C–Satiety; D-Thirst. Closed and open arrows represent meal completion for the fast (~8min) and slow (~26min) conditions, respectively. All appetite ratings, except thirst, showed a significantly time-by-condition interaction effect. AUC for hunger and desire-to-eat was significant different between conditions. * Means at given time points were significantly different between conditions (p < 0.006). # Desire-to-eat (p = 0.008) and satiety (p = 0.017) ratings were marginally significant different at 60min. Palatability ratings for the ad libitum meal did not differ significantly between conditions at the beginning or after consumption. Pleasantness and tastiness ratings were approximately 80%, suggesting participants found the meal to be highly palatable. Ratings of saltiness were approximately 30% in both conditions (data not shown). BODY.DISCUSSION: The results of this study show that when water intake is controlled, combining the techniques of small bites, pauses between bites, and chewing thoroughly is associated with decreased eating rate, less hunger, and higher satiety, but not with decreased energy intake. This has interesting implications for recommending these techniques in the context of behavioral energy intake regulation. Eating slowly in order to reduce energy intake and enhance satiation appears to be most effective when ad libitum water is served with the food [14]. Martin et al. [16] also reduced participants’ habitual rate of intake, which led to decreased food intake in men but not in women. They served ad libitum water with the meals, but there was no difference in water intake, so eating slowly may not necessarily lead to drinking more water. However, eating rate in that study was slowed by having participants take a bite of food when prompted by a computer. Although the present study also took place in a laboratory setting, eating rate was manipulated in a more natural manner. Actually, thirst ratings at meal completion suggest that participants might have consumed more water under the slow condition if it was available. Water consumption may play an important role on the effects of eating rate on food intake. However, water intake was not reported in the majority of the studies on eating rate, or was controlled but with contradictory results [17,18], so empirical evidence must be provided before precise conclusions can be made with respect to synergies between eating rate and water intake. In a previous study, thirty women with similar characteristics to the women in this study consumed the same pasta meal with water consumed ad libitum as the only aspect of the protocol that differed from the present study [14]. Under the slow condition, participants consumed significantly less food than during the fast condition. However, weight of water and total weight consumed were significantly higher in the slow condition [14]. In contrast with those findings, the present study’s lack of an effect of eating rate on food intake when water intake is matched suggests that the lower energy intake previously verified may not be attributable simply to a slower eating rate. Peripheral satiety signals associated with meal termination include measures related to stomach distension, gastric emptying rate, and responses of several hormones, such as cholecystokinin, peptide YY and glucagon-like peptide 1 [20]. It has been proposed that slowing the rate of ingestion allows more time for these processes to take place, lengthening the satiety curve, and reducing total energy intake [9]. Two recent studies have explored this hypothesis, by measuring the postprandial gut peptide responses following fixed-portion meals consumed at different eating rates, and the results differed from one another [28,29]. Since these physiological signals were not measured in the current study, a relationship between food intake and the time course of post-ingestive meal termination signals cannot be established. Nevertheless, the present findings suggest that, when the water was served ad libitum, the physiological satiety signals could have been modulated by the larger quantities of water consumed during the slow condition [14]. It is possible that this might have increased stomach distension, and thus induced greater satiation. Laboratory studies testing direct manipulations of the volume in the stomach support this hypothesis [30]. Water has a major influence on energy density because it contributes weight without adding energy to foods. Rolls, et al. [21] showed that water incorporated into the food decreases energy intake, but water served as a beverage has little effect on overall energy intake. In this paradigm, the authors suggested that water in food probably suppresses hunger due to increases of weight and volume of the food and to changes in the dispersion of nutrients, while water consumed with the food would affect thirst [21], which was recently confirmed by Martens and Westerterp-Plantenga [31]. In agreement, while meal energy density affected meal intake in free-living adults, this response was not modulated by the addition of drinks to those meals [32]. Drinks added to a meal might therefore not be expected to affect overall consumption by a “diluting” mechanism. However, results from the two studies analyzing eating rate and water intake (ad libitum [14] and controlled) showed that drinking water with a meal may also play a role on food intake and satiation. Few studies have directly addressed the hypothesis that water ingestion may modulate appetite within meals. The consumption of water with a meal has shown little effect on food intake, but mean ratings of fullness after the meal were higher than the no-beverage condition [33]. In contrast, drinking water during a meal reduced subjective ratings of hunger and increased satiety, but this effect was not maintained after the meal [34]. Nonetheless, Martens and Westerterp-Plantenga [31] have lately explored this hypothesis and showed that drinking water separately with a meal vs. water consumed in the food mainly quenches thirst, but hunger is not affected. Population-based studies have also shown that daily energy intake of water consumers is lower than that of non-water consumers [35], suggesting that water consumption may play a role on energy intake and possibly body weight regulation. Despite similar ad libitum food intake when water intake was matched, appetite ratings at 60min show a potential benefit of slow eating. As shown in Figure 2, participants rated less hunger and tended to rate lower desire-to-eat and greater satiety at 60min following the slow condition. These trends support the hypothesis that changing eating behavior might have the ability to alter physiological satiety signals. Prolonging meal ingestion may increase the time of exposure of nutrients to gastrointestinal signals and this is independent of the amount of food eaten [9]. This suggests that satiation at subsequent meals or snacks could have been affected by slow eating, which is of particular interest for future analyses. More studies are needed on the effect of eating rate on inter-meal satiety and subsequent meals, since available research is limited and inconsistent [28,36]. Strengths of this study include the careful control of activity and of food and fluid intake before the tests, and testing only during the mid-follicular phase of the participants’ menstrual cycle. Limitations include the limited range of participant characteristics. Future studies should explore these hypotheses in different populations, such as males, as evidence shows that eating rate and its effects may differ by gender [4,16]. Moreover, results require further replication in real life conditions and with different foods and beverages. BODY.CONCLUSIONS: Although prospective studies are needed to corroborate the present findings, these data, together with earlier findings, suggest that recommendations directed at changing eating behavior for the purpose of energy intake reduction should be made carefully. The present evidence is consistent with the possibility that advising people to eat more slowly, take smaller bites, and chew their food thoroughly is generally helpful for appetite regulation, but especially when water is available for ad libitum consumption. Overall, allowing time to drink sufficient quantities of water along with a given meal while slowing down eating rate appears to be the most advisable strategy to maximize satiation and regulate energy intake. BODY.COMPETING INTERESTS: PJT is a member of the scientific committee of the Institute for Hydration and Health (Portugal) for which he receives no financial or other compensation. All other authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: KJM was responsible for the conception, design and direction of the project. AMA collected the data and wrote the first draft of the manuscript. AMA and DLK performed the statistical analysis. PJT and FB reviewed the entire manuscript and made important contributions to various sections. All authors provided significant advice, contributed to and approved the final manuscript.

TITLE: Inhaled Steroids Modulate Extracellular Matrix Composition in Bronchial Biopsies of COPD Patients: A Randomized, Controlled TrialLong-Term Steroids and Airway Remodeling in COPD ABSTRACT.RATIONALE: Smoking and inflammation contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD), which involves changes in extracellular matrix. This is thought to contribute to airway remodeling and airflow obstruction. We have previously observed that long-term treatment with inhaled corticosteroids can not only reduce bronchial inflammation, but can also attenuate lung function decline in moderate-severe COPD. We hypothesized that inhaled corticosteroids and current smoking modulate bronchial extracellular matrix components in COPD. ABSTRACT.OBJECTIVE: To compare major extracellular matrix components (elastic fibers; proteoglycans [versican, decorin]; collagens type I and III) in bronchial biopsies 1) after 30-months inhaled steroids treatment or placebo; and 2) between current and ex-smokers with COPD. ABSTRACT.METHODS: We included 64 moderate-severe, steroid-naive COPD patients (24/40 (ex)-smokers, 62±7 years, 46 (31–54) packyears, post-bronchodilator forced expiratory volume in one second (FEV1) 62±9% predicted) at baseline in this randomized, controlled trial. 19 and 13 patients received 30-months treatment with fluticasone or placebo, respectively. Bronchial biopsies collected at baseline and after 30 months were studied using (immuno)histochemistry to evaluate extracellular matrix content. Percentage and density of stained area were calculated by digital image analysis. ABSTRACT.RESULTS: 30-Months inhaled steroids increased the percentage stained area of versican (9.6% [CI 0.9 to 18.3%]; p = 0.03) and collagen III (20.6% [CI 3.8 to 37.4%]; p = 0.02) compared to placebo. Increased collagen I staining density correlated with increased post-bronchodilator FEV1 after inhaled steroids treatment (Rs = 0.45, p = 0.04). There were no differences between smokers and ex-smokers with COPD in percentages and densities for all extracellular matrix proteins. ABSTRACT.CONCLUSIONS: These data show that long-term inhaled corticosteroids treatment partially changes the composition of extracellular matrix in moderate-severe COPD. This is associated with increased lung function, suggesting that long-term inhaled steroids modulate airway remodeling thereby potentially preventing airway collapse in COPD. Smoking status is not associated with bronchial extracellular matrix proteins. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT00158847 BODY.INTRODUCTION: Chronic Obstructive Pulmonary Disease (COPD) is characterized by an abnormal inflammatory response and structural alterations of the bronchial wall and parenchyma [1]. This pulmonary remodeling has been linked to airflow limitation in COPD [2], [3]. Changes in the extracellular matrix (ECM), produced by (myo)fibroblasts, epithelial cells and airway smooth muscle cells, contribute to this remodeling process and alter airway mechanics and dynamics [4], [5]. The ECM consists of three major components: elastic fibers, proteoglycans and collagens, which are involved in cell migration, proliferation, adhesion, water balance and regulation of inflammatory mediators [4]. The composition of the pulmonary ECM is different in subjects with and without COPD. Fewer elastic fibers are found in small airways and alveoli of COPD patients than in healthy controls [6], [7]. Furthermore, versican, a large proteoglycan is more abundant, while the small proteoglycan decorin is reduced in small airways in COPD compared to healthy subjects [8]–[10]. Collagens are the main component of the ECM, and collagen composition differs between COPD patients and healthy controls as shown by the observation that collagen type I is lower in the large and small airways [11] and collagen type III expression is lower in the small airways of COPD patients than in healthy controls [3]. Since smoking is a risk factor for COPD, this may also influence ECM composition. Indeed, cigarette smoke has been shown to induce secretion of several profibrotic growth factors, including transforming growth factor-beta (TGF-β), both in human lung fibroblasts and in lung tissue of COPD patients [12], [13]. Rodent models exposed to cigarette smoke had less lung elastic fibers, but more collagens than sham-smoked animals [14]. Others even reported an increased elastic fibers gene expression in lung tissue of severe COPD patients [15]. Smoke exposure decreased proteoglycan expression as demonstrated by a study with pulmonary fibroblasts from moderate and very severe COPD patients [16]. Although generally (neutrophil dominated) inflammation in COPD is considered to be resistant to steroids treatment, we recently observed that long-term inhaled corticosteroids (ICS) treatment partially decreased bronchial inflammation (CD3+, CD4+, CD8+ and mast cells) -without effects on neutrophils- and attenuated lung function decline in moderate-severe COPD patients participating in the GLUCOLD (Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease) study [17]. ICS may affect ECM through various mechanisms, including modulation of inflammation by profibrotic mediators and targeting ECM genes directly. This may explain differences in the effects of steroids that are observed in in vivo and in vitro studies. Whereas steroid treatment of asthmatics did not change elastic fibers and collagens in bronchial biopsies [18], steroids did inhibit serum-induced proteoglycan production in fetal lung fibroblasts [19]. In contrast to asthma, to the authors’ knowledge, effects of ICS on ECM composition in COPD patients have not been described. We hypothesized that inhaled steroids treatment modulates bronchial ECM components in COPD. In addition, we hypothesized that current smoking affects bronchial ECM. BODY.MATERIALS AND METHODS: BODY.SUBJECTS AND STUDY DESIGN: The current study is a substudy of the GLUCOLD (Groningen Leiden Universities Corticosteroids in Obstructive Lung Disease) study, a double-blind, placebo-controlled randomized trial in which 114 moderate-severe COPD steroid-naive patients were included [17]. The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. Clinically stable subjects participating in the GLUCOLD study were aged 45–75 years, smoked ≥10 packyears, were current or ex-smokers with ≥one month of smoking cessation and were allowed to use short-acting bronchodilators. Exclusion criteria were asthma and ICS use in the previous 6 months. Patients were randomly assigned to receive one of four treatments for 30 months: 1) fluticasone propionate 500 µg bid; 2) fluticasone/salmeterol 500/50 µg bid; 3) fluticasone 500 µg bid (6 months) and followed by placebo (24 months); or 4) placebo bid. Diskus dry-powder inhalers (GlaxoSmithKline, Zeist, The Netherlands), were used for inhalation of the study medication and placebo, and both had equal appearance. For the current study we used tissue and data of group 1 and 4. Spirometry, reversibility to salbutamol and airway hyperresonsiveness (PC20) were determined according to international guidelines [20], [21]. Approval of the medical ethics committees of both centers was obtained: all subjects provided written informed consent [17]. BODY.BRONCHOSCOPY AND BRONCHIAL BIOPSIES: A fiberoptic bronchoscopy was performed at baseline and after 30 months according to standardized protocols [22]. Six bronchial biopsies per patient per visit were collected at the 3rd–5th bronchial level, one with the best morphology being used. Tissue of 64 out of 114 patients was available due to use in previous studies [17], [22], [23]. BODY.(IMMUNO)HISTOCHEMICAL STAININGS: Processing and analysis of bronchial biopsies was performed in line with the recommendations of the ATS/ERS task force [24] by using an internal reference parameter in the analysis. We did not take specific precautions to orientate the samples during processing to assure that the orientation of the biopsies is randomized [24]. However, since biopsies tend to curl after sampling, a random orientation of the tissue structures is favored during embedding [25]. Sections of 4 µm thickness of paraffin-embedded bronchial biopsies were used for histochemistry (elastic fibers) and immunohistochemistry for proteoglycans and collagens. Elastic fibers were stained according to Weigert’s protocol [26]. Versican, decorin, collagen I and III antibodies were used after appropriate antigen retrieval, followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit EnVision system (DAKO, Glostrup, Denmark) and the chromogen NovaRed (Vector, Burlingame, CA). Images of stained biopsies are presented in figure 1, and additional information on the stainings is provided in table S1 in file S1. 10.1371/journal.pone.0063430.g001Figure 1BODY.EXAMPLES OF (IMMUNO)HISTOCHEMICAL STAININGS.: The same bronchial biopsy section is shown for the histochemical staining for elastic fibers (A) and the immunohistochemical stainings for versican (B), decorin (C), collagen type I (D) and collagen type III (E). Original magnification 200×. BODY.DIGITAL IMAGE ANALYSIS: Tissue samples were analyzed in a blinded manner by independent observers, unaware of the subjects’ clinical data (LK, JS). Total biopsy images were acquired using a color camera (200× magnification) and analyzed with image analysis software (CellD, Olympus, Zoeterwoude, The Netherlands). The lamina propria was selected per biopsy (minimum area 0.09 mm2). The percentage stained area for a specific ECM component was calculated dividing the stained area by the total selected area (volume fraction; used as an internal reference parameter; [24]). Staining intensity was further analyzed by densitometry (weighted mean per biopsy) and presented as gray value (black: gray value = 0; white: gray value = 255). Only immunohistochemical stainings can represent density, therefore density was not calculated for elastic fibers. Additional information on digital image analysis is provided in file S1. BODY.STATISTICAL ANALYSIS: Only biopsies from compliant subjects using ≥70% of the prescribed dose were analyzed (per-protocol analysis). Means with standard deviations (SD) and 95% confidence intervals (CI) or medians with interquartile range (IQR) are presented. Differences between smokers and ex-smokers were explored using Mann-Whitney tests. Paired and independent t-tests were used for evaluating the effect of ICS on ECM proteins within and between treatments, respectively. Correlations were analyzed using Spearman correlation coefficient (Rs). Statistical analysis was performed with SPSS 17.0 software (SPSS Inc., Chicago, IL). Significance was inferred at P≤0.05. BODY.RESULTS: BODY.PATIENT CHARACTERISTICS: At baseline, bronchial biopsies of 64 of 114 unselected moderate-severe COPD patients [24/40 (ex-)smokers] were included. A flow diagram of our study is presented in the figure S1. Patient characteristics of the whole group have previously been published [17], [22], [27]. 33 Patients were treated with either fluticasone or placebo for 30 months (19/19 and 13/14 adherent in fluticasone and placebo group, respectively). Mean post-bronchodilator FEV1 was 62% predicted (SD 9.9%). Ex-smokers were older at baseline compared to current smokers, as is shown in table 1. Baseline characteristics of the entire group, groups with available and unavailable bronchial biopsies, and the number of available biopsies were not significantly different between both treatment arms. During the study, six patients changed their smoking habits (balanced among groups). 10.1371/journal.pone.0063430.t001Table 1BODY.PATIENT CHARACTERISTICS AT BASELINE.: Smokers(n = 40) Ex-smokers (n = 24) Placebo(n = 13) Fluticasone (n = 19) Males [n (%)] 37 (92.5) 23 (95.8) 12 (92.3) 17 (89.5) Age (years) 60.9 (7.2) 65.1 (6.6)* 62.5 (7.9) 62.0 (7.4) Current/ex-smoker (n) 9/4 11/8 Packyears 46.8 (30.9–55.0) 37.5 (32.1–52.5) 42.0 (28.4–58.0) 44.9 (31.2–51.0) Smoking cessation (years) 5.5 (1.3–10.0) 0.0 (0.0–1.5) 0.0 (0.0–5.0) FEV1 post-bronchodilator (l) 2.05 (0.44) 1.94 (0.46) 1.95 (0.61) 2.03 (0.42) FEV1 post-bronchodilator (%pred) 63.0 (8.7) 59.6 (9.9) 59.9 (9.8) 62.5 (9.5) FEV1/IVC% post-bronchodilator 48.7 (8.9) 44.2 (8.9) 44.3 (9.5) 47.7 (8.6) Geometric mean methacholine PC20 (mg/ml) 0.76 (2.9) 0.39 (3.0) 0.67 (1.9) 0.41 (2.4) Patient characteristics for current smokers and ex-smokers with COPD and groups treated with placebo and fluticasone (only compliant patients). Bronchial biopsies were available at baseline of 64 (elastic fibers), 56 (versican), 61 (decorin), 61 (collagen I) and 64 (collagen III) patients. After 30 months, bronchial biopsies of 32 compliant patients were available, tissue from 29 (elastic fibers), 26 (versican), 27 (decorin), 28 (collagen I) and 28 (collagen III) patients had sufficient surface area for analysis (≥0.09 mm2) (fluticasone and placebo groups combined). Data are presented as mean (SD) or median (IQR), unless otherwise stated. Methacholine PC20: provocative concentration of methacholine that causes a 20% decrease in FEV1, expressed as mean doubling doses. Part of the data have been published previously [17], [22], [27]. *p<0.05 compared to current smokers (two tailed unpaired t-tests). BODY.INHALED CORTICOSTEROIDS AND EXTRACELLULAR MATRIX PROTEINS: Adjusted for baseline values, we found that ICS significantly increased percentage versican (9.6% [CI 0.9 to 18.3%]; p = 0.03) and collagen III (20.6% [CI 3.8 to 37.4%]; p = 0.02) compared to placebo (figure 2); a trend was seen for the density of decorin (3.9 [CI −0.7 to 8.6]; p = 0.09) and collagen III (8.4 [CI −1.1 to 17.9]; p = 0.09). Baseline percentage and density of versican (17% [CI 3.5 to 30.6%]; p = 0.02 and 8.0 [CI 2.7 to 13.3]; p = 0.006, respectively) and collagen III (10.7% [0.1 to 21.4%]; p = 0.03 and 7.9 [CI 0.9 to 15.0]; p = 0.05, respectively) and percentage of decorin (2.0% [CI 0.5 to 3.5%]; p = 0.02) were significantly higher in the placebo group than the fluticasone group. Change in smoking status was not included into our analysis, because current and ex-smokers with COPD had similar ECM composition. An increase in density of collagen I was associated with improvements in post-bronchodilator FEV1 (l) (Rs = 0.45, P = 0.037) when we analyzed both fluticasone and placebo treated groups combined (figure 3). No correlations were found for other ECM proteins and lung function. 10.1371/journal.pone.0063430.g002Figure 2BODY.PERCENTAGE AND DENSITY OF STAINED AREA FOR PLACEBO AND FLUTICASONE FOR ALL ECM PROTEINS.: Percentage (upper panel) and density (lower panel) of stained area in bronchial biopsies is presented. Open figures: baseline percentage stained area, closed figures: percentage stained area after 30 months. Horizontal bars represent medians. 10.1371/journal.pone.0063430.g003Figure 3BODY.CORRELATION BETWEEN CHANGE IN POST-BRONCHODILATOR FEV:1 (L) and change in density of collagen I. BODY.SMOKING STATUS AND EXTRACELLULAR MATRIX PROTEINS AT BASELINE: No significant differences in percentage of the area being positively stained and density of ECM proteins were found between current smokers and former smokers with COPD (figure 4). Long-term ex-smokers (≥5.5 years, our median value) had similar percentage and density of all ECM proteins compared to short term ex-smokers (<5.5 years) and current smokers (all P>0.05). Furthermore, no relation was found between packyears and percentage or density of all ECM proteins. 10.1371/journal.pone.0063430.g004Figure 4BODY.PERCENTAGE AND DENSITY OF STAINED AREA AT BASELINE OF EX-SMOKERS AND SMOKERS WITH COPD.: Percentage (upper panel) and density (lower panel) of stained area in bronchial biopsies is presented. Ex-smokers are presented as open circles, current smokers as closed circles. Horizontal bars represent medians. No significant differences were found for all studied extracellular matrix proteins (both percentage stained area and density). BODY.CORRELATIONS BETWEEN EXTRACELLULAR MATRIX AND LUNG FUNCTION AT BASELINE: Percentage collagen I correlated positively with FEV1 (% predicted) post-bronchodilator (Rs = 0.31, P = 0.015) (figure 5, left panel) and FEV1/IVC% (Rs = 0.38, P = 0.003). In addition, percentage collagen type I and III correlated with PC20 (Rs = 0.33, P = 0.012; Rs = 0.37, P = 0.004, respectively) (figure 5, right panel). Percentage collagen I, but not collagen III, was significantly lower in GOLD stage III (n = 9) than GOLD stage II (n = 55) (medians 5.5% and 17.7%, respectively, P = 0.01). No significant correlations were found between lung function at baseline and densities of all ECM proteins. 10.1371/journal.pone.0063430.g005Figure 5BODY.CORRELATION BETWEEN PERCENTAGE COLLAGEN TYPE I AT BASELINE AND LUNG FUNCTION PARAMETERS.: Panel A presents post-bronchodilator FEV1 (% predicted) and panel B shows PC20 (in doubling dose). BODY.DISCUSSION: Our results show that 30-month treatment with inhaled corticosteroids increases the percentage stained area of versican and collagen III, indicating that long-term treatment possibly influences the remodeling process in the airways. Furthermore, lung function is weakly, but positively correlated with collagen I both at baseline and with regard to changes in FEV1 and collagen I that occurred after treatment. In addition, we show that the content of ECM proteins in bronchial biopsies did not significantly differ between smokers and ex-smokers with moderate-severe COPD. This study shows that the content of elastic fibers, major proteoglycans and collagens in the bronchial mucosa are similar in current and ex-smokers with COPD. Our findings extend previous observations, showing no difference in the percentage elastic fibers in COPD patients and smokers without airway obstruction [11], [28]. We observed no significant difference in versican and decorin content between current and ex-smokers with COPD, which is in line with an in vitro study with cultured lung fibroblasts of moderate COPD patients and control subjects. Cigarette smoke extract (CSE) exposure of these cells did not affect versican gene expression, but decreased decorin gene expression [16]. This apparent difference with our findings could be explained by the fact that smoke-exposed fibroblasts are only selectively triggered compared to a multifactorial environment in vivo. Finally, in our study collagen type I and III were not significantly different between current and ex-smokers with COPD, which is similar to recent observations in cultured fibroblasts of COPD and non-COPD patients [29]. The percentage of versican and collagen III increased with long-term ICS treatment compared to placebo, without significant changes in elastic fibers, decorin and collagen I. In line with this, ICS for four weeks or 3.5 years did not affect elastic fibers content in bronchial biopsies of asthmatics compared to healthy controls [18]. Notably, we found a significant increase in collagen III, but not collagen I, after 2.5 years of ICS treatment compared to placebo, which was associated with lung function. Previous studies in COPD patients showed that gene expression of collagen 1α1 and collagen 3α1 in small airways and parenchyma was decreased in association with lower FEV1 [3], [30]. Thus, collagen may have stabilizing effects on the collapsible airways in patients with COPD, which could be further enhanced by long-term use of ICS. Our study has various strong points. We included only steroid-naive COPD patients, excluding possible influences of steroids on ECM components at baseline. Both the percentage and density of the stained area in bronchial biopsies were analyzed: the percentage corresponds to the presence of the ECM protein, whereas density represents the local amount of ECM protein. For the analysis of the percentage, we used the total selected tissue area for analysis as an internal reference parameter according to the recommendations of the Joint ATS/ERS Task Force [24]. We considered the possibility that part of our changes is explained by an effect of ICS on edema. However, less edema resulting from ICS treatment would probably have increased percentage and density all studied ECM proteins, whereas in our study the percentage of only some ECM proteins was affected. Furthermore, we previously found lower numbers of selected bronchial inflammatory cells after ICS treatment in the current study [17]. We did not find correlations between the effect of ICS treatment on inflammatory cells and ECM components (data not shown). There are some considerations when interpreting our results. Matched bronchial biopsies both at baseline and follow-up were available from approximately half of our COPD patients, because part of the tissue was no longer available. This could have negatively affected the power of our study. Still, the number of available biopsies was similar among both groups. Furthermore, since one biopsy per patient per visit was studied, we cannot exclude that local heterogeneity of ECM proteins has affected our results. To minimize selection bias, we only selected biopsies with the largest lamina propria. Lung tissue specimens from healthy or never-smokers were not available, but comparisons with these groups were beyond the objectives of this study. Furthermore, features of remodeling in COPD are different between large and small airways, nevertheless we evaluated the ECM in the central airways only [2] and important correlations with lung function could still be observed. Finally, despite treatment randomization, we accidentally found that the percentage and densities of versican, decorin and collagen III at baseline were significantly higher in the placebo than the fluticasone group. Not withstanding this, when still adjusted for the baseline values, we observed effect of ICS therapy. Taken together, we do not believe that the above limitations largely affected our results. How can we explain that smoking has no effect on ECM? Exposure of cultured pulmonary fibroblasts of moderate and very severe COPD patients to CSE resulted in downregulation of decorin, but not versican and collagen type I and III expression [16], [29]. In addition, collagen I and tropoelastin were dose-dependently inhibited by CSE in rat fetal lung fibroblasts [31]. Mice with long-term exposure to cigarette smoke showed a decrease in elastic fibers and collagen type III, without significant effect on collagen I [32]. We could only partially confirm these in vitro and animal studies. Inflammation and remodeling in the lung in vivo are simultaneous and complex ongoing processes and may not be mimicked by studies in isolated fibroblasts and inbred animals kept under specific conditions. Furthermore, after smoking cessation bronchial inflammation (at least) partially persists [22], which is in line with our finding of similar ECM composition between smokers and ex-smokers. We showed a positive correlation between the content of collagen and lung function after treatment with inhaled steroids. However, the current opinion of remodeling is that airway wall thickening is strongly associated with progression of COPD [2], suggesting that increased ECM deposition is related to a decreased lung function. How can we explain this apparent contradiction? In COPD, an imbalance between proteases and anti-proteases is present, as shown by an excess of matrix metalloproteinases (MMP) and a relative shortage of tissue inhibitor of metalloproteinases (TIMP) [33]. MMP degrade both collagens and proteoglycans [2], [33], [34]. Dexamethasone can reduce MMP-9 and increase TIMP-1 release from alveolar macrophages of COPD patients [35], which may result in a decreased capacity to degrade ECM. This is in line with our observation that ICS increase collagen and versican. Also the observation from Annoni et al [11], showing that patients with COPD have lower collagen I densities in their airways, is in line with the speculation that an increase in collagen I induced by ICS could stabilize the airways. Furthermore, the observed positive correlation between collagen with lung function and PC20 before and after long-term ICS therapy also suggests that increased airway wall fibrosis is actually preventing both airway collapse and attenuating airway smooth muscle contractions in COPD. Besides airway remodeling, emphysema might also influence airway collapse, which could contribute to the airflow obstruction. Unfortunately, no data were collected to quantify the extent of emphysema in our cohort of COPD patients. Although smoking cessation shows positive clinical effects [1], smoking status was not significantly correlated with our studied ECM components. Treatment with ICS increased the percentage versican and collagen III. We found positive correlations between ECM proteins and several lung function parameters at baseline and after treatment with ICS. Therefore, our data may implicate that steroids alter airway structure by increasing ECM content in COPD which is associated with preserved lung function. This suggests that increased presence ECM proteins do not by themselves lead to detrimental consequences, but instead can prevent airway collapse. In conclusion, we showed that treatment for 30 months with inhaled corticosteroids increased the relative content of versican and collagen III in the large airways of patients with moderate to severe COPD. Our data suggest that steroids not only prevent bronchial inflammation but possibly also alter airway structure by increasing specific ECM proteins in COPD that are associated with improvements in lung function. Further studies are needed to confirm these findings in other studies, and to understand the possible implications of these findings for current treatment strategies and for the development of future, targeted anti-remodeling medication in COPD. BODY.SUPPORTING INFORMATION: Figure S1 Study flow diagram. Study flow diagram of the GLUCOLD study presenting the bronchial biopsies used in this study at baseline and after 30 months treatment with inhaled fluticasone. (TIF) Click here for additional data file. Protocol S1Trial protocol GLUCOLD study. (DOC) Click here for additional data file. File S1(DOC) Click here for additional data file. Checklist S1CONSORT checklist. (PDF) Click here for additional data file.

TITLE: Effect of ivacaftor treatment in patients with cystic fibrosis and the G551D-CFTR mutation: patient-reported outcomes in the STRIVE randomized, controlled trial ABSTRACT.BACKGROUND: Cystic fibrosis (CF) is an inherited, rare autosomal recessive disease that results in chronically debilitating morbidities and high premature mortality. We evaluated how ivacaftor treatment affected CF symptoms, functioning, and well-being, as measured by the Cystic Fibrosis Questionnaire-Revised (CFQ-R), a widely-used patient-reported outcome (PRO) measure. ABSTRACT.METHODS: STRIVE, a double-blind, placebo-controlled randomized trial, evaluated ivacaftor (150 mg) in CF patients aged 12+ with the G551D-CFTR mutation for 48 weeks. Treatment effect analysis used a mixed-effects repeated measures model. Treatment benefit analyses applied the cumulative distribution function and a categorical analysis of change scores (“improvement,” “no change,” or “decline”). Content-based interpretation examined treatment effect on specific item responses. ABSTRACT.RESULTS: Data from 152 patients with a baseline CFQ-R assessment were analyzed. The treatment effect analysis favored treatment with ivacaftor over placebo on the Body Image, Eating, Health Perceptions, Physical Functioning, Respiratory, Social Functioning, Treatment Burden, and Vitality scales. Findings were supported by the analysis of categorical change. On all CFQ-R scales, the percentage of patients who improved was greater for ivacaftor. In the content-based analysis, the treatment benefit was characterized by better scores across a broad range of domains. ABSTRACT.CONCLUSIONS: Results illustrate broad benefits of ivacaftor treatment across many domains: respiratory symptoms, physical and social functioning, health perceptions, and vitality, as measured by the CFQ-R. The breadth of improvements reflects the systemic mechanism of action of ivacaftor compared to other therapies. Findings support the patient-reported value of ivacaftor treatment in this patient population. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT00909532 ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12955-015-0293-6) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Cystic Fibrosis (CF) is a rare, autosomal recessive disease affecting multiple organs, including the lungs, pancreas, sweat glands, and intestinal, biliary and reproductive tracts [1]. CF is caused by defects in the CF transmembrane conductance regulatory (CFTR) protein resulting from mutations in the CFTR gene. Some CFTR mutations produce CFTR protein channels that have defective gating (reduced channel open probability), resulting in little to no net chloride ion transport [2–4]. Patients with CF who have at least one CFTR mutation of this type are at high risk for early lung function decline and progression of other disease manifestations. The most prevalent CFTR mutation that predominantly affects CFTR channel open probability is G551D, which is found in approximately 4 % of patients with CF in the United States [5]. Ivacaftor (Kalydeco®) was approved by the FDA in January 2012 for the treatment of CF in patients 6 years of age and older who have the G551D mutation on at least one CFTR allele. The approval was expanded in the US in February 2014 and December 2014 to include 9 additional mutations [6]. All of the currently indicated mutations affect CFTR channel open probability as their primary molecular dysfunction. The Phase 3 clinical program for ivacaftor was designed to assess the efficacy and safety of ivacaftor treatment in patients with at least one of the specified CFTR mutations. Treatment-related changes in patient-reported outcomes (PROs) were evaluated and have been previously reported for children (ENVISION study), and adolescents and adults (STRIVE study) [7, 8] with at least one G551D-CFTR mutation. Developers of drugs are expected to provide direct evidence of the treatment benefit as experienced by patients. Providers, regulators, and payers seek information about how patients feel and function in daily life, with evidence from pivotal trials implementing well-defined and reliable assessments. To that end, we expand on data previously reported that show the impact of CF and its treatment on how patients feel and function in everyday life. The objective of this study was to evaluate the treatment effects of ivacaftor on patient-reported symptoms and health-related quality of life (HRQoL) as reported by patients with CF 12 years of age and older with the G551D-CFTR mutation participating in the STRIVE study. The specific aim was to interpret treatment benefit using innovative analyses of PRO data covering a broad range of HRQoL domains and CF symptoms, specifically: 1) mean changes in Cystic Fibrosis Questionnaire-Revised (CFQ-R) scale scores from baseline through 48 weeks of treatment; 2) patterns of treatment response using empirically-defined categories of change; and 3) examination of treatment using responses to salient items of the CFQ-R that characterized disease impact. BODY.METHODS: BODY.DATA SOURCE: STRIVE was a randomized, double-blind, placebo-controlled study evaluating ivacaftor, a CFTR potentiator, in subjects 12 years of age or older with CF and at least one G551D-CFTR mutation. Patients were randomly assigned in a 1:1 ratio to receive 150 mg of ivacaftor or placebo every 12 h for 48 weeks. Throughout the study, all patients continued to take their pre-study medications (with the exception of hypertonic saline). The primary endpoint was the estimated mean absolute change from baseline through week 24 in the percent predicted forced expiratory volume in 1 s (FEV1 % predicted). Secondary endpoints included the change from baseline through week 48 in the FEV1 % predicted, time to first pulmonary exacerbation, change in body weight, change in concentration of sweat chloride, as well as patient-reported scores from the CFQ-R Respiratory Symptoms Scale [9–11]. The study included a 2-week screening period, a 2-week run-in period, and a 48-week treatment period. Efficacy and safety findings from this study have been previously reported [9]. BODY.CYSTIC FIBROSIS QUESTIONNAIRE-REVISED: The CFQ-R was the first disease-specific PRO instrument developed for use by patients with CF and their caregivers [12]. The original CFQ was developed from qualitative and quantitative studies which included a conceptual framework, interviews with patients, parents, and health care professionals, cognitive testing, and psychometric evaluation [10, 12–14]. Modifications were subsequently made to the instrument leading to the current revised version, which has been identified as a widely used PRO measure for CF [15–17]. Three versions of the CFQ-R are available: a self-completed Teen/Adult version for patients with CF aged 14 and older, a Child version for children aged 6–13 years (self-report for ages 12–13 and interviewer-administered for ages 6–11), and a proxy-completed Parent/Caregiver version. The Teen/Adult and Parent/Caregiver versions include the following scales: Body Image, Digestive Symptoms, Eating Problems, Emotional Functioning, Health Perceptions, Physical Functioning, Respiratory Symptoms, Role Functioning, Social Functioning, Treatment Burden, Vitality, and Weight. The Child version does not include Health Perceptions, Role Functioning, Vitality, and Weight. Items included in each scale of the CFQ-R are summed and standardized to a 0–100 scale, with higher scores indicating better outcomes or fewer symptoms from the patient perspective. In the STRIVE study, the CFQ-R was administered at the beginning of the run-in period, baseline, day 15, week 8, and every 8 weeks thereafter, up to week 48. Patients aged 14 and older completed the Teen/Adult version while those aged 12 and 13 at baseline completed the Child version. Parents of children aged 12 and 13 at baseline also completed the Parent/Caregiver version. This analysis included pooled data from the two self-completed Teen/Adult and Child versions (data from the Parent/Caregiver version were not included). BODY.ANALYSES: BODY.TREATMENT-RELATED CHANGES IN CFQ-R DOMAINS: For each scale of the CFQ-R, mean changes from baseline through week 48 were evaluated by treatment group using a mixed-effects model for repeated measures [18] with absolute change from baseline as the dependent variable, fixed effects for study visit and treatment group, and adjustment for continuous baseline values of age, FEV1 % predicted, and baseline scale score, using an unstructured covariance matrix. Treatment effect was calculated as the difference in mean change from baseline between the ivacaftor and placebo groups. A p-value of <0.05 was employed to assess statistical significance. In addition to evaluating treatment benefit as mean change from baseline, the current analyses also included evaluating treatment group differences in terms of the percentage of patients reporting various levels of change across the different CFQ-R domains. First, to evaluate treatment group differences across the entire range of observed change, we used a method based on the cumulative distribution function (CDF). The CDF of change scores presents the proportion of patients who experienced an improvement or decrement at or below a specific value [19]. When presented separately by treatment group, CDF plots illustrate the separation between groups at each threshold of change. For evaluating treatment response, CDFs are most easily interpreted when the y-axis is reversed to show the proportion of patients with a score change > |X|, an approach known as cumulative response curves (CRCs) [20]. We evaluated the CRCs for change from baseline to week 48 by CFQ-R scale, by treatment group. Statistical significance was assessed using the two-sample Kolmogorov-Smirnov test. In addition to the CRC analysis, we used a distribution-based methodology [21] to establish threshold values that help interpret the magnitude of change observed in HRQoL scores. One-half standard deviation (SD) of the change from beginning to end of the 2-week run-in period (2 weeks prior to study drug initiation) was used as a threshold value [22] to identify minimal important change. The direction and magnitude of score changes were used to analyze treatment response. Positive or negative changes from baseline to week 48 exceeding (in absolute value) the threshold were considered “improvements” or “declines”, respectively. Change scores smaller (in absolute value) than the threshold value were considered “no change”. The Chi-square test was used to assess statistical significance of treatment group differences. Sensitivity analyses were conducted using the standard error of measurement (SEM). The SEM reflects the measurement precision and the amount of random variation from repeated assessments. One SEM has been proposed as a measure of minimal clinically important difference (MCID) and was used to identify “improvement”, “no change”, or “decline” [23], with the Chi-square test for assessment of group differences. The SEM was evaluated as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ SEM=S{D}_{\varDelta}\ast \sqrt{\left(1-ICC\right)} $$\end{document}SEM=SDΔ∗1−ICC, where SD∆ is the SD of change scores from the run-in period and the ICC is the intraclass correlation coefficient, a measure of reliability related to the repeated administration of the same test under the assumption that substantial change in the concept of interest has not occurred. BODY.CONTENT-BASED INTERPRETATION OF TREATMENT EFFECTS: These analyses examined the impact of treatment on responses to specific “sentinel” CFQ-R items empirically selected to represent each scale. Polyserial or Spearman [24] correlations between change in each item and change in its scale were calculated. The item with the strongest association with its scale change score was identified as the sentinel item. Response options for each sentinel item were collapsed into “none” vs. “any” impairment. The change in percent of patients with no impairment was evaluated by treatment group at baseline and week 48. BODY.ETHICS: The STRIVE clinical trial (“A Phase 3, Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study to Evaluate the Efficacy and Safety of VX-770 in Subjects with Cystic Fibrosis and the G551D Mutation”) protocol was reviewed and approved by the institutional review board at each participating center, and each subject provided written informed consent or written or oral assent. BODY.RESULTS AND DISCUSSION: BODY.CHARACTERISTICS OF STUDY SAMPLE: The STRIVE study randomized 167 subjects to two treatment arms. Of these, 161 subjects subsequently received at least one dose (intention-to-treat analysis). This study sample contained the 152 patients who completed a baseline CFQ-R assessment. The two treatment groups were similar in terms of gender (53.0 % female) and mean age (25.7 years). Most patients were 18 years of age or older (77.6 %). At baseline, the treatment groups also were similar in average FEV1 % predicted (64.6 %), mean sweat chloride (100.4 mmol/L), height (167 cm), body weight (78.9 kg), and body mass index (BMI) (21.9 kg/m2). BODY.TREATMENT-RELATED CHANGES IN CFQ-R SCALES: Figure 1 shows the observed mean and standard error of the change from baseline at each study visit, by scale and treatment group. Mean change on the Body Image, Eating Problems, Health Perceptions, Physical Functioning, Respiratory Symptoms, Social Functioning, Treatment Burden, and Vitality scales was consistently higher for ivacaftor vs. placebo. Favorable effects of ivacaftor were observed within the first 2 months of treatment initiation.Fig. 1Change from baseline in CFQ-R scores for each visit by treatment group (observed data). SE = standard error; means and standard error are unadjusted Table 1 presents baseline score, mean change from baseline through week 48, and treatment effects (difference between treatment groups in mean change) by CFQ-R scale and treatment group. For seven of the twelve CFQ-R scales, the mean post-baseline change was statistically significant, after adjustment for age, FEV1 % predicted, and baseline CFQ-R scale score. The largest treatment effect was found on the Respiratory Symptoms scale with a mean improvement of 5.9 points observed among patients receiving ivacaftor, exceeding the MCID value of 4 points, vs. a mean decline of 2.7 points among those receiving placebo (treatment effect of 8.6 points, P < 0.001) [12].Table 1CFQ-R adjusted change scores from baseline through week 48 by treatment group CFQ-R ScalePlaceboIvacaftor(N = 70b)(N = 80b)BaselineChangeBaselineChangeTreatment effect p-valuea Body Image 80.3 −1.2 81.0 1.5 2.7 0.086 Digestive Symptoms 85.4 0.4 85.2 0.8 0.5 0.732 Eating Problems 91.9 −1.1 91.8 2.2 3.3 0.002 Emotional Functioning 83.6 −1.4 86.0 0.7 2.1 0.096 Health Perceptions 71.7 −3.6 72.1 4.0 7.6 <0.001 Physical Functioning 80.2 −1.7 76.1 2.7 4.4 0.006 Respiratory Symptoms 68.5c −2.7 70.2 5.9 8.6 <0.001 Role Functioning 85.9 0.1 86.3 −0.5 −0.6 0.651 Social Functioning 71.9 −1.0 72.1 3.3 4.3 0.003 Treatment Burden 65.7 1.0 64.5 4.3 3.3 0.042 Vitality 64.7 −2.8 64.3 2.7 5.5 0.002 Weight 78.1 1.7 79.0 6.9 5.3 0.053 Health Perceptions, Role Functioning, Vitality and Weight are not included in the Child self-report version of the CFQ-R; results for these scales are based on the Teen/Adult version only (N = 64 for placebo and N = 76 for Ivacaftor) a P-value for overall post-baseline treatment effect, estimated using a mixed-effect model for repeated measures with fixed effects for study visit, treatment group, and adjustment for continuous baseline values of age, percent predicted FEV1 and domain score bAnalysis sample includes patients with a baseline assessment and at least one post-baseline assessment c n = 71 Figure 2 presents CRCs of change scores from baseline to week 48, by treatment group. For nearly all CFQ-R scales, lower cumulative change scores (worse outcomes) were seen for the placebo group, indicated by the dashed curve for the placebo group appearing to the left of the solid curve for ivacaftor. These findings suggest that the proportion of subjects who exceeded a particular level of change (better outcomes) was higher in the ivacaftor than placebo group. Differences between treatment groups (favoring ivacaftor) were greatest for Respiratory Symptoms (p < 0.001), Physical Functioning (p = 0.002), Health Perceptions (p = 0.019), and Vitality (p = 0.030). Using illustrative values of 5 and 10 points, the percentage of patients with change scores greater than 5 on the Respiratory Symptoms scale was 59 % for ivacaftor vs. 27 % in the placebo group. For changes greater than 10 points, percentages were 47 and 11 % for ivacaftor and placebo, respectively. For change scores of 5 and 10 points on Physical Functioning, percentages of ivacaftor-treated patients with improvement were 36 and 23 %, respectively, compared to 13 and 8 % in the placebo group.Fig. 2Cumulative response curves for CFQ-R scales by treatment group. P-values from the Kolmogorov-Smirnov two-sample test for equality of cumulative distribution functions Figure 3 presents the percentage of patients who experienced “improvement”, “no change”, or “decline” after 48 weeks of treatment with either ivacaftor or placebo, according to our change threshold of 0.5 SD. Differences favoring ivacaftor were observed for Respiratory Symptoms (P < 0.001), Social Functioning (P = 0.026), Vitality (P = 0.006), Treatment Burden (P = 0.016), Health Perceptions (P = 0.003), Physical Functioning (P < 0.001), Eating Problems (P = 0.015), and Weight (P = 0.015).Fig. 3Analysis of categorical change from baseline to week 48 by CFQ-R scale and treatment group. *p-value < 0.05. from chi-square test for differences between treatment groups in the percentage of “improvement”, “no change” and “decline” patients Results were similar under the 1 SEM criterion (see Additional file 1: Tables S1 and Additional file 2: Table S2). BODY.CONTENT-BASED INTERPRETATION OF TREATMENT EFFECTS: Table 2 shows the sentinel items by CFQ-R scale and the baseline percentage of patients reporting “no impairment”.Table 2Percentage of patients with “no impairment” in representative CFQ-R items at baseline and 48 weeks Representative items% No Impairment at baseline (pooled)CFQ-R scaleTeen-adult version/Child versionResponse optiona Corr.b with domain change scoreBody image Look different from others Very false/Not at all true 0.82 60.0 Digestive symptoms Problems with gas/stomach hurt Never 0.85c 48.1 Eating problems Force myself to eat/pushed to eat Very false/Never 0.82c 77.7 Emotional functioning Felt sad/worried Never 0.79 71.1 Health perceptions Feel healthy Very true 0.86 32.5 Physical functioning Walking as fast as others No difficulty 0.85 65.9 Respiratory symptoms Coughing Not at all/Never 0.85 3.7 Role functioning Running errands out of the house Never 0.85 41.0 Social functioning Comfortable going out/got together with friends Very true 0.66 59.2 Treatment burden Tx makes life more difficult/Tx bothered you Not at all/Not at all true 0.78 25.1 Vitality Felt exhausted/grouchy Never 0.81 47.4 Weightd Trouble gaining weight Not at all e 62.0 aIdentified to represent patient report of no impairment bPolyserial correlation unless otherwise noted cSpearman correlation dSingle item scale eNot applicable for single item scales Pooled = ivacaftor and placebo The proportion of patients treated with ivacaftor who showed “no impairment” generally increased between baseline and week 48 and decreased among patients treated with placebo (data not shown). The items with at least 10 % separation between ivacaftor and placebo at week 48 were: “feel healthy” (17.5 % separation), “walking as fast as others” (15.1 %), “coughing” (14.1 %), “going out with friends” (14.1 %), “trouble gaining weight” (14.0 %), “force myself to eat”“treatment makes life more difficult” (12.5 %). Figure 4 presents the change in percentage with “no impairment” from baseline to week 48 by treatment group. As before, the percentage with “no impairment” increased with ivacaftor and decreased with placebo between baseline and week 48 for most items, eg, “walking as fast as others”(+13.7 vs. −10.7 %, respectively), “feel healthy” (+10.2 vs. −4.5 %), “running errands out of the house” (+18.0 vs. −1.2 %), “going out with friends” (+11.2 vs. −0.7 %), [never feeling] “bothered by treatment” (+15.7 vs. −1.0 %).Fig. 4Change in the percentage of patients with no impairment after 48 weeks of treatment BODY.CONCLUSIONS: Our analyses provide empirical data describing and quantifying the benefits of ivacaftor treatment in patients aged 12 years and older with CF and the G551D-CFTR mutation. In the STRIVE study, early and sustained treatment effects from ivacaftor were observed through week 48 on the CFQ-R Respiratory Symptom scale. The magnitude and significance of the improvements in Respiratory Symptoms was consistently observed across the analyses in line with improvements in % predicted FEV1 in the study population. Of interest, sustained improvements were also observed on other scales of the CFQ-R, including Physical Functioning, Social Functioning, Eating Problems, Treatment Burden, Health Perceptions, and Vitality. Trends also were favorable for ivacaftor-treated patients on the Body Image, Digestive Symptoms, Role Functioning, and Emotional Functioning scales, but their magnitude was smaller and failed to reach statistical significance. These positive changes across multiple CFQ-R scales are supported by findings from categorical change analyses. Using change scores classified into “improvement”, “no change”, or “decline”, the treatment effects favoring ivacaftor over placebo became even more apparent. On all 12 CFQ-R scales, the percentage who improved by week 48 was greater for ivacaftor-treated patients. Further, the percentage who declined was greater for placebo-treated patients, although differences across the three categories of change were not statistically significant for Role Functioning, Body Image, Emotional Functioning, and Digestive Symptoms. For the Respiratory Symptoms and Physical Functioning scales, the ivacaftor group included at least five times more patients with improvements compared to placebo. For the Social Functioning, Vitality, Treatment Burden, and Role Functioning scales, the ivacaftor group included three to five times more patients with improvements compared to placebo. Worsening scores through week 48 were more prevalent among patients on placebo. Of particular interest are the Weight scores, with a three-fold difference between ivacaftor and placebo in the percentage of patients who declined during the study (9 vs. 28 %). The content-based analysis of sentinel items-showed that ivacaftor led to improvements in a broad range of functional outcomes, including the respiratory, nutritional, physical, social, and treatment-related domains. Although several trial-based publications have documented treatment benefits using the CFQ-R, they have focused on selected scales: Respiratory Symptoms, Physical Functioning, and Vitality [25–30]. Additional data, particularly from observational studies, would provide needed real-world evidence to complement trial-based data on the patient-reported benefits associated with ivacaftor treatment. In the PERSIST open-label extension study sample, including 75 % (n = 144) of the STRIVE sample, the effect of ivacaftor on the CFQ-R Respiratory Symptoms scale was maintained over an additional 96 weeks of treatment [31]. In an observational study of ivacaftor (US GOAL), clinically meaningful and statistically significant gains (7.4 points, p < 0.0001) were observed in the Respiratory Symptoms scale after 6 months of treatment [32]. Clinicians, patients, and other stakeholders will benefit from understanding the impact of ivacaftor treatment on patients with selected CF mutations using a patient-centered point-of-view. We have presented direct evidence of therapy effects, extending the interpretation of treatment benefit beyond the previously reported clinical markers. These results complement the clinical results, providing evidence of how patients feel and function in daily life. Our findings suggest that ivacaftor treatment led to significant improvements that were substantial, sustained over 48 weeks, and spanned a wide range of symptoms, functioning, and well-being in patients with the G551D-CFTR mutation in the STRIVE study. Results from the STRIVE study illustrate broad benefits of ivacaftor treatment across highly salient aspects of HRQoL: respiratory symptoms, physical and social functioning, health perceptions, and vitality as measured by the CFQ-R in patients with CF 12 years of age and older with the G551D-CFTR mutation. The breadth of improvements reflects the systemic mechanism of action of ivacaftor compared to other (symptomatic) therapies. Our results are supportive of the patient-reported value of ivacaftor treatment in this patient population. Limitations: The analyses reported here are post-hoc in nature (not pre-specified). Although an MCID has been established for the Respiratory Symptoms scale [12], no such benchmarks for interpretation of change exist for the remaining CFQ-R scales. Further, we used distribution-based methods to identify thresholds of change [21] because use of an anchor-based method was not possible using these data. The STRIVE study included only patients with the G551D-CFTR mutation. As noted, our findings are based on results of a clinical trial and the magnitude of treatment effects of ivacaftor on patient-reported outcomes may differ in actual clinical practice.

TITLE: Economic evaluation of an experience sampling method intervention in depression compared with treatment as usual using data from a randomized controlled trial ABSTRACT.BACKGROUND: Experience sampling, a method for real-time self-monitoring of affective experiences, holds opportunities for person-tailored treatment. By focussing on dynamic patterns of positive affect, experience sampling method interventions (ESM-I) accommodate strategies to enhance personalized treatment of depression―at potentially low-costs. This study aimed to investigate the cost-effectiveness of an experience sampling method intervention in patients with depression, from a societal perspective. ABSTRACT.METHODS: Participants were recruited between January 2010 and February 2012 from out-patient mental health care facilities in or near the Dutch cities of Eindhoven and Maastricht, and through local advertisements. Out-patients diagnosed with major depression (n = 101) receiving pharmacotherapy were randomized into: (i) ESM-I consisting of six weeks of ESM combined with weekly feedback regarding the individual’s positive affective experiences, (ii) six weeks of ESM without feedback, or (iii) treatment as usual only. Alongside this randomised controlled trial, an economic evaluation was conducted consisting of a cost-effectiveness and a cost-utility analysis, using Hamilton Depression Rating Scale (HDRS) and quality adjusted life years (QALYs) as outcome, with willingness-to-pay threshold for a QALY set at €50,000 (based on Dutch guidelines for moderate severe to severe illnesses). ABSTRACT.RESULTS: The economic evaluation showed that ESM-I is an optimal strategy only when willingness to pay is around €3000 per unit HDRS and around €40,500 per QALY. ESM-I was the least favourable treatment when willingness to pay was lower than €30,000 per QALY. However, at the €50,000 willingness-to-pay threshold, ESM-I was, with a 46% probability, the most favourable treatment (base-case analysis). Sensitivity analyses confirmed the robustness of these results. ABSTRACT.CONCLUSIONS: We may tentatively conclude that ESM-I is a cost-effective add-on intervention to pharmacotherapy in outpatients with major depression. ABSTRACT.TRIAL REGISTRATION: Netherlands Trial register, NTR1974. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12888-017-1577-7) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: Depression consistently ranks high worldwide in terms of disability [1–3] and societal costs due to health care consumption and productivity loss [4]. In the Netherlands, twelve-month prevalence of a depressive disorder is 5.2% [5], health care costs are estimated at 1592 million euros (1.8% of the total health care costs in 2011) [6], and disability days are eight times higher compared with the general population [7]. Because of the high disease burden of depression [1–5], non-pharmacological interventions that can enhance (psychopharmacological) treatment effects have the potential to be cost-effective. Although clear evidence exists for the effectiveness of combined pharmacotherapy with psychotherapy in the treatment of depression [8], face-to-face psychological treatment is cost-intensive and may, unfortunately, not be routinely available. Furthermore, it is estimated that optimal use of cognitive-behavioural therapy, counselling, and medication would lower the disease burden of depression by 35% at most [9]. Thus, efforts to improve the efficacy of pharmacotherapy combined with psychotherapy are considered a priority. Moment-to-moment ambulatory monitoring tools ―designed to collect real-life data with easy and immediate availability to both patients and professional caregivers― pave the road for potential low-cost strategies to improve and personalize mental health care. In particular, digitalized experience sampling method (ESM) tools incorporating repeated in-the-moment assessments of affective experience and context seem to be an acceptable and feasible strategy to provide unique person-tailored insights about affective patterns in daily life [10–12]. Interventions using the Experience Sampling Method (Experience Sampling Method-Interventions or ESM-I) may, therefore, provide possibilities for mobile health (mHealth) interventions in depression [13]. These interventions could be directed at increasing positive affect, as ESM studies have shown that a high ability to experience positive affect may predict development, course, and recovery of depression [14–16]. A first effect study showed that ESM-I as add-on intervention to psychopharmacological treatment, with feedback focussed on positive affect, was efficacious in reducing symptoms in patients with depression [17]. Although some evidence exists that ambulatory self-assessments may be cost-effective tools to manage health conditions [18, 19], to our knowledge, no randomized controlled trials have investigated the cost-effectiveness or cost-utility of any ESM-interventions in patients with depression. The present paper presents a trial-based economic evaluation using data from a randomised controlled trial. The purpose is to evaluate the cost-effectiveness and cost-utility of ESM-I as add-on intervention to psychopharmacological treatment as usual, from a societal perspective. Because the hypothesis was that ESM-derived feedback on daily life patterns is an essential ingredient, ESM-I was compared with two control conditions: (1) ESM self-monitoring without feedback, hereafter pseudo-intervention; and (2) treatment as usual (hereafter control group). BODY.METHOD: BODY.SETTING: For the current randomized controlled trial [17], participants were recruited between January 2010 and February 2012 from out-patient mental health care facilities in or near the Dutch cities of Eindhoven and Maastricht, and through local advertisements. Patients were considered eligible if they were aged between 18 and 65 years; diagnosed with major depression according to DSM-IV [20] with current or residual symptoms (score of >7 on the 17-item Hamilton Depression Rating Scale (HDRS) [21]); and treated with antidepressants or mood stabilizers. Patients were excluded if they met criteria for a non-affective psychotic disorder according to DSM-IV or if they met criteria for a manic, hypo-manic or mixed episode within the past month. The study was approved by an institutional review board (Medical Ethics Committee of Maastricht University Medical Centre); all participants provided written informed consent before enrolment. The trial was registered in the Netherlands Trial Register (ID: NTR1974). The study was performed according to the declaration of Helsinki. The original protocol and a CONSORT checklist are provided (see Additional files 1 and 2). BODY.TREATMENT ARMS: A randomized controlled trial was conducted with three treatment arms [17, 22]. All participants were asked to complete a five-day ESM baseline assessment. After baseline, patients were randomly allocated to the ESM-I, pseudo-intervention, or control group. Randomization (allocation ratio 1:1:1) was stratified for duration of pharmacological treatment (use of a particular antidepressant for shorter vs. longer than 8 weeks prior to study entry) and psychotherapy (yes/no). After all baseline assessments were performed, allocation took place using opaque, sealed, sequentially numbered envelopes (prepared by an independent research coordinator) with a number sequence produced by an electronic random sequence generator (http://www.random.org), in blocks of six. Envelopes were opened by the researcher (CS, PH, IK, CML, JH) or a research assistant. Allocation was not blinded. The ESM-I group participated in an ESM procedure (three days per week over a six-week period; see below), as addition to treatment as usual. This group received weekly standardised feedback on personalized patterns of positive affect. The pseudo-intervention group participated in the same ESM procedure but received no feedback. The control group received no additional intervention (treatment as usual). BODY.EXPERIENCE SAMPLING METHOD: ESM was carried out in accordance with previous studies [11, 23–25]: participants received a dedicated electronic ESM device (‘PsyMate’, [26]) which emitted a signal at a random moment in each of ten 90-min time blocks between 07:30 am–10:30 pm, prompting participants to fill in self-assessments including current positive and negative affect, activities, and context (7-point Likert scale ratings and forced-choice questions). BODY.INTERVENTION: For 6 consecutive weeks, ESM-I participants engaged in ESM self-monitoring for three consecutive days within each week. Each ESM week was followed by a face-to-face feedback session with one of the researchers (a psychologist or psychiatrist, n = 5). These six sessions were held at the participating mental health institutions or at Maastricht University. In these sessions, the researcher provided the participant verbal, graphical, and written feedback using the participant’s ESM data, delivered according to a fixed format, in a fixed order. Feedback showed actual levels of positive affect in the context of daily life activities, events, and social situations. In addition, changes in positive affect level and depressive feelings over the course of the ESM intervention were visualized (see [17] for examples). A bullet-point summary report of the feedback was also given to both the participant and his/her mental health professional using a fixed template. The procedure in the pseudo-intervention group was identical to the procedure in the ESM-I group except that no feedback was given. In the pseudo-intervention group, sessions were filled with an alternative activity (an HDRS interview) to keep duration of contacts equivalent to the ESM-I group. BODY.TREATMENT AS USUAL: Treatment as usual consisted of psychopharmacological treatment as usual, either in primary or ambulatory specialized care, that is, patients were treated with antidepressants or mood stabilizers, either as stand-alone treatment (e.g., with supportive counselling) or in combination with psychotherapeutic treatment. BODY.PROCEDURE: Full screening occurred two weeks before randomization to treatment. Severity of symptoms was assessed to determine eligibility. In addition, self-report instruments were completed assessing costs and quality adjusted life years (QALYs). One week later, the ESM procedure was explained and all participants engaged in a five-day ESM procedure after which baseline assessment took place. At baseline, symptoms were assessed and participants were subsequently randomized to treatment. Assessment of costs and QALYs was not repeated at baseline, thus full screening was used as a proxy for baseline. The 6-week intervention period (week 1–6) was followed by an immediate post-assessment, including symptom assessments and a five-day ESM post-assessment (week 7), and a first follow-up (week 8). Other follow-up assessments were conducted at 4 (week 12), 8 (week 16), 12 weeks (week 20), and 24 weeks (week 32) after this first follow-up assessment. At follow-up, symptoms (all follow-up assessments), costs, and QALYs (follow-up at week 20 and 32) were assessed. BODY.OUTCOME MEASURES: In the cost-effectiveness analyses, the Hamilton Depression Rating Scale-17 (HDRS) [21] was used as the primary outcome measure. The HDRS is a semi-structured interview measuring the severity of depressive symptoms over the past week. A higher HDRS score indicated higher levels of depression. In the bootstrapping models (see Statistical analysis paragraph), the HDRS was reversed (higher score is better outcome, as is obligatory in economic evaluations). Symptomatic remission was obtained using the HDRS; participants with a HDRS score ≤ 7 were considered to be in symptomatic remission [27]. In the cost-utility analyses, QALYs were used as the primary outcome. QALYs were generated for each participant, based on health states. These health states were obtained using the EuroQol-5D-3 L (EQ-5D; [28]), a generic, self-report instrument. At the start of the trial, this was the most recent version of the EQ-5D. Utilities for each possible health state were available from a UK general population survey, which is the international standard to valuate the EQ-5D [29, 30]. Those utilities scores were used as weights to obtain quality adjusted life years (QALYs) [31]. BODY.ECONOMIC EVALUATION: BODY.STUDY PERSPECTIVE AND TIME HORIZON: The economic evaluation was performed from a societal perspective, including intervention costs, health care costs, as well as productivity losses. The time horizon (i.e. the period of time evaluated in the analyses [32]) for this study was 32 weeks, equalling the full assessment period (eight weeks until end of intervention plus 24 weeks follow-up). As the time horizon was <1 year, no discounting of costs and effects was necessary (future costs and benefits were not valued to the present). All costs were presented in Euro’s and calculated to their 2012 value using price index figures from Statistics Netherlands [33]. The most recent cost prices that were available in 2012 (the year the trial ended) were used to calculate costs. BODY.COSTS MEASURES AND VALUATION: In the cost-assessment, we a priori identified health care costs (See Additional file 3: Tables S1 and S2 for details), absence from work (absenteeism), and productivity loss at work (presenteeism) as relevant. Information on costs was monitored with two self-report instruments assessing health care consumption, absence from work, and productivity loss at work in the past three months. Health care consumption and medication use were assessed using the Trimbos/Institute for Medical Technology Assessment questionnaire for Costs associated with Psychiatric Illness (TiC-P; [34]); the Productivity and Disease Questionnaire (PRODISQ; [35, 36]) was used to measure costs of absence from work and cost of productivity loss at work. The valuation of health care costs was based on the updated Dutch Manual for Cost Analysis in Health Care Research [37]. This manual contains methods and standard cost prices for economic research in health care. The costs of medication were based on the Dutch medication prices [38]. Costs of absenteeism were calculated using the human capital method by multiplying the number of days absent with an estimation of the productivity costs per hour of each participant (obtained from age and gender specific productivity costs also including an elasticity factor of 0.8; elasticity to account for the proportional reduction in productivity resulting from absence from work) ([37], Table 6.1). Productivity loss (presenteeism) was calculated using the QQ method, that is 100% − [quantity of work] × [quality of work] [39, 40]. This percentage was multiplied by the number of working hours in 3 months (385 h [37]), because the PRODISQ was assessed every three months (or data were imputed; see Statistical analysis). BODY.INTERVENTION COSTS: Cost calculations of the intervention were based on a psychologist salary (to translate the researchers time delivering the intervention to the salary of a health care professional), which was €173 per hour in 2009 [37], calculated to the 2012 value of €183.73. ESM-I and pseudo-intervention group participants completed on average 5.3 (SD = 1.7) and 5.7 (SD = 0.94) intervention sessions, respectively. Mean duration of these sessions was 48.9 and 39.5 min. Thus, total time spent per patient was 4.3 h (€792.62) for ESM-I, and 3.7 h (€689.45) for the pseudo-intervention. BODY.WILLINGNESS-TO-PAY THRESHOLD: In the Netherlands, proportional upper limits exist for various levels of severity: €20,000 (mild condition), €50,000 (moderate severe condition), €80,000 (severe condition) [41]. It is unclear which of these limits would be most appropriate to set as a maximum willingness to pay for the QALY for patients with major depression. In most previous economic evaluations, the willingness-to-pay threshold for depression was set above this threshold for mild conditions toward the threshold for moderate severe conditions (e.g., [42–44]). Therefore, we set the willingness-to-pay threshold for ESM-I in major depression at €50,000 ($59,115). BODY.STATISTICAL ANALYSIS: Power calculations using the STATA SAMPSI command were based on previous work [45], and led to an initial sample size of 120 with a power of 84% to detect a 3-point difference in the score on the 17-item HDRS [46, 47], the primary effectiveness outcome [17]. However, because many participants were excluded, the inclusion rate was lower than expected. The eventual number of patients who participated in the trial was 102. The economic evaluation consisted of a base-case cost-effectiveness and cost-utility analysis, and sensitivity analyses. For the main analysis (base-case), intention-to-treat (ITT) data were used. All analyses in step 1–4 were performed using Stata version 13 [48], the economic evaluation in step 5 was performed using Microsoft Excel (version 2010). First, missing observations at the 32-weeks assessment were replaced by Last Observation Carried Forward; missing observations at the 20-weeks assessment were replaced by Next Observation Carried Backward. Second, costs and QALYs of week 1–8 were estimated by individual mean imputation of the baseline and the week 20 assessment (corrected for length of the period by multiplying by 0.667) to obtain information of the full 32-week period [47]. Subsequently, a total cost variable was generated, being the sum of all above-mentioned costs in the full 32 weeks (base-case societal perspective). Similarly, QALYs were summed for the full 32-week period. Third, costs and QALYs over 32-weeks and HDRS at 32 weeks were analysed using linear regression analysis to provide background information to the economic evaluation results. These regression models included treatment arm as well as baseline values of the dependent variable (costs, QALYs, and HDRS scores, respectively) as independent variables and included all assessments of all participants (intention-to-treat). When costs were the dependent variable, assumptions for linear regression were not met and p49], i.e., baseline costs were subtracted from total costs post-assessment. Coefficients obtained from a regression analysis corrected for baseline costs could not be used in the present data, because the data failed to meet the assumption of a normal distribution of the cost residuals, even after applying methods to address outliers [49] (see also Methodological considerations). HDRS scores and QALYs were exported to Excel without further transformations. For the fifth step, cost and effect pairs per participant were imported in a previously designed Excel file. Because residuals in the analysis were not normally distributed, non-parametric bootstrap resampling techniques were used to explore sample uncertainty around estimates of the cost-utility and cost-effectiveness analysis, using the original data of the three treatment groups. Using this Excel file, 5000 replications were generated. We calculated incremental cost-effectiveness ratios (ICER) by dividing the incremental costs by the incremental effects (HDRS scores); by dividing the incremental costs by the differences in QALYs, we calculated incremental cost-utility ratios (ICUR). Using the 5000 replications, cost-effectiveness acceptability curves (CEAC) were generated [31, 50]. For QALYs, an a priori willingness-to-pay threshold of €50,000 was defined (see above). BODY.SENSITIVITY ANALYSES: Several one-way sensitivity analyses for deterministic variables were performed to assess how sensitive results are to different input values in a priori selected parameters. First, QALYs were based on the Dutch tariff [51] rather than the UK tariff [29]. Second, costs for a standard GP-contact (€29.73 in 2012) were replaced by costs of a psychiatric GP contact (€60.53) as obtained from the cost manual [37]. Third, the economic evaluation was performed from a health care perspective, rather than a societal perspective. Fourth, complete cases were analysed (as opposed to intention-to-treat). Finally, analyses were performed when NOT adjusting for baseline costs. BODY.RESULTS: BODY.PARTICIPANTS AND BASELINE CHARACTERISTICS: A total number of 102 participants were randomized to one of the three treatment arms (see participant flow in Fig. 1). The intention-to-treat sample consisted of 101 participants (n = 33 ESM-I, n = 35 pseudo-intervention, n = 33 control group), given that one participant randomized to the pseudo-intervention did not fill in any of the assessments needed for the present analyses, not at baseline, nor at follow-up. At the 20-week and 32-week follow-up assessments, 86 (85%) and 80 (79%) participants responded, respectively. Table 1 presents baseline characteristics (see [17] for more details). At baseline, mean HDRS score was 2 units lower in the ESM-I group than in the control group, and total costs (societal perspective over 3 months) were about €450 lower (Table 1). Control group participants more often had a bipolar disorder, more often had a recent switch in their antidepressant medication, and less often received psychotherapy compared with the other two groups; pseudo-intervention group participants more often had a comorbid axis I disorder and were more often treated in primary health care (Table 1).Fig. 1Flow diagram of the study. ESM = Experience Sampling Method; TAU = treatment as usual HDRS – 17-item Hamilton Depression Rating Scale; EQ-5D – EuroQol-5D-3 L; TiC-P – Trimbos/IMTA questionnaire for Costs associated with Psychiatric Illness; PRODISQ – The Productivity and Disease Questionnaire Table 1Baseline characteristics ESM-I (n = 33)Pseudo-intervention (n = 35)Control group (n = 33)Gender, male: n (%) 17 (51.5) 14 (40.0) 15 (45.5) Age, years: mean (SD) 49 (10.2) 47 (9.7) 49 (10.9) Education  low (no/primary/low secondary) 6 (18.1) 9 (25.7) 10 (30.3)  medium (high school/low vocational) 12 (36.4) 14 (40.0) 12 (36.3)  high (higher vocational/university) 15 (45.5) 12 (34.3) 11 (33.3) Depressive symptoms (HDRS): mean (SD) 13.5 (5.6) 15.1 (6.9) 15.5 (5.4)  range 2–30 2–30 5–27 Treated in primary care 7 (21) 6 (17) 8 (24) Bipolar disorder 2 (6) 2 (6) 5 (15) DSM-IV axis I comorbidity 12 (36) 16 (46) 12 (36) Psychotherapy yes/no 4 (15) 4 (13) 2 (7) Use of antidepressant medicationa  New 2 (6) 3 (9) 1 (3)  Switch 3 (9) 3 (9) 7 (21)  Maintenance 28 (85) 29 (83) 25 (76) QALYs, last 12 weeks, mean (SD)  EQ-5D, UK tariff (Dolan) (range − 0.04; 0.23) 0.15 (0.07) 0.14 (0.06) 0.12 (0.07) Health care costs (last 12 weeks), mean (SD)  Health care use €2230 (€7186) €3150 (€4369) €1999 (€4348)  Medication €42 (€145) €18 (€59) €32 (€79)  Total €2273 (€7179) €3168 (€4363) €2032 (€4342) Societal (last 12 weeks), mean (SD)  Absence from work €2541 (€5983) €2154 (€4852) €2785 (€5066)  Productivity loss at work €2156 (€3910) €2135 (€3486) €2637 (€4224)  Total (incl. health care costs) €6828 (€12817) €7458 (€10201) €7291 (€9360) HDRS 17-item Hamilton Depression Rating Scale, QALY quality adjusted life year, EQ-5D EuroQol-5D-3L aNew and switch are defined as shorter than 8 weeks on this medication; the rest is maintenance BODY.COSTS AT THE 32-WEEKS ASSESSMENT: Intention-to-treat analysis showed that total costs (over the total 32 weeks; societal perspective) were higher in the ESM-I group (€17,957) than in the control group (€16,216) and the pseudo-intervention group (€16,816; Table 2). However, these differences were not statistically significant after adjustment for baseline costs (Table 2, last two columns; Additional file 3: Table S1). There were also no statistically significant differences between ESM-I and the control groups in any of the cost categories separately (Table 2).Table 2Costs over 32 weeks (intention-to-treat) ESM-I (n = 33)Pseudo-intervention (n = 35)Control (n = 33)Regression coefficients; B (p-value obtained from permutation analyses)M (SD)M (SD)M (SD)ESM-I vs ControlESM-I vs Pseudo-interventionHealth care use (total)a €6751 (€19420) €6510 (€7315) €6520 (€14082) -€165 (p = 0.96) €1818 (p = 0.66) Medication €104 (€296) €25 (€56) €75 (€171) €12 (p = 0.82) €36 (p = 0.54) Total health costsb €7648 (€19402) €7225 (€7304) €6596 (€14072) €640 (p = 0.80) €1957 (p = 0.61) Societal  Absence from work €6067 (€13691) €4732 (€9372) €5379 (€11889) €945 (p = 0.74) €857 (p = 0.75)  Productivity loss at work €4234 (€7721) €4858 (€8438) €4241 (€7627) €605 (p = 0.76) -€594 (p = 0.80)  Total €17957 (€31329) €16816 (€17595) €16216 (€27756) €2483 (p = 0.74) €2152 (p = 0.75) aSeveral types of health care consumption see Additional file 3: Table S1 bIncluding intervention costs in ESM-I and pseudo-intervention group BODY.HDRS AND QALYS AT THE 32-WEEKS ASSESSMENT: At 32 weeks, mean HDRS score was three units lower in the ESM-I group than in the control group (after adjustment for baseline HDRS scores), which was statistically imprecise by conventional alpha (Table 3; B = −3.1, p = 0.051, intention-to-treat analysis). The ESM-I and the pseudo-intervention group did not differ (B = −1.13, p = 0.47). There was no evidence that ESM-I participants were more often in symptomatic remission compared with control group participants (OR = 2.65, p = 0.12); ESM-I participants did not differ in the rate of symptomatic remission compared with the pseudo-intervention participants (OR = 1.84, p = 0.29).Table 3Outcomes (intention-to-treat) ESM-I (n = 33)Pseudo-intervention (n = 35)Control (n = 33)Regression coefficients; B (95% confidence interval)a ESM-I vs ControlESM-I vs Pseudo-interventionHDRS (at 32 weeks)  HDRS scorea 10.8 (7.1) 13 (7.12) 15.3 (8.3) −3.1 (−6.2; 0.01) −1.13 (−4.18; 1.92)  Improvement since baseline 2.7 (6.1) 2.1 (5.9) 0.24 (7.5) 3.1 (−0.01; 6.2) 1.13 (−1.92; 4.18)  Symptomatic remissionb 11 (33.3) 9 (25.7) 6 (18.2) OR = 2.65 (0.79; 8.9) OR = 1.84 (0.59; 5.7) QALYs (over 32 weeks)  EQ-5D, UK tariff 0.45 (0.17) 0.38 (0.18) 0.32 (0.18) 0.08 (0.02; 0.10)* 0.04 (−0.02; 0.10) HDRS 17-item Hamilton Depression Rating Scale, QALY quality adjusted life year, EQ-5D EuroQol-5D-3L *p < 0.05 aControlled for baseline values bSymptomatic remission = HDRS score ≤ 7 QALYs were higher in the ESM-I group than in the control group (B = 0.08, p = 0.01, Table 3), but the difference between ESM-I and pseudo-intervention group was not statistically significant (B = 0.04, p = 0.15). BODY.COST-EFFECTIVENESS AND COST-UTILITY ANALYSIS (TIME HORIZON 32 WEEKS): In the cost-effectiveness analysis (outcome: HDRS), ESM-I had the highest probability of being cost-effective compared with treatment as usual and pseudo-intervention when willingness to pay was over €4000 ranging from a probability of 10 to 86% (when willingness to pay is €0 and €37,500 respectively; Fig. 2). Note that the treatment with the highest probability of cost-effectiveness is the upper line in the figure at each level of willingness to pay.Fig. 2Cost-effectiveness acceptability curve of the base-case analysis, assessing the Hamilton Depression Rating Scale. The analysis was controlled for baseline costs. The lines per treatment indicate the probability (y-axis), i.e., the proportion of replications this treatment has the highest net monetary benefit given various levels of willingness to pay (x-axis). At those willingness-to-pay levels where ESM-I has the higher probability compared with the other two treatments (summing up to 100%), ESM-I is the most cost-effective option. TAU = treatment as usual (control group); PSEUDO = pseudo-intervention group; ESM-I = ESM-intervention group In the cost-utility analysis (outcome: QALYs), the CEAC curve showed that ESM-I had the highest probability of being the most optimal of the three treatments when willingness to pay was over €40,500 (Fig. 3). At the a priori willingness-to-pay threshold of €50,000, ESM-I was the intervention with the highest probability of being cost effective (ESM-I 46%, pseudo-intervention 34%, treatment as usual 20%).Fig. 3Cost-effectiveness acceptability curve of the base-case analysis, assessing the EQ-5D. The analysis was controlled for baseline costs. The analysis was controlled for baseline costs with the bootstrapped societal costs and EQ-5D-based QALYs. The lines per treatment indicate the probability (y-axis), i.e., the proportion of replications this treatment has the highest net monetary benefit, given various levels of willingness to pay (x-axis). At those willingness-to-pay levels where ESM-I has the higher probability compared with the other two treatments (summing up to 100%), ESM-I is the most cost-effective option. At the willingness-to-pay threshold of €50,000, the probability is 46% for ESM-I, which is higher compared with TAU (20%) and Pseudo (34%). TAU = treatment as usual (control group); PSEUDO = pseudo-intervention group; ESM-I = ESM-intervention group BODY.SENSITIVITY ANALYSES: Table 4 presents both the base-case and sensitivity cost-effectiveness and cost-utility results. When willingness to pay levels were higher than between €30,000 and €40,000, ESM-I was the most optimal treatment (Table 4, Fig. 2, Additional file 4: Figure S1, Additional file 5: Figure S2, Additional file 6: Figure S3 and Additional file 7: Figure S4) in the cost effectiveness analyses (HDRS). The sensitivity analysis from the health care perspective and the complete cases analysis were more optimistic than the base-case analysis, being most cost effective from €3000 and €3750, respectively.Table 4Results of base-case and sensitivity analyses of the cost-utility and cost-effectiveness analyses: willingness to pay when compared with both control conditions Level of willingness to pay when ESM-I is more cost effective thanPercentage at WtP thresholda treatment as usualpseudo-interventionHDRS € €  Base-case analysis 3000 4000  Different GP cost calculation 3000 4000  Health care perspective 1500 3750  Complete cases 2250 3000  NOT controlling for baseline costs 1250 0b QALY € € %  Base-case analysis 31, 500 40, 500 46%  Dutch instead of UK tariff 32, 500 43, 000 44%  Different GP cost calculation 30, 500 39, 500 45%  Health care perspective 16, 500 36, 000 64%  Complete cases 23, 500 30, 500 65%  NOT controlling for baseline costs 11, 500 0b 58% aThe probability that ESM-I is most cost-effective at the willingness-to-pay threshold of €50,000 bA value of €0 indicates that ESM-I is dominant compared with the other intervention, i.e., ESM-I shows more improvement in outcomes at lower costs compared with the other treatment At the willingness-to-pay threshold of €50,000, the probability that ESM-I is most cost-effective was between 44 and 65% (cost utility analysis, Table 4, Figs. 3 and 4 and Additional file 8: Figure S5, Additional file 9: Figure S6, Additional file 10: Figure S7 and Additional file 11: Figure S8). Again, the sensitivity analysis from the health care perspective and the complete cases analysis were most optimistic with percentages of 64 and 65% at the willingness-to-pay threshold of €50,000.Fig. 4Cost-effectiveness acceptability curve of a sensitivity analysis assessing the EQ-5D: unadjusted for baseline costs BODY.DISCUSSION: BODY.KEY FINDINGS: The present study provides, to our knowledge, the first economic evaluation of an intervention using ESM in patients with major depression. The results suggest that ESM-I is more expensive, but also more clinically effective than both treatment as usual and pseudo-intervention. In the cost-effectiveness analysis and cost-utility analysis, ESM-I was the most optimal strategy when willingness to pay was over €3000 and €40,500, respectively. All sensitivity analyses except one were similar to the base-case analysis. That one exception, that is the analysis unadjusted for baseline costs, had lower willingness to pay, and a probability of cost-effectiveness at €50,000 of 58%. In addition, CEAC showed that ESM-I cost-effectiveness probability increased rapidly towards the most favourable treatment. Furthermore, although costs are below the threshold set for a QALY (€50,000), such a threshold could not be defined for the HDRS. Therefore, we can only tentatively conclude that ESM-I is cost-effective. BODY.COST-EFFECTIVENESS OF ESM-I IN REAL LIFE MAJOR DEPRESSION TREATMENT: The present trial shows that ESM-I consisting of protocolled feedback delivered by a researcher has the potential to be cost-effective. When implementing ESM-I in real life treatment, feedback can be delivered directly to the patient and professional caregiver. Feasibility and cost-effectiveness are hypothesized to increase when the option of feedback provided by a third person (the researcher) is replaced with ESM-I feedback that forms an integral part of the treatment. ESM-I could then also be used to enrich psychological treatments such as cognitive behaviour therapy [52] with daily life contextual information and to bring that therapy out of the mental health care setting into daily life. Our six-week ESM intervention has been shown feasible in outpatients with major depression [17], but the feasibility of implementation in routine clinical practise is not yet established [13]. Web-based feedback systems for ESM-I applications are under development. If such a web-based system allows individuals to navigate through their own feedback, this may facilitate implementation of the current ESM intervention by promoting easy access to and flexible use of feedback for patients as well as professional caregivers. This should be backed up by appropriate resources for professional caregivers including training, monitoring, and technical support [53]. In addition, withdrawal of the professional caregiver and patient disengagement may be an important issue, requiring research to improve sustained use [54]. BODY.EFFECTS OF ESM-I ON DEPRESSIVE SYMPTOMS: The ESM-I group showed lower HDRS scores at 32-weeks than the two control groups, suggesting that ESM-I reduced depressive symptoms. However, although the economic evaluation showed that ESM-I may be cost-effective, in the accompanying regression analyses (HDRS and QALYs; Table 3), the difference between the ESM-I and the pseudo-intervention group was not statistically significant while the difference between ESM-I and control group was statistically imprecise by conventional alpha. The effect study, accompanying the present economic evaluation [17], did show that allocation to ESM-I was associated with a statistically significant linear decrease in HDRS depressive symptoms over time that lasted throughout the study. This decrease was significantly stronger than in the control group to a degree that can be considered clinically relevant (difference > 3 HDRS units; [46, 47]). The difference with the pseudo-experimental group was clinically relevant and borderline significant [17]. For the regression analysis results accompanying the cost effectiveness results in the present paper, less data were used than in the original analyses which included all follow-up assessments. In addition, the original paper analysed subjects as randomized with available data while the present paper imputed data (using last observation carried forward). BODY.COST-EFFECTIVENESS AND SEVERITY OF DEPRESSION: The study sample consisted of patients with a major depression with current symptoms in the mild to severe range, including residual depressive states. Given that meta-analytic evidence suggests that the efficacy of psychotherapeutic interventions may be larger in patients with higher levels of pre-treatment depressive symptoms [55], a subgroup analysis only including patients with severe or very severe depressive symptoms was warranted. However, in the present data, the number of patients in subgroups (e.g. only 20 patients with HDRS ≥ 19) was too low to obtain valid results. Future economic evaluations of ESM-I should include sufficient numbers of patients at each level of severity to enable subgroup analysis in patients with mild/moderate and with severe/very severe symptoms separately. BODY.METHODOLOGICAL CONSIDERATIONS: The present study was limited to patients aged between 18 and 65 years (mean age 48 years) and more than 90% of the sample was from Dutch origin. ESM-I is designed to obtain insights in everyday life and, therefore, we recruited outpatients that could engage in ESM self-monitoring in their home environment. Outpatients were included in the study if they scored above remission level (HDRS > 7) at study entrance. This mild inclusion criterion, coupled with the time intensive nature of the study protocol (multiple visits to the researcher on top of an intensive intervention consisting of 6 weeks of self-monitoring), may have led to recruiting mainly participants in a mild to moderate depressive state. However, this may be a rather accurate representation of the population of patients with major depression, of which the majority experiences mild to moderate symptoms, and using higher HDRS cut-offs would compromise the external validity of the trial [56, 57]. On the other hand, our sample was mostly recruited from specialised mental health care settings (approximately 20% was treated in primary care only), and had a diagnosis of major depression as well as current symptoms for which they were using antidepressants. Although the results may not be generalizable to all outpatients with major depression, they may be generalizable to outpatients with complex mental problems who are using antidepressants. The present paper has several limitations. First, owing to the nature of the intervention, it was not possible to blind participants and the use of envelopes could potentially have led to biased allocation. However, given that care-providers were not involved in the randomization process and most envelopes were drawn from a distance, with one researcher drawing an envelope for another researcher, it is unlikely that subversions to the procedure took place. Researchers conducting the post-intervention assessments were also not blind to treatment allocation due to resource constraints. Thus findings may reflect a placebo response. However, the effect study [17] showed that directly after the six-week intervention, the decrease in HDRS ratings was similar in the ESM-I group and the pseudo-intervention group, while in the pseudo-intervention group effects did not appear to persist during the full 32-weeks of the trial. It is often assumed that placebo effects in depression do not persist in the long run [58]. Although, this belief has been falsified [58], the difference in persistence between the pseudo-intervention group and the ESM-I group may evidence that it is unlikely that our findings are completely attributable to a placebo effect. The improvement in the ESM-I group showed a persistent, steady and clinically relevant growth over time in the full 32 weeks, further making the possibility of a placebo effect even more unlikely. Second, all three treatment arms were embedded in an extensive research protocol, including regular assessment of depressive symptoms and two five-day ESM assessments. Besides treatment effects, patients may have had non-specific benefits from self-monitoring. Therefore, what has been called treatment as usual in the present paper, strictly is not. ESM-I may be even more cost-effective when compared with true treatment as usual. Third, we used the human capital method rather than the friction costs method to calculate work absence costs, because the PRODISQ absenteeism module only asked number of absent days during a period of 3 months, while friction period was longer at the time of data collection (approximately 5 months) [37]. Therefore, end of the friction period could not be identified. Fourth, sampling uncertainty was estimated using the non-parametric bootstrapping approach. Alternatively, another common approach for the handling of trial-based data would have been to estimate the mean total costs per treatment condition using a GLM that assumes a Gamma distribution for costs (i.e., to accommodate the skewness in the distribution of costs). This would also allow for the regression-based adjustment of cost estimates through the inclusion of possible covariates in the GLM. It could therefore be considered a limitation that non-adjusted costs were reported. 49] resulted in non-normal distribution of residuals, even after transformation to the natural logarithm. Several methods to deal with the problem of outliers have been advocated [49]. However, removing various percentages (2, 5, 10, 20, or 30%) of observations at the extremes, resulted in non-normally distributed residuals and in inconsistent regression coefficients of baseline costs (B = 0.84, 0.82, 0.79, 0.72, 0.66, respectively; base-case analyses: B = 0.86). Therefore, the best option to correct for baseline costs [49] was impossible in the present data, and it is most prudent to perform the delta method to control for baseline costs rather than regression-based adjustment [49] (see also Methods). Furthermore, we chose for easy methods to deal with missing data because the number of missings was limited. The proportion of missing values was not significantly associated with treatment allocation, nor with baseline and previous observed depression scores or baseline demographics. Last observation carried forward, next observation carried backward, and mean imputation have been shown to perform as good as multiple imputation [59]. Finally, of all parameters that we varied in the sensitivity analyses, correction for baseline costs was the only factor that changed the willingness to pay, but probability of cost-effectiveness at the a priori threshold of €50,000 remained similar to the base-case analysis. Correction for baseline costs is relatively new in economic evaluations, in contrast to epidemiology and statistics, were controlling for baseline differences is standard practise to get valid results [60]. The present results show that the impact of controlling for baseline may be considerable and suggest that, as in other fields of research, results without baseline correction may be invalid. BODY.CONCLUSION: We may tentatively conclude that ESM-I is cost-effective in outpatients with major depression. Only tentatively because the probability that ESM-I was cost effective was only 44% at the predefined threshold of €50,000, while no threshold for the HDRS could be defined. Future studies are needed to replicate the present findings and to study patients with severe depressive symptoms separately. If future research replicates effectiveness and cost-effectiveness, we would recommend ESM-I as an addition to psychopharmacological treatment as usual. Integration of ESM-I in psychological treatment is also a possibility. BODY.ADDITIONAL FILES: Additional file 1:CONSORT Checklist. (DOC 217 kb) Additional file 2:Protocol. Original study protocol. (PDF 245 kb) Additional file 3: Table S1. Costs at baseline and costs over 32 weeks (intention-to-treat) per type of consultation. Table S2. Unit costs per cost category. Costs were obtained from a Dutch cost manual (2009-prices, Hakkaart van Roijen 2010) and calculated to their 2012 value (Statline). (DOCX 20 kb) Additional file 4: Figure S1.Cost-effectiveness acceptability curve assessing HDRS, sensitivity analysis: GP costs based on psychiatric tariff. (DOCX 93 kb) Additional file 5: Figure S2.Cost-effectiveness acceptability curve assessing HDRS, sensitivity analysis: health care perspective. (DOCX 94 kb) Additional file 6: Figure S3.Cost-effectiveness acceptability curve assessing HDRS, sensitivity analysis: completers only. (DOCX 93 kb) Additional file 7: Figure S4.Cost-effectiveness acceptability curve assessing HDRS, sensitivity analysis: unadjusted for baseline costs. (DOCX 95 kb) Additional file 8: Figure S5.Cost-effectiveness acceptability curve, sensitivity analysis, assessing EQ-5D: Dutch valuation of the EQ-5D. (DOCX 96 kb) Additional file 9: Figure S6.Cost-effectiveness acceptability curve, sensitivity analysis, assessing EQ-5D: GP costs based psychiatric tariff. (DOCX 92 kb) Additional file 10: Figure S7.Cost-effectiveness acceptability curve, sensitivity analysis, assessing EQ-5D: health care perspective. (DOCX 96 kb) Additional file 11: Figure S8.Cost-effectiveness acceptability curve, sensitivity analysis, assessing EQ-5D: completers only. (DOCX 95 kb)

TITLE: Sustained treatment effect in attention-deficit/hyperactivity disorder: focus on long-term placebo-controlled randomized maintenance withdrawal and open-label studies Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that often persists throughout life. Approximately two-thirds of patients with a childhood diagnosis of ADHD continue to experience clinically significant symptoms into adulthood. Nevertheless, most of these individuals consider themselves “well,” and a vast majority discontinue medication treatment during adolescence. As evidence concerning the adult presentation of ADHD becomes more widely accepted, increasing numbers of physicians and patients will face decisions about the benefits and risks of continuing ADHD treatment. The risks associated with psychostimulant pharmacotherapy, including abuse, dependence, and cardiovascular events, are well understood. Multiple clinical trials demonstrate the efficacy of psychostimulants in controlling ADHD symptoms in the short term. Recent investigations using randomized withdrawal designs now provide evidence of a clinically significant benefit with continued long-term ADHD pharmacotherapy and provide insight into the negative consequences associated with discontinuation. Because many patients lack insight regarding their ADHD symptoms and impairments, they may place a low value on maintaining treatment. Nevertheless, for patients who choose to discontinue treatment, physicians can remain a source of support and schedule follow-up appointments to reassess patient status. Medication discontinuation can be used as an opportunity to help patients recognize their most impairing symptoms, learn and implement behavioral strategies to cope with ADHD symptoms, and understand when additional supportive resources and the resumption of medication management may be necessary. BODY.INTRODUCTION: In the United States, an estimated 4.4% of adults meet diagnostic criteria for attention-deficit/hyperactivity disorder (ADHD).1 The prevalence of syndromic ADHD is estimated to be lower (between 2.8% and 3.3%) in elderly adults aged 60 years and older from countries in the European Union.2,3 Among adults who received a diagnosis of ADHD in childhood, approximately 65% continued to experience significant ADHD symptoms and functional impairments, although persistence estimates vary widely.4–6 Also, recent findings suggest that, in addition to those who continue to meet full diagnostic criteria for ADHD, a number of adults display persistence of a subset of ADHD symptoms. They continue to experience substantial functional deficits associated with those symptoms.7 The factors that determine ADHD persistence are under active investigation. Recent studies suggest that a childhood ADHD symptom profile, along with psychiatric comorbidity, may be predictive of ADHD persistence. This profile includes predominant inattentive symptoms; more severe symptoms during childhood; and the presence of psychiatric comorbidities, executive function deficits, and some maternal psychopathologies.5,8,9 The behavioral pathology of ADHD is associated with abnormalities in neuroanatomy and neurophysiology in both children and adults.10–15 ADHD appears to be a highly heritable disorder marked by the presence of familial neuropathologic patterns and specific genetic polymorphisms associated with ADHD pathology.16–22 Neuroimaging studies of individuals with ADHD have also identified delayed neurodevelopment, volumetric differences in specific brain areas, and differences in neural activation for tasks when compared to those without ADHD.23,24 Although some of these abnormalities may lessen or resolve as the brain matures, other central neurologic abnormalities persist in adulthood. The functional implications of persistent neurologic abnormalities have not been clearly defined. However, compared with normal controls, cognitive deficits in untreated adults with ADHD appear to persist throughout life.25 ADHD, across the lifespan, is marked by varying degrees of hyperactivity, impulsivity, and attentional symptoms across settings.14 Patients may also exhibit poor emotional control and motivational problems.15 Recommended first-line pharmacotherapy for ADHD in children and adults includes psychostimulants such as methylphenidate and amphetamines, as well as nonstimulant medications such as atomoxetine.6,26–28 The nonstimulants guanfacine extended-release and clonidine extended-release are US Food and Drug Administration-approved for the treatment of ADHD in children and adolescents.29 BODY.NEED FOR A LONG-TERM APPROACH TO ADULT ADHD MANAGEMENT: In the past, ADHD treatment was often routinely discontinued during adolescence; it was unclear whether older patients still exhibited clinically significant symptoms or functional impairments. Moreover, it was uncertain if older patients derived benefits from continued treatment. However, the medical community is now better aware of the changing clinical presentation of ADHD through life transitions and of the need for a longitudinal, developmental approach toward ADHD detection, reassessment, and management.30 During childhood, ADHD can be readily identified by marked overt physical hyperactivity and impulsivity, especially in boys, whereas inattention was often overlooked; in adulthood, such hyperactivity and impulsivity wane and may be internalized as restlessness or impatience, although inattentiveness and disorganization persist and may become the predominant impairing symptoms.31,32 Moreover, with time, ADHD symptoms and impairments take on a progressively more distinct adult presentation.32–35 Impulsivity may be evidenced by sexual promiscuity, financial problems, high job turnover, and/or a short temper; inattention may be evidenced by a high number of traffic citations/accidents, disorganization, chronic tardiness, difficulty finishing projects, forgetfulness, and/or procrastination. Hyperactivity in adults may be experienced as an internal feeling of restlessness or being on edge and expressed outwardly through fidgeting or an inability to sit for long periods of time. Many patients may fail to associate these behaviors with ADHD and may instead consider them to be character traits or part of their personality. Such patients may consider themselves “well” because their childhood symptoms (the reduction of hyperactivity) seem to have resolved; therefore, these patients often discontinue treatment during the transition from adolescence to young adulthood.36 Persisting symptoms in adults with ADHD, although less evident than those in childhood, are associated with relatively greater functional impairments.32 Older adolescents and adults who do not receive treatment for ADHD may suffer lasting consequences related to uncontrolled symptoms and impaired functioning (eg, low occupational/educational attainment, arrest, unintended pregnancy, sexually transmitted diseases, marital problems, and injury).37 Possibly associated with such functional impairments, the development of certain psychiatric comorbidities (ie, conduct disorders or depression) may also exacerbate poor ADHD symptom control.38,39 The socioeconomic and personal burdens of ADHD experienced by patients, their families, and the community may be mitigated through appropriate long-term treatment.40–42 In addition, in a published systematic review of outcomes, increased substance use disorders or suicidality, compared to untreated ADHD, were not seen with long-term treatment.42 Some evidence suggests that, aside from reductions in core ADHD symptoms, quality of life may be improved with pharmacotherapy.43 Although treatment may not “normalize” functioning to the level seen in non-ADHD individuals,42 large numbers of observational studies worldwide suggest that maintained ADHD treatment over time tends to have a significant beneficial impact on aspects of a patient’s life, including driving, obesity, self-esteem, social functioning, and academic performance when compared to individuals with untreated ADHD.42,44,45 Thus, it is reasonable to examine how the need to continue therapy can be assessed, weighing relative risks and benefits. These risks and benefits include a balance between the economic impact of long-term pharmacotherapy weighed against the potential for lower occupational attainment and wages, potential increased legal and insurance costs, and overall quality of life for patients and family members. BODY.INVESTIGATING TREATMENT MAINTENANCE AND LONG-TERM EFFICACY: Because ADHD persists into adulthood in about two-thirds of patients who have a childhood diagnosis and is associated with significant functional impairments, it would seem that long-term treatment maintenance is necessary and – as evidence shows in many cases – beneficial. In about one-third of these patients, persistent ADHD symptoms may not meet current diagnostic thresholds.5 As with any pharmacotherapy, medications indicated for ADHD are associated with certain risks. In the case of psychostimulants, these include abuse, misuse, addiction, diversion, cardiovascular safety risks such as elevated blood pressure and rare but life-threatening events/cardiac problems (ie, sudden death, myocardial infarction, and stroke); with long-term use, decreases in height and weight have also been a concern with pediatric patients.46–48 However, there is a lack of high-quality evidence from controlled trials in adults regarding the possible benefits of long-term treatment or the consequences of treatment discontinuation.49 Randomized, controlled clinical trials in subjects with ADHD have been conducted in children and have been short-term. Most long-term ADHD treatment trials have been observational and open-label in nature (Table 1).50–64 Open-label trials are valuable because they often approximate real-world clinical practice, marked by flexible dosing, to address changes in efficacy or treatment tolerability. In all available open-label trials with durations of 1 year or more, a high level of symptom control has been observed throughout the entire study period, in some cases extending up to 24 months (Table 1). Open-label evidence, however, does not rigorously demonstrate maintained treatment efficacy or the consequences of treatment discontinuation. There is a need to more thoroughly examine the value of long-term ADHD pharmacotherapy to support the more widespread use of a lifelong care management approach toward ADHD. Because there are well-established, effective treatments available for ADHD, it is considered unethical to randomly assign subjects to long-term placebo treatment. Adequate subject retention in long-term trials is also problematic. The randomized withdrawal study design is one research approach aimed at addressing these challenges. This design has been used to rigorously investigate the value of continued treatment in other lifelong illnesses, such as major depressive disorder65 and psoriasis.66 For withdrawal trials, subjects with a clinically significant response to active treatment in a prolonged lead-in phase are randomly assigned to double-blind treatment with a placebo or continued active treatment (Figure 1). For ethical reasons, subjects randomized to a placebo who exhibit significant symptom recurrence and impairments are discontinued from the study and given the opportunity to re-establish active treatment. In this manner, the value of continued treatment may be established; effective treatment is not withheld for an extended period of time. Randomized withdrawal studies can be used to examine maintenance of response, but it should be noted that they do not describe efficacy versus placebos. BODY.LONG-TERM TREATMENT EFFECTIVENESS IN ADHD: A small number of randomized, placebo-controlled withdrawal trials have examined the maintenance of efficacy in adults with ADHD (Table 2).67–70 These investigations add to the literature by examining the duration, extent, and nature of symptom control that remain when active treatment is continued compared to when it is discontinued. For these trials, investigators examined ADHD symptom ratings and classroom behaviors69 or rates and time to ADHD symptom relapse or loss of response, defined as a deterioration of ≥ 2 points on the Clinical Global Impressions-Improvement (CGI-I) scale and a 50%–90% decline in ADHD symptoms from baseline.67,68,70 In the earliest of these trials, Nolan et al examined the impact of randomized medication discontinuation in a small sample of children (aged 6–18 years, n = 19) with ADHD and comorbid chronic tic disorder or Tourette disorder.69 All subjects were on a stable psychostimulant regimen for at least 1 year prior to enrollment (n = 17 on methylphenidate; n = 2 on dextroamphetamine). Using a two-period crossover design, each subject experienced a randomized, double-blind placebo treatment for 2 weeks and double-blind, continued active treatment for 2 weeks. Analysis of 4-week withdrawal-phase data showed a significant advantage of continued medication treatment compared with a placebo based on all ADHD symptom ratings with mean (SD) Child Symptom Inventory-3R scores of 10.5 (9.7) and 5.5 (6.4) for placebos and active treatment (P = 0.0004), respectively. Core ADHD symptoms and aggression by Mother’s Method for Subgrouping (MOMS) and continuous performance tests, as well as observed simulated classroom behaviors such as time on task and worksheet completion, also demonstrated significant advantages for continued treatment.69 In another randomized withdrawal trial,68 children (aged 6–15 years; n = 416) with ADHD who showed adequate clinical response to the nonstimulant atomoxetine were re-randomized to continued active treatment or a placebo after 12 weeks. At the 9-month study endpoint, 22.3% of subjects who continued with atomoxetine exhibited relapse (defined as symptom return ≥ 90% of baseline in ADHD Rating Scale (ADHD-RS) total scores and ≥ 2 points on the CGI-Severity [CGI-S] scale) compared to 37.9% of those given a placebo (P = 0.002). A Kaplan–Meier analysis further showed that subjects switched to a placebo relapsed in a significantly shorter time than subjects who continued with atomoxetine (Figure 2).68 Of note was the significant worsening of psychosocial functioning detected among subjects randomized to a placebo, based on the Child Health Questionnaire. In a more recent trial, Biederman et al67 conducted a three-phase investigation of methylphenidate (MPH) delivered via an osmotic-release oral system (OROS®, Alzo Corporation, Mountain View, CA, USA) in adults (aged 19–60 years; n = 223) with ADHD. For the first phase, subjects were randomized to receive either a placebo or clinically optimized doses of OROS MPH for 6 weeks. Subjects who showed an adequate therapeutic response in the first phase (CGI-I of much or very much improved [ie, ratings of 1 or 2] and a reduction in ADHD Investigator Symptom Report Scale [AISRS] score > 30%) were then eligible to continue on to the second phase, in which Phase I treatment (OROS MPH or a placebo) was continued in double-blind fashion for an additional 24 weeks. In Phase III, OROS MPH responders were either re-randomized to a placebo or continued on OROS MPH for 4 weeks; placebo responders were not re-randomized. Figure 3 shows mean AISRS scores for the OROS MPH and placebo groups throughout each phase of the trial.67 During the randomized withdrawal phase (ie, Phase III), subjects who were re-randomized to a placebo showed a gradual small worsening of ADHD symptoms, but these symptoms did not return to baseline severity, whereas subjects who continued with OROS MPH showed a small improvement. No significant changes in AISRS scores were seen during double-blind withdrawal. The investigators noted that a robust therapeutic response among subjects treated with a placebo was indistinguishable from the responses among subjects given OROS MPH. No significant difference in relapse rate (defined as a deterioration in CGI-I rating of ≤ 2 points or an AISRS improvement < 15% from baseline) was detected between the subjects who continued active treatment (0%) versus those who were discontinued (18%). Biederman et al propose that, with up to 30 weeks’ treatment with OROS MPH, ADHD symptom control may have allowed subjects to develop more effective, adaptive coping skills.67 In the most recently published randomized withdrawal study, Brams et al enrolled adults (aged 18–55 years; n = 116) with ADHD who had been receiving a stable dose of lisdexamfetamine dimesylate (LDX) (30, 50, or 70 mg/day) in a community setting for at least 6 months with an acceptable safety profile.70 During the first study phase, all subjects received their usual prestudy doses of LDX for 3 weeks. All subjects were then randomized for 6 weeks to receive either a placebo or the same dose of LDX as that given in the open-label phase. At the endpoint of the open-label treatment period, nearly all subjects were rated as not at all or mildly ill based on the CGI-S scale. During the randomized withdrawal phase, symptom relapse (defined as ≥ 50% increase in ADHD-RS-IV score and an increase in CGI-S rating ≥ 2, indicating deterioration of symptom control) was seen in 8.9% of subjects who continued on LDX, compared with 75% of subjects assigned a placebo (P < 0.0001) (Figure 4).70 For subjects maintained on LDX, least squares (LS) mean changes in ADHD-RS-IV scores from baseline of the randomized withdrawal phase to the endpoint showed only a small change (+1.6 points), compared to larger changes in the placebo group (+16.8 points).70 BODY.CONCLUSION AND SUMMARY: Despite clear evidence that approximately two-thirds of adults with a childhood ADHD diagnosis continue to experience persistent symptoms and functional impairments, many older adolescent and adult patients believe they are “well” and do not adhere to treatment over the long term. Patients, family members, and some physicians who believe the patient is “well enough” may consider the use of medication to treat ADHD as “unnecessary” due to safety concerns. “Well enough” needs to be operationally defined by the patient and physician. Patients who lack insight into the presence of symptoms may agree that they are still underachieving at school, at their job, and at home even while denying ongoing ADHD symptoms. A growing body of evidence from high-quality clinical trials demonstrates that many patients with ADHD continue to benefit from long-term therapy, whereas discontinuation is often accompanied by a return of symptoms. Continued clinical investigation is needed regarding factors that affect the risk of symptom relapse on discontinuation of pharmacotherapy. Findings will assist clinicians and patients in considering the balance of potential risks and benefits. Long-term consistent use of medication may increase a patient’s awareness of ADHD impairments independent of the symptoms experienced. When symptom insight is lacking, daily underachievement may encourage the development of effective compensatory skills that persist after medication is stopped.67 On a clinical note, patients may be aware of their symptoms and the resulting daily impairments. These patients may readily adopt behavioral skills to compensate. Patients who have little insight into their ADHD symptoms may acknowledge daily underachievement and be receptive to behavioral techniques. When patients have little insight into their ADHD symptoms and deny daily underachievement, pharmacologic and behavioral treatments are often rejected. Identifying which of these three categories a patient falls into will help a physician determine the best approach when discussing treatment options. Ultimately the discontinuation of medication is likely to lead to a fairly rapid recurrence of the symptoms and worsening of the functional impairments of the disorder. Brams et al demonstrated that, when physicians and patients discontinue medication, follow-up visits within the first few weeks may allow the physician and patient to assess the return of symptoms.70 Such an assessment will allow the physician to understand the degree of insight the patient has gained about changing ADHD symptoms and daily productivity, and may also enhance long-term management of the disorder. Given that ADHD symptoms – particularly hyperactivity/impulsivity symptoms – tend to decrease with age in adolescents and young adults,71 and also based on clinical experience, some patients may be able to successfully discontinue ADHD pharmacotherapy. Brief drug holidays under clinician supervision may help patients become more aware of their symptoms and functioning when they are on medication compared to when they are off. The odds of these holidays being successful may also be increased by timing the discontinuation of medication. A period when environmental demands are reduced or when patients may have increased support from a spouse or family member may limit the impact of ADHD-impaired functioning. Conversely, a patient’s request to discontinue medication during environmentally demanding times is very likely to result in a severe escalation of ADHD impairments and should be delayed for “quieter” times. It is important for clinicians to discuss the risks/benefits of discontinuing medication with their patients who have ADHD and highlight important symptoms and impairments that should be monitored following discontinuation. However, if the patient wishes to stop medication for reassessment or as an act of asserting control over treatment, the physician needs to remain supportive so as to maintain the therapeutic relationship and not discourage patients’ active participation in managing their health care plan. Performing adequate follow-up allows clinicians to act as a resource upon which patients can rely should they experience a setback or have questions. Encouraging those patients already engaged in psychotherapy or behavioral therapy to continue that form of treatment may help them develop insight into their symptoms and learn behavioral strategies to manage impairments. In addition, participation in therapy may allow patients to see the strengths and limitations of behavioral techniques. If follow-up assessment indicates the return of symptoms or impairments, physicians should facilitate the discussion of resuming treatment. If the patient has not relapsed, it is important to discuss sentinel “red flag” symptoms the patient can easily recognize that may return at a later date. For patients who have successfully discontinued medication, it is crucial to maintain regular periodic follow-up, even if no treatment is prescribed. Patients may be relieved to know their physician remains available when life transitions or stressful events require additional support consisting of a number of options (eg, organizational or goal coaching, cognitive behavioral therapy, psychological counseling, and/or a resumption of pharmacotherapy).

TITLE: The Impact of Caloric and Non-Caloric Sweeteners on Food Intake and Brain Responses to Food: A Randomized Crossover Controlled Trial in Healthy Humans Whether non-nutritive sweetener (NNS) consumption impacts food intake behavior in humans is still unclear. Discrepant sensory and metabolic signals are proposed to mislead brain regulatory centers, in turn promoting maladaptive food choices favoring weight gain. We aimed to assess whether ingestion of sucrose- and NNS-sweetened drinks would differently alter brain responses to food viewing and food intake. Eighteen normal-weight men were studied in a fasted condition and after consumption of a standardized meal accompanied by either a NNS-sweetened (NNS), or a sucrose-sweetened (SUC) drink, or water (WAT). Their brain responses to visual food cues were assessed by means of electroencephalography (EEG) before and 45 min after meal ingestion. Four hours after meal ingestion, spontaneous food intake was monitored during an ad libitum buffet. With WAT, meal intake led to increased neural activity in the dorsal prefrontal cortex and the insula, areas linked to cognitive control and interoception. With SUC, neural activity in the insula increased as well, but decreased in temporal regions linked to food categorization, and remained unchanged in dorsal prefrontal areas. The latter modulations were associated with a significantly lower total energy intake at buffet (mean kcal ± SEM; 791 ± 62) as compared to WAT (942 ± 71) and NNS (917 ± 70). In contrast to WAT and SUC, NNS consumption did not impact activity in the insula, but led to increased neural activity in ventrolateral prefrontal regions linked to the inhibition of reward. Total energy intake at the buffet was not significantly different between WAT and NNS. Our findings highlight the differential impact of caloric and non-caloric sweeteners on subsequent brain responses to visual food cues and energy intake. These variations may reflect an initial stage of adaptation to taste-calorie uncoupling, and could be indicative of longer-term consequences of repeated NNS consumption on food intake behavior. BODY.1. INTRODUCTION: Excess sugar consumption, in particular in the form of sugar-sweetened beverages (SSBs), has been repeatedly identified as a major factor contributing to weight gain, overweight and obesity prevalence as well as associated metabolic disorders [1,2,3]. In order to fight increasing rates of overweight and obesity and help body weight management, many non-caloric molecules with a high sweetening power, called non-caloric or non-nutritive sweeteners (NNS), were developed and introduced into our daily diet. That is, hedonic properties of sweet taste can be enjoyed without consuming excess liquid calories. Yet, epidemiological studies have shown a link between NNS consumption and an increased prevalence of overweight and obesity risk on the long-run [4,5,6]. Although risks of reverse causality cannot be excluded when interpreting the results of observational cohort studies, one other reason for such a link could be functional properties of the central food intake regulation system discussed below. In healthy humans, food intake is regulated by a fine-tuned balance between drives towards palatable items (‘hedonic’ processing and reward valuation), physiological needs (‘homeostatic’ processing), and inhibitory control. While inhibitory control is supported by brain areas of the executive function network, i.e., dorsal, ventrolateral prefrontal and parietal regions [7], reward valuation is assisted by cortico-limbic networks comprising basal ganglia, the anterior cingulate and orbitofrontal cortices [8]. Reward is integrated with homeostatic signals in the hypothalamus and insula. Gastro-intestinal hormones secreted before (e.g., ghrelin) and after a meal (e.g., insulin), nervous afferents as well as sweet taste receptor activation in the mouth (and possibly along the digestive track) all provide feedbacks to the brain and thereby inform these networks on physiological states and consequences of food ingestion [9,10,11]. This regulatory system likely evolved with a caloric value assigned to sweet taste, as naturally occurring sweeteners generally contain about 4 kcal/g. Due to their sweet but non-caloric properties NNS might thus provide erroneous information to the food intake regulatory system, potentially inducing maladaptive food choices as compensatory mechanism [12,13]. In animals, findings support this hypothesis by showing detrimental effects of NNS consumption on food intake behavior and increased adiposity and body weight longitudinally [14,15]. So far, the impact of NNS consumption on food choices and body weight control in humans remains controversial, showing either beneficial, detrimental, or no effects [16,17,18,19]. From a neurophysiological perspective, some studies have shown differences in the cerebral processing of taste information between NNS and sucrose stimuli of equal sweetness intensity, highlighting the capacity of the human brain to readily discriminate between caloric and non-caloric sweet taste [20,21,22]. However, these studies assessed immediate gustatory responses and neural activity, but not subsequent food cravings and intake of excess solid calories. Neuroimaging studies in humans have further shown that food cravings and choices often result from exposure to visual food cues [23,24]. However, only one study, to our knowledge, has investigated brain response modulations to food cue exposure following NNS consumption in humans, i.e., in the context of a 3-month replacement of sucrose-sweetened beverages by artificially sweetened equivalents [25]. This former study of our group highlighted the potential implication of central cognitive control mechanisms in weight loss failure. However, that study did not directly compare the impact of consuming caloric vs. non-caloric sweeteners on responses. Further, participants had not been blinded to the type of beverage they consumed. Thus, how NNS influence subsequent drives towards certain types of foods (in particular sweet foods) and how this relates to modulations in food intake behavior remains largely unknown. The goal of our current study was thus to investigate whether, as compared to water consumption, activation of sweet taste receptors with NNS or with sucrose, as part of a standardized meal, exert different acute effects on (a) postprandial brain responses to food viewing, (b) postprandial gastro-intestinal hormone secretion known to impact hunger and satiety feelings and (c) subsequent food intake behavior, both in terms of quantity and quality of choices (ad libitum buffet). BODY.2. MATERIALS AND METHODS : BODY.2.1. PARTICIPANTS: Eighteen healthy, normal-weight men were recruited. All volunteers used to drink on average ≥3 cans of 33 cL of SSBs per week. None of the participants had current, prior, or family history of diabetes, cardiovascular, kidney, hepatic, neurological or psychiatric disease. Further exclusion criteria were color blindness, particular diets (e.g., vegetarianism), any food intolerance or allergy, arterial blood pressure > 140/90 mmHg at rest, exercising for more than 3 h per week, current medication, drug-taking or smoking habits, and consuming more than 10 g of alcohol per day. Only infrequent consumers of NNS-sweetened beverages (≤1 can of 33 cL per week) were included. All volunteers were weight-stable, right-handed according to the Edinburgh Handedness Inventory [26], and had normal or corrected-to-normal vision. To ensure medical safety, volunteers were not included when hemoglobin and ferritin levels were below 13.5 g/L and 50 µg/L respectively, when weighing less than 50 kg, and when having donated blood or participated to another clinical trial in the prior three months. Recruitment was done by means of advertising at local university campuses. Recruitment, screening, and follow-up of participants is shown in Figure 1A. Potential participants were first screened by email and then invited to a screening visit. Nineteen volunteers met all eligibility criteria and were enrolled in the study. One participant had to be excluded due to medical discomfort during the first test day; so that 18 volunteers completed the entire protocol. BODY.2.2. GENERAL PROCEDURE: The study consisted of three in-center test days for each volunteer, on which one of the three beverage conditions (i.e., Water, Sucrose, and NNS consumption, further referred to as WAT, SUC, and NNS) was tested in a randomized crossover controlled design. The beverage conditions were separated by a wash-out period of three weeks. Each test day was preceded by a 5-day nutritional and lifestyle recommendation period followed by a 2-day run-in period. During this run-in period, volunteers received a controlled weight-maintenance diet (55% carbohydrates (including 10% as sugars), 30% lipids and 15% proteins) calculated from the Harris–Benedict equation with a physical activity factor set at 1.5. Participants were instructed to consume all meals and snacks at specified times of the day (7 a.m., 10 a.m., 12 a.m., 3 p.m., 7 p.m.), and to drink only water. Detailed procedures for the in-center test days are provided in Figure 1B. On each test day, volunteers reported to the Metabolism, Nutrition, and Physical Activity unit from the Clinical Research Center of the Lausanne University Hospital at 6.30 a.m. They were fasting since 10 p.m. the evening before the test. They were asked to void, and body weight was measured thereafter (Seca 708, Seca GmbH, Hamburg, Germany). Body composition was assessed using bio-electrical impedancemetry (Biacorpus, Medical Healthcare GmbH, Germany). Each volunteer was then placed in a bed, and a catheter was inserted into an antecubital vein of the left forearm to allow for repeated blood collection throughout the test day. The venous path was kept open with a slow perfusion of saline solution (NaCl 0.9%). Two blood samples were collected in fasted state at T = −60 and 0 min before standardized meal and beverage ingestion. At those time points, participants were also asked to fill visual analog scales (VAS; 0–100) for hunger, thirst, and satiety levels as well as a Likert scale (LS; 1–9) for taste cravings. Each volunteer was then accompanied to a light-proof room where the electroencephalographic (EEG) recording system was installed. A cap with 64 active electrodes was placed on the participants’ head and prepared so that electrical impedance between electrical sensor and scalp were kept below 40 kΩ. A first EEG recording took place between T = −60 and 0 min (i.e., further referred to as the pre-prandial recording session), while participants completed an online continuous recognition task. The EEG recording procedure, as well as visual stimuli and task used are detailed below (Section 2.8). At T = 0 min, a standardized meal and the 350 mL test drink (T0-beverage; WAT, SUC, or NNS) were given to each volunteer. Five blood samples were collected over the post-prandial period, at T = 30, 60, 90, 150 and 210 min after meal and beverage consumption had started. At T = 30, 60 and 210 min, participants also filled VAS and LS for hunger, thirst, satiety, and taste cravings. A second EEG recording took place 45 min after meal and beverage ingestion (i.e., further referred to as the post-prandial recording session), that followed the same procedure as the pre-prandial session. In order to avoid ceiling effects and further strengthen the impact of beverage type on spontaneous food intake, participants received a 200 mL pre-buffet drink 210 min after the meal ingestion (T210-beverage; SUC, WAT or NNS), its composition repeating the beverage condition. This drink served as a preload for further quantitative and qualitative assessments of spontaneous food intake at an ad libitum buffet taking place 20 min after the preload ingestion. The primary study outcomes were pre- to post-prandial changes in brain responses to food viewing across beverage conditions. All behavioral and physiological parameters were considered as secondary outcomes. Study sample size was determined by assuming the same effect size on the spatio-temporal brain dynamics as in [25] (1 − β: 80%; α: 5%). The randomization sequence of treatment allocation was determined before the start of recruitment by random generation of blocks using the R software version 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria). To ensure double-blinding of both participants and experimenters to the beverage condition, a third person (J.C.) prepared the beverages. The experimental protocol was conducted according to the Declaration of Helsinki and was approved by the Ethics Committee of the Canton de Vaud in September 2015 (protocol number 353/15). The protocol is registered in the international and national registers for clinical trials (clinicaltrials.gov: NCT02853773 and kofam.ch: SNCTP000001882). Participants were enrolled between February 2016 and April 2017. All test days were performed between March 2016 and July 2017. All experimental visits took place in the Metabolism, Nutrition and Physical Activity unit from the Center for Clinical Research of the Lausanne University Hospital. All volunteers were informed about the procedures during the screening visit and signed a written consent. BODY.2.3. MEAL AND TEST BEVERAGE COMPOSITION: The T0-beverage consisted of five 70 mL-glasses (i.e., 350 mL in total) corresponding to one of the three beverage conditions (WAT, SUC, NNS). Beverage composition was based on commercialized concentrations, as determined by Ordoñez and colleagues [27]. The SUC T0-beverage provided 149 kcal (37.1 g of sucrose). The WAT and NNS T0-beverages provided 0 kcal, with 137.2 mg cyclamate, 63.35 mg acesulfame K and 40.6 mg aspartame for the NNS. The standardized meal provided at T = 0 min was identical for all three beverage conditions. It corresponded to 30% of the estimated individual 24-h energy requirements calculated from the Harris–Benedict equation, and was low in sugars and sweet taste (55% carbohydrates (2% sugars), 30% lipids and 15% proteins). Participants were asked to drink one 70 mL-glass every five minutes and consumed the provided meal in-between, i.e., starting and ending the meal with the consumption of a 70 mL-glass. To maximize the sweet taste receptor stimulation by sucrose or NNS, each mouthful of liquid was to be kept in the mouth for ten seconds before swallowing. The T210-beverage was of the same composition as the T0-beverages. The SUC T210-beverage thus provided 85 kcal (21.2 g of sucrose). The WAT and NNS T210-beverages provided 0 kcal, with 78.4 mg cyclamate, 36.2 mg acesulfame K and 23.2 mg aspartame for the NNS. All beverages were provided at room temperature. BODY.2.4. QUALITATIVE AND QUANTITATIVE ASSESSMENTS OF SPONTANEOUS FOOD INTAKE: The ad libitum buffet presented at the end of the test day comprised 12 snacks, subdivided into 4 categories (3 snacks each) based on the fat content and taste quality of the foods provided, i.e., Low Fat/Non-Sweet (LF/NSW), Low Fat/Sweet (LF/SW), High Fat/Non-Sweet (HF/NSW), and High Fat/Sweet (HF/SW). The threshold for Low-Fat/High-Fat and Non-Sweet/Sweet subdivisions was set at 10 g of lipids/sugars per 100 g of food based on the nutritional information available on the packaging. The textures were matched as much as possible between food categories. The presentation context was kept as identical as possible between all beverage conditions, and always took place in the kitchen of the Clinical Research Center. Snacks were consistently prepared by the same experimenters (C.C. and L.C.), weighed and presented following the same protocol, and served on identical white dishes. Each snack was available in larger quantity than the expected average intake. The environment was kept neutral (e.g., no visible food packaging and no particular odor), and each participant was either accompanied by the experimenter or left alone for periods of five minutes. Participants remained uninformed of their food intake being measured, and were told to eat until feeling comfortably sated. Questions regarding food type were answered, but no nutritional information was given. Water was provided ad libitum with the snacks. All snack leftovers were carefully weighed after consumption. Total energy intake and energy intake per food category was calculated based on the nutritional information available on the packaging. BODY.2.5. ANALYTICAL PROCEDURES FOR PLASMA SAMPLES: Plasma was separated from blood cells immediately after sampling by centrifugation during 10 min at 4 °C and 3500 rotations per minute. Aliquots of plasma were stored at −20 °C until analysis. Plasma glucose concentrations were measured by enzymatic methods (Randox Laboratories Ltd., Crumlin, UK). Plasma insulin and ghrelin concentrations were determined by radioimmunoassay (Merck Millipore Merck KGaA, Darmstadt, Germany). BODY.2.6. BEHAVIORAL RATINGS: VAS for hunger, thirst, and satiety consisted of 15-cm long lines with ‘0′ and ‘100′ anchored to the left and right side, respectively, presented with the written indication “Please indicate how hungry/thirsty/satiated you are at present by drawing a point on the line below”. Individuals’ responses were converted to % of the scale maximum. Taste cravings were assessed with a 9-point LS with ‘salty’ and ‘sweet’ anchored to the left and right side, presented with the written indication “Please indicate how much you crave for a rather salty or sweet food item at present by ticking the correct box on the scale below”. BODY.2.7. STATISTICAL ANALYSES OF FOOD INTAKE, BEHAVIORAL RATINGS AND PLASMATIC PARAMETERS: Data distribution was controlled for normality and homoscedasticity using the Shapiro–Wilk and Bartlett tests, respectively. Data that were not normally distributed were transformed using the BoxCox algorithm. All anthropometric parameters (body weight, body mass index (BMI), and body composition), plasma concentrations and behavioral parameters (VAS and LS ratings) were tested for differences between beverage condition baselines with a one-way repeated-measure ANOVA including the independent within-subject factor of Beverage (three levels: WAT, SUC, and NNS). The impact of beverage type on food intake at the ad libitum buffet (total energy intake) was investigated first by a one-way repeated-measure ANOVA with the within-subject factor of Beverage. In a second step, the impact of the beverage type was detailed for food categories ingested by a two-way repeated-measure ANOVA with the within-subject factors of Beverage and Food category (four levels: LF/NSW, LF/SW, HF/NSW and HF/SW). Whenever a significant main effect of Beverage or an interaction Beverage × Food category was found, post-hoc paired t-tests (two-tailed) were conducted between Beverage conditions and/or Food categories. The effect of beverage type on the kinetics of plasma concentrations and behavioral scales was assessed using two-way repeated-measure ANOVAs with the within-subject factors of Beverage and Time. Whenever an interaction Beverage × Time was found, post-hoc paired t-tests (two-tailed) were conducted between Beverage conditions at each time point. Further, one-way repeated-measure ANOVAs with the within-subject factor of Beverage were conducted on plasma concentrations and behavioral scales at T = 210 min, irrespective of the kinetic results, to investigate the pre-buffet state. Whenever a main effect of Beverage was found, post-hoc paired t-tests (two-tailed) were conducted between beverage conditions. All data are expressed as mean ± SEM (standard error of mean). All analyses were performed with R version 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria), and p-values ≤ 0.05 were considered as significant. BODY.2.8. ELECTROENCEPHALOGRAPHY (EEG) STIMULI PRESENTATION PROCEDURE, EEG ACQUISITION AND PREPROCESSING: During both EEG recording sessions, color photographs showing foods that differed in fat content and in taste quality were presented to participants. This image database (240 items) has been used in several studies investigating food perception [25,28,29,30] and pictures were controlled for low-level visual features [31]. Stimulus presentation took place in a light-proof room, using the E-prime 2 software (Psychology Software Tools, Inc., Pittsburgh, PA, USA). Images were presented centrally for 500 ms each on a 19″ computer screen, in 3 consecutive blocks lasting 5 min. Each block contained 120 items, i.e., 80 initial encounters and 40 repeated items. Participants were asked to categorize initial from repeated image encounters via button-press. This behavioral task served to ensure participants’ attention to food images, and they were instructed to perform as quickly and accurately as possible. Following participants’ button press, the Inter-Trial-Interval (ITI) randomly varied between 250 and 750 ms to avoid anticipatory responses. During the ITI, a fixation cross was centrally displayed on screen to avoid eye movements. Number of trials between initial and repeated items were controlled across blocks and recording sessions to ensure similar difficulty of the recognition task. Continuous EEG was recorded while participants viewed the images and performed the online recognition task. EEG was acquired at a sampling rate of 500 Hz using a 64-channel BrainProducts ActiCAP system. Details of the electrode montage can be found on the BrainProducts website (http://www.brainproducts.com/products_apps.php). All pre-processing analyses were performed using the CarTool software version 3.51 (2268) (https://sites.google.com/site/fbmlab/cartool). Only the responses to initially encountered images were used to compute visual evoked potentials (VEPs). VEPs were computed over the period from −100 ms to +500 ms peri-stimulus epoch for each image. During single subject averaging, EEG epochs were cleaned from artifacts with a semi-automatic procedure using a 80 μV rejection criterion and visual trial-by-trial inspection. Epochs containing eye blinks or other motor artifacts were manually removed. During averaging, baseline correction was applied on the peri-stimulus period (i.e., −100 ms to +500 ms), and data was band-pass filtered at 0.1–30 Hz. An additional notch filter of 50 Hz was applied. First, VEPs were averaged for each single subject, recording session (Pre- and Post-prandial) and beverage condition (WAT, SUC, and NNS). Electrodes with artefactual signals were then interpolated [3233]. BODY.2.9. EEG ANALYSES AND ESTIMATIONS OF NEURAL SOURCE ACTIVITY: Time windows of interest in brain responses to food viewing in the SUC, WAT, and NNS conditions were determined around GFP peaks in the group-average Global Field Power (GFP) waveform. The GFP is a reference-independent measure of the global amplitude of the electric field (VEPs), i.e., calculated as the standard deviation of the electric field amplitude across all 64 electrodes at a given time point [34]. These peak periods in the GFP represent the time windows of highest synchronized neural activity underlying distinct steps in sensory and cognitive processes, and thus served as a rationale for further investigation of beverage type-induced modulations in source activity [25,35]. The center and width of each time windows of interest were determined by the average peak timing and the standard deviation across participants’ individual GFP peaks, across sessions and beverage conditions. Over each time window of interest, we estimated the neural source activity based on the head-surface recorded VEPs using a local autoregressive average (LAURA) distributed a linear inverse solution [36]. That is, mean amplitudes of neural activity were calculated for each of the 4350 solution points of an inverse solution matrix based on a realistic 3D head model. The output of this algorithm is one scalar value (µA/mm3) per solution point, per viewing condition and time window. As the goal of our study was to investigate the differential effects of three beverages on the meal-induced modulations in brain responses to food viewing, we focused our analyses on the relative change in neural activity from pre- to post-prandial recording sessions [25]. For each time window of interest, statistical analyses first comprised of whole-brain repeated-measure one-way ANOVAs with the within-subject factor of Beverage, computed on the % neural activity change from pre- to post-prandial session on each node of the 4350 solution point matrix. Only regions showing a significant main effect of Beverage in a cluster of ≥10 neighboring nodes were considered for post-hoc region-of-interest (ROI) analyses. Results of these analyses were rendered on the Montreal Institute template brain (MNI) and Talairach coordinates of the node showing the maximal statistical difference between beverage conditions are given for each statistically determined brain region. In each ROI showing a significant main effect of Beverage, neural activity of the source node revealing maximal statistical differences plus the 6 neighboring nodes was extracted and averaged in each individuals’ data, for each beverage condition. These results are visualized as bar plots, indicating pre-to-post changes in neural activity to food viewing. Statistical outliers (< or >3 standard deviations from the mean) were removed from further analyses. Post-hoc paired t-tests (two-tailed) were then conducted on the respective pre-to-post change in neural activity (in %) between beverage conditions. Additionally, orthogonal one-sample t-tests (two-tailed) assessed, within each ROI and in each beverage condition, whether the relative pre-to-post % change in signal significantly differed from baseline (i.e., pre-prandial activity; [25]). Overall, only results with p ≤ 0.05 were considered significant. All analyses were conducted using customized Python scripts, the software R version 3.3.1, and the STEN toolbox version 2.0 developed by Jean-François Knebel and Michael Notter (http://doi.org/10.5281/zenodo.1164038). BODY.3. RESULTS: Participants’ body weight, BMI, body composition, plasma concentrations of glucose, insulin and ghrelin and behavioral ratings for hunger, thirst, satiety, and taste cravings are shown in Table 1 for each beverage condition. None of the parameters showed differences between beverage conditions at baseline (all p = ns). BODY.3.1. SPONTANEOUS FOOD INTAKE AT THE AD LIBITUM BUFFET: Total energy intake and energy intake by food category at the ad libitum buffet are shown in Table 2. Regarding total energy intake, we observed a main effect of Beverage (F2,17 = 3.62; p < 0.05), i.e., participants on average ingested significantly less energy in SUC than in WAT (Δ = −151 ± 59 kcal; t17 = −2.58; p < 0.05) and NNS (Δ = −126 ± 56 kcal; t17 = −2.26; p < 0.05). However, no significant difference was observed between WAT and NNS (Δ = 25 ± 66 kcal; t17 = 0.38; p = ns). Further analyses on energy intake segregated by food categories did not show an interaction between Beverage × Food category (F2,17 = 0.54; p = ns), i.e., participants did not modify their food choice pattern as a function of the beverage type ingested. BODY.3.2. PLASMA CONCENTRATIONS OF METABOLITES AND GASTRO-INTESTINAL HORMONES: Plasma concentrations of glucose, insulin, and ghrelin in response to meal and test beverage ingestion are shown in Figure 2. The main effect of Beverage was significant for insulin (Figure 2B; F2,17 = 8.29; p < 0.05; i.e., plasma insulin yielded overall higher values in SUC as compared with both WAT and NNS) and ghrelin (Figure 2C; F2,15 = 4.56; p < 0.05; i.e., plasma ghrelin yielded overall lower values in SUC as compared with both WAT and NNS), but not for plasma glucose (Figure 2A; F2,17 = 0.15; p = ns). More importantly, a significant interaction between Beverage × Time was observed for all parameters (glucose: F2,17 = 2.83; insulin: F2,17 = 5.31; ghrelin: F2,15 = 2.41; all p < 0.05). Plasma glucose and insulin concentrations were significantly higher in SUC at T = 30 min (glucose: t17 = −3.77 and t17= −2.46; insulin: t17 = −3.94 and t17 = −5.11; all p < 0.05) as compared to WAT and NNS, respectively. Plasma ghrelin concentration, on the other hand, was significantly lower in SUC, as compared to WAT; this difference being significant at T = 30 min (t15 = −2.77; p < 0.05) and T = 60 min (t15 = −2.48; p < 0.05). No differences were observed between WAT and NNS for any of the parameter kinetics. Before the buffet (T = 210 min), plasma glucose concentrations were similar in all beverage conditions (F2,17 = 0.70; p = ns). By contrast, a significant main effect of Beverage was observed for plasma insulin (F2,17 = 13.04; p < 0.05) and ghrelin concentrations (F2,16 = 9.44; p < 0.05). Plasma insulin concentration was most elevated in SUC (t17 = 2.55 and t17 = 4.77; both p < 0.05 against WAT and NNS, respectively), and the lowest in NNS (t17 = 2.74; p < 0.05 against WAT). Plasma ghrelin concentration was lower in SUC as compared with WAT (t17 = 2.88; p < 0.05) and NNS (t17 = 3.89; p < 0.05), but there was no difference between WAT and NNS (t17 = 0.81; p = ns). BODY.3.3. RESULTS OF BEHAVIORAL RATINGS: No main effect of Beverage nor interaction between Beverage × Time were observed for any of the parameter kinetics (Supplementary Figure S1A–D for hunger, satiety, thirst ratings and taste cravings). Before the buffet (T = 210 min), a significant main effect of Beverage was found on hunger ratings (F2,17 = 5.68; p < 0.05). Hunger ratings were lower in SUC as compared with WAT (t17 = −2.71; p < 0.05) and NNS (t17 = −2.66; p < 0.05). No difference in hunger ratings was found between WAT and NNS (t17 = 0.28; p = ns). No significant main effect of Beverage was found for thirst ratings (F2,17 = 0.07), satiety ratings (F2,17 = 1.10) and taste cravings (F2,17 = 0.37; all p = ns). BODY.3.4. PRE- TO POST-PRANDIAL CHANGES IN NEURAL SOURCE ACTIVITY TO FOOD VIEWING: Two time periods of interest were defined around the peaks of the group-average GFP waveform. A first period of interest ranged from 120 to 150 ms, and a second period of interest ranged from 250 to 320 ms after image onset (Figure 3A). Over the first time window of interest (TW1: 120–150 ms post-image onset), whole brain analyses revealed a main effect of Beverage on the pre- to post-prandial % change in neural activity in the left dorsolateral prefrontal cortex (DLPFC; Max: x = −36, y = 4, z = 33) and in the left ventrolateral prefrontal cortex (VLPFC; Max: x = −49, y = 47, z = −10). That is, the beverage type differentially modulated the neural activity to food viewing within these brain areas (Figure 3B, left panel). With WAT, meal intake led to decreased neural activity within the DLPFC (t16 = −2.16; p < 0.05 for t-test against baseline), but did not impact neural activity within the VLPFC (t17 = −0.25; p = ns for t-test against baseline) (Figure 3C, left panel). Unlike WAT, there was no modulation in the neural response within the DLPFC with SUC (t16 = 1.66; p = ns for t-test against baseline; t16 = 3.31; p < 0.05 for paired t-test between WAT and SUC responses). Like WAT however, SUC did not lead to modulated neural activity within the VLPFC (t17 = −1.22; p = ns for t-test against baseline). NNS also did not impact neural activity within the DLPFC (t16 = 0.39; p = ns). Yet, in contrast to WAT and SUC, NNS led to increased neural activity within the VLPFC (t17 = 2.42; p < 0.05 for t-test against baseline; t17 = −3.20 and t17 = −4.19; both p < 0.05 for paired t-tests between NNS-WAT and NNS-SUC, respectively). Over the second time window of interest (250–320 ms post-image onset), a main effect of Beverage on the pre- to post-prandial % change in neural activity was observed in the right insula (Ins; Max: x = 42, y = −22, z = 10), in the left (l) and right (r) DLPFC ((l)DLPFC Max: x = −36, y = 36, z = 25 and (r)DLPFC Max: x = 42, y = 12, z = 51), and in the right middle temporal gyrus (MTG; Max; x = 49, y = −48, z = 0) (Figure 3B, right panel). With WAT, meal intake led to increased neural activity within the insula (t17 = 2.55; p < 0.05), (l)DLPFC (t17 = 2.82; p < 0.05) and (r)DLPFC (t16 = 2.60; p < 0.05), but did not impact neural activity within the MTG (t16 = 0.85; p = ns; all t-tests against baseline) (Figure 3C, right panel). Like WAT, SUC also led to increased neural activity within the insula (t17 = 2.48; p < 0.05 for t-test against baseline). Unlike WAT however, there were no pre-to-post changes in neural activity in SUC in the (l)DLPFC (t17 = −1.09; p = ns for t-test against baseline; t17 = −4.94; p < 0.05 for paired t-test between SUC and WAT) and the (r)DLPFC (t16 = 0.04; p = ns for t-test against baseline). In addition, SUC led to decreased neural activity within the MTG (t16 = −3.21; p < 0.05 for t-test against baseline; t16 = −3.74; p < 0.05 for paired t-test between SUC and WAT). In contrast to WAT and SUC, NNS did not impact neural activity within the insula (t17 = −1.72; p = ns for t-test against baseline; t17 = 3.11 and t17 = 2.86; both p < 0.05 for paired t-tests between NNS-WAT and NNS-SUC, respectively). Like in SUC, there were no pre-to-post changes in neural activity in NNS within the (l)DLPFC (t17 = −0.01; p = ns for t-test against baseline) and the (r)DLPFC (t16 = −1.49; p = ns for t-test against baseline; t16 = 2.56; p < 0.05 for paired t-test between NNS and WAT). Like WAT, but unlike SUC, NNS consumption did not impact neural activity within the MTG (t16 = 0.55; p = ns for t-test against baseline; t16 = −2.46; p < 0.05 for paired t-test between NNS and SUC). BODY.4. DISCUSSION: Our study aimed at investigating the acute impact of consuming caloric (sucrose) and non-caloric sweeteners (NNS), as compared to water, on the subsequent brain responses to visual food cues and spontaneous food intake behavior. As expected, we found neurophysiological and physiological markers of satiety following the ingestion of the standardized meal with water. We further observed that sucrose consumption impacted the responses in brain areas associated with cognitive control (prefrontal cortices) and food categorization (temporal cortices), and led to decreased subsequent food intake, indicating an adequate compensatory behavior. In contrast, NNS consumption did not alter spontaneous food intake when compared to water, but altered postprandial brain responses to visual food cues, most pronounced in prefrontal areas and in the insula. BODY.4.1. BRAIN RESPONSES TO FOOD VIEWING FOLLOWING WATER OR SUCROSE CONSUMPTION: Meal ingestion combined with water (i.e., control beverage condition lacking sweet taste and caloric load) impacted brain responses to visual food cues in bilateral dorsal prefrontal areas and in the right insula. The neural activity in dorsal prefrontal areas has long been linked with the capacity to exert cognitive control over food intake when exposed to palatable food cues, as part of the executive function network. Tataranni and colleagues [37] were the first to highlight differences in brain responses between hunger and satiety beyond hypothalamic areas using functional neuroimaging, and found increased neural activity in the dorsolateral prefrontal cortex. Since then, many other studies have found dorsolateral prefrontal regions to be involved in top-down cognitive control over food intake [38,39,40]. A study of Camus and colleagues [41] could even attest causality in the role of dorsolateral prefrontal regions in control and decision-making using transcranial magnetic stimulation. Using the high temporal resolution of EEG, Harris and colleagues [42] were able to provide further insights on the dual role of DLPFC in cognitive control, showing that early response modulations (around 150 ms post-stimulus onset) were associated with top-down filtering of sensory input, whereas later ones (from 450 ms post-stimulus onset) were associated with reward value modulation. In our study, we observed decreased activity in the left dorsal prefrontal region over an early time window following food viewing (120–150 ms) and increased activity in the bilateral dorsal prefrontal region over later timing (250–320 ms). Our findings thus likely reflect elevated cognitive control following meal ingestion, which is rather due to value integration than to response modulation by the sensory input per se. We also observed increases in neural activity in the insula following meal ingestion accompanied by water. Insular responses to visual food cues have consistently been associated with interoception, i.e., awareness of bodily energy states. Also, the insula does contain molecular receptors for several gastro-intestinal hormones relaying this peripheral information to central nervous responses [11,43,44]. Furthermore, other studies have found increased insular activity subsequent to PYY infusion mimicking satiety [45], to mouth rinsing with a glucose drink mimicking food intake anticipation [46], but also in response to calorie ingestion as such [20,47]. The insula, being a hub between salience, homeostatic and control networks, is generally involved in signal integration, and thought to perform flavor-nutrient conditioning, too [48]. In accordance with these findings, we also found a higher postprandial insular activity following sucrose ingestion. Altogether, increases in insular activity both in the sucrose and water conditions thus likely reflect the adequate adaptation of participants’ responses as a function of the beverage consumed when taste properties and caloric load were congruent. Sucrose drinking (i.e., the beverage condition combining sweet taste and caloric load) elicited partially different modulations in brain responses to visual food cues as compared to meal ingestion with water. In particular, the postprandial response to visual food cues in cognitive control related areas was found blunted in the sucrose condition. Sucrose consumption also led to markedly decreased neural activity in the middle temporal lobe. This brain area is involved in the categorization and optimization of visual stimulus processing by attention [49], and is generally more active when participants are exposed to palatable food over neutral stimuli [50], as well as when responses to food are compared between hunger and satiety [24,51]. These differential patterns of brain responses to food cues following sucrose vs. water ingestion likely show that when sweet taste is coupled to a caloric load, brain responses shift from a rather reflective (usually involving prefrontal brain areas) to a more reflexive processing of food cues [52]. BODY.4.2. BRAIN RESPONSES TO FOOD VIEWING FOLLOWING NNS CONSUMPTION: NNS consumption (i.e., the beverage condition with discrepant sweet taste and caloric load) also yielded differential neural activity in response to subsequent exposure to visual food cues. In contrast to the ingestion of the meal with water or a sucrose drink, we observed early enhanced ventrolateral prefrontal cortex activity (120–150 ms after food image onset), but no changes in insular activity to food viewing over the later time window of interest (250–320 ms). Increases in neural activity following NNS tasting in ventrolateral prefrontal cortices has been highlighted in gustatory processing when neural responses were assessed at the time of or immediately after tasting. For instance, Smeets and colleagues [53] have shown greater activation of the ventrolateral prefrontal cortex directly after the ingestion of an artificially sweetened beverage as compared to sucrose. Ventral prefrontal regions have been widely associated with hedonic integration and reward valuation of (visually) perceived stimuli, including food cues [54,55,56]. However, these functions were mostly attributed to medial parts of the ventral prefrontal cortex, whereas we show enhanced activity within the ventrolateral prefrontal cortex in response to visual cues following NNS consumption. Ventrolateral regions of the prefrontal cortex are part of the executive function circuitry, supporting decision-making adjustments, in particular related to motor response inhibition when exposed to cues associated with high reward, as well as targeting attention to behaviorally significant stimuli (reviewed in [57]). Thus, this area is proposed to be responsible for altering behavior as a function of estimated changes in the reward value of (viewed) stimuli. Our results show increased neural activity to visual food cues within the ventrolateral prefrontal area, likely related to greater (need for) impulse retaining and control over anticipated food intake. Although no study so far investigated the impact of NNS consumption on brain responses to food cues longitudinally, research in the gustatory modality showed differences in neural activation to NNS tasting between non-diet soda drinkers and frequent consumers of diet soda [58]. The study of Green & Murphy found increased responses to saccharin vs. sucrose in the ventrolateral prefrontal cortex in non-diet soda drinkers, whereas this difference was absent in frequent diet soda drinkers. These findings were interpreted as reflecting ‘fading’ neural activity in this region with repeated consumption of NNS, impacting impulse control over time. In line, we previously found decreased activity in the more posterior part of the ventrolateral prefrontal cortex following a 3-month replacement of SSBs by non-calorically sweetened equivalents [25]. Our current results thus provide additional evidence as to a target region for future longitudinal studies on the longer-term impact of sweet taste stimulation by NNS, and on responses to tempting visual food cues following NNS ingestion. With NNS consumption, on the other hand, we did not observe pre-to postprandial changes in insular activity. This suggests that congruent caloric and taste signaling is required to elicit adequate response adaptation to food cues, and that incongruences between taste information and caloric load may impair nutrient-flavor conditioning [48]. Rudenga and Small [59] have further shown that the neural response to sucrose tasting in the insula (and also in the amygdala) decreased as a function of NNS consumption habits of participants, implying that this region might be more vulnerable to chronic dissociations between sweet taste signaling and metabolic consequences. BODY.4.3. INTEGRATION OF POSTPRANDIAL BRAIN RESPONSES TO FOOD VIEWING WITH GASTRO-INTESTINAL HORMONE SECRETION AND FOOD INTAKE BEHAVIOR: Sucrose drinking during meal ingestion, as compared to water, led to subsequent decreased food intake at the ad libitum buffet indicative of a compensatory food intake behavior. In parallel, we observed elevated plasma concentrations of insulin (an anorexigenic hormone) and decreased plasma concentrations of ghrelin (an orexigenic hormone), likely promoting some of the brain response alterations. Thus, the effects of sucrose intake may be related to hormonal signaling and/or to sweet taste receptor activation coupled with other peripheral satiety signals (e.g., vagal afferents). Yet, whether the observed differences are sucrose-specific effects or more general ones driven by an extra caloric load cannot be concluded from our current study, as there was no condition with a caloric load from another nutrient source (e.g., maltodextrin or fat). NNS consumption did not lead to pronounced modulations of glucose, insulin, and ghrelin concentrations, nor to higher caloric consumption or variation of the food choice pattern at the ad libitum buffet. Thus, the observed changes in brain activity to food viewing post-meal and food intake pattern cannot be attributed solely to differential signaling of gastro-intestinal mediators. Although we did not measure other anorexigenic hormones such as leptin or PYY, the observed differences in brain responses between the water and NNS condition are congruent with the idea that discrepant information between sweet taste receptor activation and gastrointestinal hormone signaling leads to changes in brain response patterns [12]. BODY.4.4. LIMITATIONS: Several limitations of our work need to be considered. First, the study design likely pronounces the impact of the meal ingestion stronger than the impact of the test beverage. However, we aimed at designing this study with the highest ecological validity, i.e., having volunteers consuming standard amounts of beverages concomitant with a meal (quantity close to a commercially available can size). For this reason, we cannot exclude that the design was not sensitive enough to detect all secondary outcome differences, especially between the water and NNS conditions, and in terms of qualitative analyses on food choice patterns. Second, while we used a double-blinded design, participants could still detect the absence of sweet taste in the water condition, as opposed to both sweet taste conditions. Thus, some differences in brain response patterns might have arisen from these perceptual properties [60]. Finally, using electroencephalographic recording and electrical neuroimaging analyses, we are not able to detect deeper activity changes, e.g., in the basal ganglia (dopaminergic origin of the reward system), that might occur together with response modulations in cortices associated with higher-level functions. BODY.5. CONCLUSIONS: To our knowledge, this is the first study to assess the impact of NNS consumption on neural activity to food viewing, and the relationship with food intake behavior. We did not observe an acute effect of NNS consumption on immediate food intake in humans who are not frequently drinking NNS beverages. Yet, we observed imminent changes in brain response patterns in brain areas that are key players in food intake regulation. The responsiveness of these brain areas to sweet taste has been shown to ‘fade’ as a function of longer-term NNS consumption [58,59]. Thus, it remains to be investigated whether such longer-term brain response alterations can also be observed to visual food cues, often mediating pre-ingestive food choices. Given such longer-term alterations, the brain response modulations observed under the NNS condition in our study might reflect an initial stage of adaptation to taste-calorie uncoupling, possibly indicating that longer-term alterations of food intake regulation (via responses to tempting visual cues) take place when NNS are repeatedly consumed over time. Our study thus provides first insights linking neuroimaging research in the gustatory modality and behavioral research on the impact of non-caloric sweetener consumption on food intake, by investigating the neural correlates of drives towards visually conveyed food cues.

TITLE: Comparison between the EX-PRESS P-50 implant and trabeculectomy in patients with open-angle glaucoma ABSTRACT.PURPOSE: To evaluate the EX-PRESS P-50 implant compared to standard trabeculectomy (TBC). ABSTRACT.METHODS: Single-center prospective randomized study; 20 eyes of 20 patients were treated with the EX-PRESS P-50 implant, and 20 eyes of 20 patients with TBC, over a 19-month period. Records of all patients were reviewed and compared. Success was defined as intraocular pressure (IOP) <21 and >5 mmHg or a decrease of 30% of IOP. Failure was defined as >21 mmHg or decline in visual acuity. Statistical analysis was made with Student’s t-test and χ2 test analyzed with SPSS version 13.0. ABSTRACT.RESULTS: The average follow-up was 8.6 months (±4.9 months) for the EX-PRESS P-50 group and 9.6 months (±5.3 months) for the TBC group. The postoperative visual acuity and IOP were not significantly different. We report more complications in the EX-PRESS P-50 group. At 3, 6, and 12 months follow-up, the control group was found to be free of complications, whereas multiple complications were observed in the EX-PRESS P-50 group at 3 and 6 months follow-up. We found no differences in either group with respect to success. ABSTRACT.CONCLUSION: Both procedures are equally effective for the treatment of glaucoma, with 80% success in the EX-PRESS P-50 group and 72.7% in the control group. BODY.INTRODUCTION: Glaucoma is defined as a chronic, progressive, and irreversible neuropathy with loss of ganglion cells and nerve fibers, along with characteristic structural changes in the optic nerve.1 With an estimated 64.3 million people affected worldwide in 2013, and an expected increase to 76 million by 2020 and 111.8 million by 2040, glaucoma is the second leading cause of blindness in the world and the leading cause of irreversible blindness.2 Filtering surgery is indicated when medical or laser treatments are not enough to control intraocular pressure (IOP) to prevent progression of the damage caused by glaucoma. Trabeculectomy (TBC) was introduced by Cairns in 1968 as a surgical procedure for glaucoma, and it is the procedure of choice for surgical open-angle glaucoma treatment.3 Early postoperative complications associated with this condition include hyphema, excessive filtration, wound leak, flat chamber, choroidal detachment, hypotony, suprachoroidal hemorrhage, and cataract.4,5 These complications have motivated the development of alternative techniques and devices, including the EX-PRESS® glaucoma filtration device.3,6 BODY.EX-PRESS P-50 IMPLANT: The EX-PRESS glaucoma filtration device was originally developed by Optonol Ltd. (Neve Ilan, Israel). It is an alternative procedure to TBC filtration surgery. It is made of stainless steel, is valveless, is 2–3 mm long, has a 0.4 mm external diameter and 50 μm internal diameter,7,8 is placed under a scleral flap,4 and shunts the aqueous humor from the anterior chamber to the intrascleral space and subsequently to the bulla. It is similar to TBC, but with the following benefits. The conjunctival incision is smaller, allowing the implant to be placed in eyes with previous scars, since it only needs 2-hour zones of healthy conjunctiva to properly place the device.6 The 3 mm sclerectomy made with the scleral punch in conventional TBC is replaced by the 27-gauge needle foramen that makes a self-sealing ostium, maintaining the stability of the anterior chamber during the procedure.6 The fistula has a 50 μm lumen that is more resistant to flow compared to the 750 μm of the sclerectomy in TBC. In both, the scleral sutures are flow resistant, but it is greater in the implant due to the added inherent resistance of the lumen,5 which theoretically would reduce the number of patients with flat chamber.6 With the EX-PRESS P-50 implant, it is not necessary to perform iridectomy, thus reducing the risk of bleeding, inflammation, pigment release, and vitreous collapse, thereby decreasing the number of revisions.5,7,8 The goal of this study was to compare surgical results of the EX-PRESS P-50 implant to TBC in patients with open-angle glaucoma, as well as IOP, visual capacity, and surgical complications in the short and medium term. BODY.METHODS: This study was a clinical, experimental, single-center prospective randomized study, including patients consulting at the Glaucoma Department, Institute of Ophthalmology ‘Conde de Valenciana’, with the following inclusion criteria: patients over 18 years of age with open-angle glaucoma diagnosis, intolerant to topical medications, or those who had poor compliance to topical treatment and were scheduled for surgery. The Institution’s Ethics Committee of Conde de Valenciana approved this study. After signing an informed consent form and expressing their willingness to attend the follow-up visits, these patients were enrolled in the study over a 19-month period. Surgical treatment was indicated when it was not possible to reach the IOP goal, that is, the pressure measurements under which it is thought that there would be no progression of the disease, in spite of the maximum tolerated or recommended dose, considered as two first-line medications and one second-line medication. Subjects with a history of eye surgery, except cataract phacoemulsification, or with a single functional eye, aphakic, with eye diseases in the last 6 months such as active blepharitis, severe dry eye, uveitis, autoimmune diseases, cicatricial conjunctivitis, and cheloid scarring, were excluded. BODY.SAMPLE SIZE: The sample size was calculated using a formula that adjusts sampling from finite populations,9 requiring 31 patients with 5% alpha error and 20% beta error, with a 95% confidence interval. Forty eyes were studied, which were randomly assigned to two surgical treatment groups: TBC with mitomycin C. EX-PRESS P-50 implant with mitomycin C. BODY.PROCEDURE: Data were obtained from the clinical files; subsequently, a baseline evaluation of the visual capacity was performed, IOP was measured using a Goldmann tonometer (Haag-Streit AG, Könis, Switzerland) twice, and a third time if there was more than 2 mmHg difference from the two previous measurements. The eye surface and the anterior segment were examined; gonioscopy and fundoscopy were performed and increased eye pressure or open-angle glaucoma was diagnosed. Topical treatment was adjusted. BODY.TBC SURGICAL PROCEDURE: The surgical procedure reported by Skuta et al10 was used. BODY.EX-PRESS P-50 IMPLANT SURGICAL PROCEDURE: The surgical procedure reported by Sarkisian6 was used. BODY.POSTOPERATIVE FOLLOW-UP: A first postoperative visit was at 24 hours, and topical antibiotic was prescribed (Ciprofloxacin), one drop four times a day; 1% prednisolone acetate, one drop six times a day; and dilator and/or cycloplegic agent, one drop two times a day. Subsequently, the patients were checked at 1 week, and first, third, sixth, and 12th postoperative month. The following outcomes were defined: Complete success: When the IOP was equal to or <21 mmHg but >5 mmHg or showed a 30% decrease in pressure from preoperative IOP, with no hypotensive medical treatment at 3 months follow-up. Qualified success: IOP ≤21 mmHg or showing a 20% decrease from preoperative IOP, with hypotensive medical treatment at 3 months follow-up. Failure: IOP >21 mmHg, or ≤5 mmHg; or less visual acuity than in the preoperative period. BODY.STATISTICAL ANALYSIS: Descriptive statistics were used. Subsequently, bivariate analysis of the variables of interest (Student’s t-test) and nonparametric tests (χ2 test) were done. The significance for testing the hypothesis was P<0.05. The data were analyzed using the SPSS version 13.0 (SPSS Inc., Chicago, IL, USA) statistical program. BODY.RESULTS: The EX-PRESS P-50 implant was placed in 20 eyes in 20 patients, and compared to a control group of 20 eyes in 20 patients who underwent TBC, excluding one patient in the TBC group who did not comply with follow-up. The mean follow-up was 8.6 months (standard deviation [SD] ±4.9 months) for the EX-PRESS P-50 group and 9.6 months (SD ±5.3 months) for the TBC group. The subjects from the first group, on average, were slightly younger, 63.45 years (SD ±14.9 years), than the control group, 65.2 years (SD ±14.6 years); this was not a statistically significant difference (Table 1). There was greater prevalence in male subjects and left-eye procedures, 61.9% in males and 38.1% in females. So, a bivariate analysis was performed with the operated eye and sex of the patient as variables, and no significant differences were found (P=0.75). There was also no difference between the operated eye and the surgical procedure (P=0.621) since out of the left eyes, 47.6% were from the EX-PRESS P-50 group and 52.4% were from the TBC group. BODY.VISUAL ACUITY PER TREATMENT GROUP: The mean of visual capacity measured in logarithm of the minimum angle of resolution (logMAR), per group of treatment during the study period (1 day, 1 week, 1 month, 3, 6, and 12 months), showed on the first day a significant difference in visual acuity between the EX-PRESS P-50 and TBC groups, with P=0.049. There was no significant difference in visual capacity in subsequent controls (Figure 1 and Table 2). BODY.INTRAOCULAR PRESSURE: There were no significant differences in the mean IOP evaluated with Student’s t-test at the established time points (baseline, 1 day, 3, 6, and 12 months). In both groups, the IOP reduction on the first day was 0–34 mmHg and in the first week was 2–30 mmHg, with no significant differences (P=0.189 and P=0.357, respectively). In the first month, the range of pressure reductions in the control group were 5–42 and 6–30 mmHg (P=0.380) in the EX-PRESS P-50 group and control group, respectively; in the third month, the pressures were 8–22 mmHg in the control group and 5–23 mmHg (P=0.145) in the EX-PRESS P-50 group; and in the sixth month, pressures were 8–19 mmHg in the control group and 5–44 mmHg (P=0.783) in the EX-PRESS P-50 group, with no significant differences between groups. Finally, pressures were 7–19 mmHg in the control group and 9–18 mmHg in the EX-PRESS P-50 group, with no significant differences (P=0.627; Table 3 and Figure 2). BODY.SURGICAL SUCCESS: Regarding surgical success per treatment group, in the first month, 75% of subjects in the EX-PRESS P-50 group were successful, while 78.9% in the control group were successful (P=0.347); in the third month, 90% in the EX-PRESS P-50 group were successful, while 84.2% with TBC were successful (P=0.297); and in the sixth month, 75% of subjects with EX-PRESS P-50 were successful and 85.7% with TBC were successful (P=0.200). Finally, 80% were still successful with EX-PRESS P-50, while 72.7% with TBC were successful (P=0.867; Table 4 and Figure 3). BODY.SURGICAL COMPLICATIONS: No significant differences were found in the bivariate analysis, among the complications in both groups in the first week (P=0.247), with 45% in the EX-PRESS P-50 group and 42.1% in the TBC group. Among the complications in the EX-PRESS P-50 group, we found hypotony 15%, but no choroidal detachment, whereas in the TBC group, there was no hypotony but 10% of patients had choroidal detachment. This could be associated with the sudden changes in IOP during the surgery of patients who underwent TBC. In terms of the Seidel test and resuture, the TBC group more often had a positive Seidel test, with 26.3% versus 20.0%; however, 4.7% more subjects had resuture in the EX-PRESS P-50 group. In the first month, there was no significant difference in complications between both groups (P=0.502), 25% in the EX-PRESS P-50 and 15.8% in the TBC group. In the first group, 5% had hypotony and 5% choroidal detachment. In contrast, the second group had no hypotony, but 10.5% of the subjects had choroidal detachment. Both groups continued with the same positive Seidel test rate. The positive Seidel test and resuture is still greater in the EX-PRESS P-50 group found in 10% of cases. In the third month, there were no complications in the TBC group, but 5% in the EX-PRESS P-50 group required resuture. The difference was not significant between the groups (P<0.513). Neither was there any significant difference found (P=0.405) in the sixth month. The TBC group still had no complications, and the other group presented hypotony, Seidel, resuture, and choroidal detachment. At 1-year follow-up, there were no complications in any of the groups (Table 5 and Figures 4 and 5). BODY.DISCUSSION: In terms of visual acuity, this study shows a significant reduction of vision from baseline levels of logMAR on the first day after surgery, but an improvement of vision to near baseline levels of logMAR by the first month in the EX-PRESS P-50 group, and by the third month in the TBC group. Vision continued to improve in both groups, and after the sixth month, slight improvements above baseline levels of logMAR were observed. These results are in accordance with those of Netland et al,11 who also found a reduction of visual acuity on the day following the procedure and recovery by the first month in the EX-PRESS patients and by the third month in TBC patients. Similarly, Seider et al12 noted a return to baseline levels of vision at the third month in both groups. Likewise Good and Kahook13 reported a very fast recovery of vision in the EX-PRESS group where vision near baseline levels of logMAR were achieved 1 week postoperation. In our study, the TBC group returned to baseline vision within 1 month. BODY.INTRAOCULAR PRESSURE: In assessing IOP, our study found similar changes in IOP between the two groups, without significant differences at any point. The mean preoperatory IOP in the EX-PRESS P-50 was 22.95 mmHg (SD 9.22), in the trabeculectomy (TBC) group 23.79 mmHg (SD 12.46); at the final follow up visit the IOP was 13.09 mmHg (SD 3.36) in the EX-PRESS P-50 group and 13.4 mmHg (SD 3.2) in the TBC group. These results are similar to the ones reported by Maris et al,8 in which preoperative pressure was 26.2±10.5 and 25.5±9.9 mmHg. In the EX-PRESS and TBC groups, at the final follow-up visit, IOP was 13.7±6.4 and 12.9±8.5 mmHg, respectively. In addition, the mean IOP in both groups did not differ statistically from the third month to the end of their study. Our results are consistent with previous published work about comparable success rates for each treatment group. At the last follow-up visit, the success rate of this study for EX-PRESS P-50 group was 90% (80% complete success and 10% qualified success), while the TBC success rate was 90.9% (72.7% complete success and 18.2% qualified success). Our results confirm those of Maris et al,8 who had 90% success in the EX-PRESS group and 92% in the TBC group. Marzette and Herndon5 also documented surgical success rates of 82% for EX-PRESS and 71% for TBC, but without statistical significance (P=0.182). The lower success rates in their study compared to this study may be explained by the sample of patients that were included, among them previous failed glaucoma surgeries, which according to Mariotti et al14 is one of the principal risk factors for failure (P=0.02). Presumably the reason for the lower success within previously failed glaucoma surgeries is the increased earlier conjunctival scarring near the second filtration surgery. Furthermore, Good and Kahook’s13 study did not find statistically significant differences in the success rates between the groups, but his study revealed slightly lower rates of success in both groups, with 82.85% success rate for EX-PRESS and 82.86% for TBC (unqualified success of 77.14% for EX-PRESS versus 74.29% for TBC [P=1.00], and qualified success of 5.71% and 8.57% [P=0.99], respectively). A plausible reason for the different rates of success might be the definition of success, considered to be an IOP of more than 5 mmHg but less than 21 mmHg. In contrast to previous studies, de Jong et al15 declared that control of the IOP in the first year was more effective with an EX-PRESS device than with TBC (86.6% success versus 61.5%, respectively), although the definition of success used by de Jong et al was different than ours, with a threshold pressure of 15 mmHg instead of 21 mmHg. The difference in success rates between his groups was statistically significant (P=0.01). With regard to complications, the hypotony rate was higher in the EX-PRESS group than in the control, even though the rate of hypotony of this study was within the values previously reported for EX-PRESS in the range from 4%5,8 to 47.2%.12 This reduction of hypotony rates on TBC group compared with previous studies could be explained by the use of ophthalmic viscosurgical devices during the TBC procedure. An alternative explanation for this finding includes less suture tension on the flap in the EX-PRESS group, or a looser seal of the flap against the EX-PRESS implant. On the other hand, similar to other studies, choroidal detachment was more frequent after TBC. The rate of this complication was also in the range previously published (from 3%5 to 38%).8 BODY.CONCLUSION: According to the study and the results obtained, we can conclude that both procedures for open-angle glaucoma treatment are equally efficacious, since there was no significant difference in IOP control or surgical success at 1 year, which was 80% in the EX-PRESS P-50 group and 72.7% in the TBC group.

TITLE: Femoral nerve block-sciatic nerve block vs. femoral nerve block-local infiltration analgesia for total knee arthroplasty: a randomized controlled trial ABSTRACT.BACKGROUND: The use of femoral nerve block (FNB) combined with sciatic nerve block (SNB) after total knee arthroplasty (TKA) has recently become controversial. Local infiltration analgesia (LIA) has been reported to be effective for postoperative TKA pain control. We aimed to assess whether LIA with continuous FNB is as effective as SNB combined with continuous FNB. ABSTRACT.METHODS: This was a prospective, randomized, single-center, observer-blinded, parallel group comparison trial of 34 American Society of Anesthesiologists (ASA) physical status 1–3 patients who underwent TKA and fulfilled the inclusion and exclusion criteria. Patients were randomized into two groups: a periarticular LIA and FNB group (group L, n = 17), and an SNB and FNB group (group S, n = 17). In both groups, participants received FNB with 20 mL of 0.375 % ropivacaine, and 5 mL h−1 of 0.2 % ropivacaine after surgery. In group L, participants received 100-ml injections of 0.2 % ropivacaine and 0.5 mg epinephrine to the surgical region. In group S, participants received SNB with 20 ml of 0.375 % ropivacaine. After TKA, Numeric Rating Scale (NRS) scores for the first 24 h post-operation were compared via repeated-measures analysis of variance (ANOVA) as the primary outcome. Other outcome measures included NRS score changes within groups, area under the curve for the NRS scores, total analgesic dose, change in knee flexion and extension, pain control satisfaction, nausea and vomiting, and hospital stay duration. ABSTRACT.RESULTS: NRS score changes were greater in group L than in group S (P < 0.01, ANOVA) and greater in group L than in group S at three postoperative time points: 3 h (P < 0.01), 6 h (P < 0.01), and 12 h (P = 0.013; Mann–Whitney U test). Changes in the mean NRS score were observed in each group (P < 0.01, Friedman test). No significant differences were detected in the other outcome measures (Mann–Whitney U, Wilcoxon signed-rank, and chi-squared tests). ABSTRACT.CONCLUSIONS: Sciatic nerve block with femoral nerve block is superior to local anesthetic infiltration with femoral nerve block for postoperative pain control within 3–12 h of total knee arthroplasty. ABSTRACT.TRIAL REGISTRATION: UMIN-CTRID:000013364R:000015591 BODY.BACKGROUND: Total knee arthroplasty (TKA) is a common operative procedure performed to improve mobility and quality of life [1]. Femoral nerve block (FNB) was recently reported to be useful for postoperative pain control after TKA [2, 3]. FNB can be performed more safely than epidural block in the presence of an anticoagulant [4, 5]. In contrast, continuous FNB with sciatic nerve block (SNB) was found to reduce analgesic requirements after TKA [6]. The pain and discomfort in the posterior aspect of the knee associated with TKA were reported to be reduced by SNB, and a single SNB injection was found to reduce severe pain on the day of surgery [7]. We have thus used continuous FNB combined with an SNB procedure for several years for postoperative pain control after TKA. However, the benefit of adding SNB to FNB to improve analgesia after TKA has recently become controversial [8]. Additionally, it has been reported that local infiltration analgesia (LIA) is superior to both FNB and epidural anesthesia for postoperative TKA pain control [9–11]. LIA is also reported to reduce pain in the early postoperative period [12–17]. The objective of this study was to compare the analgesic effect of LIA with that of SNB in combination with a continuous FNB for postoperative pain control after TKA. Our null hypothesis was that there would be no difference in NRS score changes between the groups. BODY.METHODS: This prospective, single-center, randomized, observer-blinded, parallel group comparison study was conducted in accordance with the ethical principles of the Helsinki Declaration, and was approved (authorization number A2405) by the Regional Ethical Committee of Saitama City Hospital (Research Ethics Committee No. 11000176) in September 2012. The registry number of this RCT is UMIN-CTR ID: 000013364 R: 000015591. The patients were recruited from October 2012 to July 2013. Thirty-four subjects were randomly assigned to two groups: an SNB group (group S) or a local anesthesia group (group L). All subjects provided written informed consent to participate in this study. Anesthesiologists who did not perform the anesthesia enrolled the participants and assigned them to the groups. The allocation ratio was 1:1. In total, 34 numbered cards assigned as L1-L17 and S1-S17 in an opaque envelope were used for random allocation to groups. The envelope was concealed in a safe and was opened on the morning of the operation. The patients and evaluators were blinded to the group assignments throughout the study. The inclusion criteria were as follows: Scheduled for unilateral TKA for degenerative arthritis, American Society of Anesthesiologists (ASA) physical status 1–3, weight 40–99 kg, and fully able to understand the study contents from oral and written descriptions. The exclusion criteria were: Scheduled for bilateral TKA, regular narcotic use, allergies to any study drug, neuromuscular disease, sensory disturbances of the legs, severe diabetes, heart failure (New York Heart Association classification greater than 2), renal impairment with an estimated glomerular filtration rate of < 60 mL min−1 1.73 m−2 or liver dysfunction (Child–Pugh classification greater than class B), and inability to be assessed via the Numeric Rating Scale (NRS). All patients received instruction in the use of the NRS, where the number 0 on the ruler represented the absence of pain and the number 10 represented the most severe pain they had ever experienced, and they were asked to indicate the number that best reflected their pain on that scale. The same team of orthopedists performed all surgeries, and the same anesthesiologist performed the anesthesia for all cases. All patients underwent general anesthesia induced by 2 mg kg−1 propofol and 50 μg of fentanyl, followed by insertion of a laryngeal airway. General anesthesia was maintained with inhaled 1–2 % sevoflurane in oxygen and air. Additionally, remifentanil administration was started at incision from 0.1 μg kg−1 min−1, increased or decreased when the blood pressure or heart rate changed by approximately 10 %, and stopped at least prior to skin closure. No additional narcotics or analgesics including fentanyl were administered during the operation. The operation was performed using the midvastus approach. None of the participants underwent patellar resurfacing. The resurfacing criterion in our facility is International Cartilage Repair Society Grade IV. The implant models used in this study were of the Triathlon (Stryker, Mahwah, NJ, USA) or Verilast (Smith & Nephew, Andover, MA, USA) type. The implant was fixed with cement (Simplex P Bone Cement with cefazolin) on the cut bone surfaces after confirmation of the joint gap, and balanced by trial and error. A vacuum drain was not used. Edoxaban tosilate hydrate (Lixiana®, Daiichi-Sankyo, Tokyo, Japan; 30 mg) was administered orally for 10 days from postoperative day 2. BODY.GROUP S: Subjects were moved to a lateral recumbent position after insertion of the laryngeal airway. An ultrasound-guided (M-Turbo Fujifilm SonoSite, Tokyo, Japan) SNB approach was utilized in combination with a 0.5–1.0 mA nerve stimulus for 0.1 ms (Stimplex HNS12, B. Braun, Melsungen, Germany). By setting the echo-probe (C60x/5-2 MHz Transducer Fujifilm SonoSite, Tokyo, Japan) parallel to the line created by the posterior superior iliac spine and ischial tuberosity, we inserted a needle (Stimplex D Needle STD-2280, B. Braun, Melsungen, Germany) with an in-plane approach from the lateral aspect of the ultrasound probe and injected 20 mL of 0.375 % ropivacaine after the position of the needle tip was confirmed by a motor response from the ankle. After SNB, the patient was moved into the supine position, and ultrasound-guided FNB was performed in combination with 0.5–1.0 mA nerve stimulation for 0.1 ms (Stimplex HNS12, B. Braun, Melsungen, Germany). We placed the echo-probe (HFL38x/13-6 MHz Transducer, Fujifilm SonoSite, Tokyo, Japan) parallel to the inguinal ligament region to identify the femoral nerve and then inserted a needle (Contiplex Touphy B. Braun, Melsungen, Germany) using an in-plane approach from the lateral aspect of the ultrasound probe. We then injected 20 mL of 0.375 % ropivacaine after the position was confirmed by contraction of the quadriceps muscle. After the ropivacaine bolus injection, a catheter tip (Aesculap, B. Braun, Melsungen, Germany) was placed 3 cm from the needle tip. A continuous FNB (5 mL h−1) of 0.2 % ropivacaine was commenced at the end of the operation. BODY.GROUP L: Subjects in group L underwent FNB similarly to those in group S. The local anesthetic mixture was prepared from 100 mL of 0.2 % ropivacaine by adding 0.5 mL (0.5 mg) of adrenaline. The local anesthetic mixture was administered three times in measured doses as follows:20 mL of the local anesthetic mixture was administered intracutaneously to the surgical region at the start of the operation. 50 mL of the local anesthetic mixture was administered as infiltration anesthesia to sites posterior to the articular capsule near the incised part, namely the vastus intermedius, vastus lateralis, and lateral collateral ligament, before the injection of cement. 30 mL of the local anesthetic mixture was administered intraarticularly at the end of the operation. A continuous femoral block of 0.2 % ropivacaine at 5 mL h−1 was commenced at the end of the operation, as in group S. The participants were administered a 25-mg diclofenac suppository if their NRS score was > 3 upon exiting the operating room, and if at 3, 6, 12, and 24 h after that time or at any other time they requested an analgesic and had an NRS score higher than 3. Repeated administrations of 25-mg diclofenac suppositories were allowed after a 3-h interval. If this method was inadequate and the patient could not cope with the pain (NRS score > 3), 15 mg of pentazocine was injected intramuscularly. We did not apply a strict fast-track recovery program in this study. BODY.OUTCOME MEASURES: The primary outcome was the change in NRS scores between groups L and S at five time points: upon exiting the operating room and 3, 6, 12, and 24 h later. Nurses in the operating room and on the ward who were blinded to the group allocations recorded the NRS scores. Other outcome measures included NRS score changes within groups, the area under the curve of the NRS scores, total dose of diclofenac, change in knee flexion and extension, pain control satisfaction, nausea and vomiting, and hospital stay duration. Physiotherapists measured knee flexion and extension using an angle meter every postoperative day. The Knee Society Score (The 2011 Knee Society Knee Scoring System) [18] was recorded before the operation and at discharge. Furthermore, the participants were asked about numbness or unusual sensations in their tongue and mouth, metallic tastes, dizziness, tinnitus, and agitation, and they were also checked for possible ropivacaine side effects by evaluating their level of consciousness and convulsions upon exiting the operating room and at 30 min, 1 h, and 3 h after that time. Nausea and vomiting were evaluated at the same time by inquiring about the presence or absence of symptoms. Nurses on the ward who were blinded to the group allocations checked these items. In addition, during the first 24 h after the operation, the nurses and physicians in charge observed the participants closely on the ward every 4 h. If symptoms of ropivacaine toxicity were detected, the continuous infusion of ropivacaine was discontinued, and symptomatic treatment was performed accordingly. In cases of unanticipated side effects, the details were recorded. Analgesic administration times were recorded, and the total doses were calculated. On the third postoperative day, a survey was administered relating to pain control satisfaction, consisting of a five-point scale (1 = extremely severe pain; 2 = quite severe pain; 3 = neutral; 4 = generally content; 5 = extremely content). No changes to trial outcomes or methods occurred after the trial commenced. BODY.STATISTICS: Repeated measures analysis of variance (ANOVA) was used to compare the changes in NRS scores between groups S and L. The differences in NRS scores at each time point and the area under the curve of the NRS scores were analyzed using the Mann–Whitney U test. The changes in NRS scores within each group were analyzed by the Friedman test. The values at 3, 6, 12, and 24 h were compared to those at 0 h by the Wilcoxon signed-rank test with Bonferroni correction in both groups, after differences within groups were confirmed. The P value for the primary outcome measure was determined using a two-tailed test. Other ordinal variables and continuous variables were analyzed by the Mann–Whitney U test or Wilcoxon signed-rank test. Categorical variables were analyzed by a chi-squared test for independence. Continuous variables are presented as the mean and standard deviation. Ordinal variables are presented as the median and interquartile range. Categorical variables are presented as percentages. The sample size of the primary outcome measure was calculated by G*Power 3 (Erdfelder & Buchner, 2007). With an alpha error of 0.05, a beta error of 0.2, and an effect size, f, of 0.25, the required sample size for detecting a significant difference between the groups was calculated to be 28. We enrolled 34 cases (17 to each group), anticipating that some patients may be lost to follow-up. Statistical analyses were performed using SPSS (SPSS Statistics 19 for Windows; SPSS Inc, Chicago, IL). Significance was defined as P < 0.05. BODY.RESULTS: BODY.RANDOMIZED TRIAL PROGRESS AND PARTICIPANT BACKGROUND: Thirty-eight patients were assessed as competent. Four patients were excluded, and 34 participants were randomized into the study groups. Seventeen participants were allocated to each group. In group L, one participant was excluded after allocation, as an NRS score could not be obtained because of delirium. A patient flow diagram generated in accordance with the CONSORT 2010 statement guidelines is shown in Fig. 1.Fig. 1Flow diagram generated in accordance with CONSORT 2010 guidelines No hospital readmissions or infections occurred during the first 60 postoperative days. The patients’ characteristics were similar in both groups. No significant difference was detected in side, sex, age, weight, body mass index, ASA status, operation time, anesthetic time, intraoperative blood loss, intraoperative fluids, remifentanil dosage, implant choice, knee flexion and extension, or Knee Society Score (Table 1).Table 1Patient characteristics and clinical variables Group LGroup SP value(n = 16)(n = 17)Side, right : left, n (%) 10 : 6 (63 : 38) 8 : 9 (47 : 53) 0.37 Sex, men : women, n (%) 3 : 13 (19 : 81) 2 : 15 (12 : 88) 0.58 Age, years (range) 73 (5.9) 72 (10) 0.97 Weight, kg (range) 62 (12.5) 55 (8.2) 0.14 Body mass index (range) 27 (5.0) 25 (3.5) 0.10 ASA status 1/2/3 2 (0) 2 (0) 0.27 Surgical time (min) (range) 81 (20) 71 (15) 0.09 Anesthetic time (min) (range) 159 (25) 147 (19) 0.09 ROM (preoperative extension) (degrees) 10 (9.6) 8.4 (8.5) 0.65 ROM (preoperative flexion) (degrees) 122 (6.7) 128 (13) 0.08 KSS (score 0–100) 36 (20) 35.5 (19) 0.69 Intraoperative blood loss (mL) 3.5 (12) 20 (36) 0.14 Intraoperative fluids (mL) 1156 (210) 1309 (231) 0.06 Remifentanil dosage (mcg) 148 (45) 117 (52) 0.08 Implant T : V, n (%) 5 : 11 (31 : 69) 4 : 13 (24 : 76) 0.62 Data are expressed as the mean (SD), median (interquartile range: value at 75 %–value at 25 %), or n (%), and were compared using the Mann–Whitney U test or chi-squared test T Triathlon, V Verilast, KSS Knee Society Score BODY.POSTOPERATIVE OUTCOMES: A significant difference was detected in NRS score changes between groups S and L (P < 0.01). The NRS scores were higher in group L than in group S at three time points, i.e., 3 h (P < 0.01), 6 h (P < 0.01), and 12 h (P = 0.013) after patients exited the operating room (Fig. 2). Significant NRS score changes were apparent within each group. Significant changes from the mean NRS score at 0 h were recorded at 12 h (P < 0.01) and 24 h (P < 0.01) in group S, and at 3 h (P = 0.011), 6 h (P < 0.01), 12 h (P < 0.01), and 24 h (P < 0.01) in group L after the patients exited the operating room (Fig. 2). A significant difference in the area under the curve for the NRS scores was detected between the groups (Table 2).Fig. 2Changes in NRS scores from 0–24 h in groups L and S after TKA. The groups were compared by repeated-measures analysis of variance and Mann–Whitney U tests. Comparisons within the groups were analyzed by the Friedman test and Wilcoxon signed-rank test with Bonferroni correction (versus the 0-h value). The mean and SD are indicated. *P < 0.05, comparisons between the groups; †P < 0.05, comparisons within the groups. NRS, numerical rating scale; TKA, total knee arthroplasty Table 2Comparison of NRS scores and area under the curve of NRS scores between the groups Time after exiting roomGroup LGroup SP value(n = 16)(n = 17)0 h 0 ± 0 0 ± 0 3 h 2.13 ± 2.66 0.06 ± 0.24 <0.01 6 h 2.5 ± 2.34 0.18 ± 0.39 <0.01 12 h 3.5 ± 1.83 2 ± 2.12 0.013 24 h 3 ± 1.86 2.47 ± 1.59 0.3 Area under the curve of NRS 66.5 ± 27.1 33.4 ± 19.4 <0.01 Data are expressed as mean ± SD, and compared by Mann–Whitney U test No significant differences were detected in nausea or vomiting frequency, degree of pain control satisfaction, dose of diclofenac, or time of first administration of an analgesic after patients exited the operating room. There was no occurrence of local anesthetic toxicity in either group. Hospital stay duration, changes in knee flexion and extension measured by the physiotherapists, and changes in the Knee Society Score were also similar between the groups (Table 3). Comparable results were observed at discharge between the groups. No other side effects or unanticipated postoperative complications were recognized in either group. No participant in either group received intramuscularly injected pentazocine.Table 3Comparison of postoperative factors or changes between the two groups Group LGroup SP value(n = 16)(n = 17)Nausea or vomiting (%) 4/16 (25) 4/17 (24) 0.92 Symptoms of local anesthetic poisoning (%) 0/16 (0) 0/16 (0) 1.00 Satisfaction 4 (1) 4 (1) 0.75 Dose of diclofenac (mg) 0 (25) 25 (25) 0.28 Time until first analgesic usage (hour) 20 (5.4) 18.6 (7.7) 0.59 Hospital stay (days) 25 (7.75) 24 (11.00) 0.69 ROM (extension, temporal change) (degrees) −8 (11) −6.9 (10) 0.89 ROM (flexion, temporal change) (degrees) −6.1 (13) −6.3 (13) 0.59 KSS (temporal change) 42 (42.5) 49.5 (24.3) 0.86 Data are expressed as the mean (SD), median (interquartile range: value at 75 %–value at 25 %), or n (%) and were compared using the Mann–Whitney U test, Wilcoxon signed-rank test, or chi-squared test. Satisfaction was rated using a five-point evaluation system ROM range of motion, KSS Knee Society Score BODY.DISCUSSION: In this study, group S showed superior postoperative pain control to group L within 3–19]. There was no difference between the groups in the total analgesics required, time to first analgesic administration, nausea, vomiting, patient satisfaction, or hospital stay duration. The lack of an observed difference may result from FNB alone providing sufficient analgesia, such that the effect of the adjunctive technique is masked. The maximum pain in both groups was still relatively mild, even at 24 h, which supports this explanation. Notably, there are reports challenging the importance of SNB for TKA [20, 21]. The administration method for the local anesthetic mixture was decided by referring to the techniques of Kerr et al. [22]. We did not insert a catheter into the joint because of the risk of infection. Even though the infection risk is low, concerns of infection still exist [23–26]. Incidentally, mobility impairment of the area supplied by the peroneal nerve after plasty of the nerve was reported to occur in approximately 0.3–10 % of TKA cases, irrespective of the presence of nerve block [27]. In our study, one patient in group L experienced a fall on the day after surgery. There are limitations to our study. First, a better ropivacaine dosage and injection site for the LIA method may be available. An increase in the ropivacaine dose and limitation of the administration area to the posterior site of the knee may lead to different results. Regarding local anesthetic doses, we decided that the total ropivacaine dose for local infiltration should be 200 mg (0.2 % 100 ml), considering that 75 mg (0.375 % 20 ml) of ropivacaine was used as an FNB shot before operation, and approximately 300 mg of ropivacaine was considered to be safe by several papers [10, 28–30]. However, a study similar to our study reached different conclusions [31]. In that study, the doses of FNB and SNB were the same as ours, but the ropivacaine dose for LIA was 300 mg, and injections were only performed in the posterior part of the knee. However, the validity of increasing the ropivacaine dose is controversial [32, 33]. Second, our mixture of local anesthetics did not contain ketorolac or corticosteroids. It was previously reported that inclusion of ketorolac in LIA resulted in reduced pain intensity [34]. Corticosteroids have also been reported to provide effective analgesia [35–39]. Although the possibility of infection exists if administration is performed prior to TKA [40–43], administration during operation as LIA has been reported as safe with regard to infection [35–38]. Devandra et al. demonstrated that there is no difference between SNB and LIA as adjuncts to FNB in terms of postoperative opioid requirement [44]; however, pain scores were higher in the LIA group in the first 12 h. This is compatible with our study, and the periatricular injection did not include ketorolac and corticosteroids as in our study. Thus, the LIA contents of our study should be reconsidered. BODY.CONCLUSION: Our results suggest that postoperative pain control within 3–12 h of TKA is better attained through SNB than LIA as a supplement to continuous FNB, without any significant differences in complications, functional outcomes, anesthetic time, or hospitalization. This knowledge will be of value to individuals responsible for determining the anesthesia strategy to control intense postoperative pain after TKA.

TITLE: Smart Care™ versus respiratory physiotherapy–driven manual weaning for critically ill adult patients: a randomized controlled trial ABSTRACT.INTRODUCTION: A recent meta-analysis showed that weaning with SmartCare™ (Dräger, Lübeck, Germany) significantly decreased weaning time in critically ill patients. However, its utility compared with respiratory physiotherapist–protocolized weaning is still a matter of debate. We hypothesized that weaning with SmartCare™ would be as effective as respiratory physiotherapy–driven weaning in critically ill patients. ABSTRACT.METHODS: Adult critically ill patients mechanically ventilated for more than 24 hours in the adult intensive care unit of the Albert Einstein Hospital, São Paulo, Brazil, were randomly assigned to be weaned either by progressive discontinuation of pressure support ventilation (PSV) with SmartCare™. Demographic data, respiratory function parameters, level of PSV, tidal volume (VT), positive end-expiratory pressure (PEEP), inspired oxygen fraction (FiO2), peripheral oxygen saturation (SpO2), end-tidal carbon dioxide concentration (EtCO2) and airway occlusion pressure at 0.1 second (P0.1) were recorded at the beginning of the weaning process and before extubation. Mechanical ventilation time, weaning duration and rate of extubation failure were compared. ABSTRACT.RESULTS: Seventy patients were enrolled 35 in each group. There was no difference between the two groups concerning age, sex or diagnosis at study entry. There was no difference in maximal inspiratory pressure, maximal expiratory pressure, forced vital capacity or rapid shallow breathing index at the beginning of the weaning trial. PEEP, VT, FiO2, SpO2, respiratory rate, EtCO2 and P0.1 were similar between the two groups, but PSV was not (median: 8 vs. 10 cmH2O; p =0.007). When the patients were ready for extubation, PSV (8 vs. 5 cmH2O; p =0.015) and PEEP (8 vs. 5 cmH2O; p <0.001) were significantly higher in the respiratory physiotherapy–driven weaning group. Total duration of mechanical ventilation (3.5 [2.0–7.3] days vs. 4.1 [2.7-7.1] days; p =0.467) and extubation failure (2 vs. 2; p =1.00) were similar between the two groups. Weaning duration was shorter in the respiratory physiotherapy–driven weaning group (60 [50–80] minutes vs. 110 [80–130] minutes; p <0.001). ABSTRACT.CONCLUSION: A respiratory physiotherapy–driven weaning protocol can decrease weaning time compared with an automatic system, as it takes into account individual weaning difficulties. ABSTRACT.TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT02122016. Date of Registration: 27 August 2013. BODY.INTRODUCTION: A large proportion of intensive care unit (ICU) patients require mechanical ventilation for more than 24 hours [1]. Automatic ventilator control and ventilation modes designed for weaning from mechanical ventilation can be particularly helpful, as 40 % of mechanical ventilation time is spent on this procedure [2]. Owing to the complexity of the decision-making process for weaning, the use of computers has been increasingly favored to ensure that protocols are followed completely and safely [3]. Automated weaning programs were developed to bring better control to this process and to make it faster and more secure [4]. Patients’ information is entered into the program for monitoring, then, through the program’s decision-making process defined by the weaning protocol, the computer suggests changes or maintenance of ventilator parameters [5]. This happens through the direct inclusion of monitored data into the program by the ICU team, whereby the computer defines the action to be taken and the caregivers act accordingly, with the program dictating the protocol. This process can also occur automatically, through the so-called closed-loop ventilation system, when this software is integrated with a mechanical ventilator. The ventilator monitors the patient and, through an internal algorithm, changes ventilation parameters according to the ventilation program in a constant feedback ventilation [5]. In general, ventilation modes in closed loop target the lowest possible ventilatory support needed to properly ventilate the patient, which is why the most popular forms of closed-loop ventilation are those used for weaning from mechanical ventilation. Automated systems use closed-loop control to enable ventilators to perform basic and advanced functions while supporting respiration [6]. A recent meta-analysis showed that weaning with SmartCare™ (Dräger, Lübeck, Germany) significantly decreases weaning time in critically ill patients [7]. SmartCare™ is a unique automated weaning system that measures selected respiratory variables, adapts ventilator output to individual patient needs by operationalizing predetermined algorithms and automatically conducting spontaneous breathing trials (SBTs) when predetermined thresholds are met. ICU weaning protocols driven by respiratory therapists and/or physiotherapists can also be effective in shortening weaning duration [8, 9]. In the adult ICU of Albert Einstein Hospital, São Paulo, Brazil, there is a weaning protocol [10] that consists of a daily assessment for the possibility of an SBT. The ICU SBT is performed in pressure support ventilation (PSV), which allows for better patient monitoring, a greater control of inspired oxygen fraction (FiO2) and the maintenance of positive end-expiratory pressure (PEEP). The SBT is performed as soon as the daily assessment indicates that a weaning trial is feasible. If the SBT is successful, the patient is extubated. Otherwise, patients return to the previous ventilation mode and another daily assessment of readiness for weaning is performed the next day (24-hour interval). If it succeeds, another SBT is attempted [10]. We hypothesized that the weaning strategy with SmartCare™ ventilatory mode would be as effective as our respiratory physiotherapy–driven weaning protocol in the ICU patients receiving invasive mechanical ventilation for more than 24 hours. Therefore, the objective of this study was to compare SmartCare™ ventilatory mode with our validated respiratory physiotherapy–driven weaning protocol [10] in critically ill patients receiving invasive mechanical ventilation for more than 24 hours. We sought to evaluate the following:Primary outcome: duration of weaning or weaning time (from randomization to extubation) Secondary outcomes:Rate of extubation failure (the need to return to invasive mechanical ventilation within 48 hours of extubation) Mechanical ventilation duration or mechanical ventilation time (from intubation to extubation) Physiologic measurements: respiratory rate (f), tidal volume (VT), rapid shallow breathing index (RSBI; calculated as frequency of breaths [respiratory rate] divided by tidal volume in liters [f/VT {L}]), forced vital capacity (FVC), level of PSV, PEEP, FiO2, end-tidal carbon dioxide concentration (EtCO2) and airway occlusion pressure at 0.1 second (P0.1) at the beginning of weaning and before extubation BODY.METHODS: A randomized, unblinded, prospective, controlled clinical trial was carried out in 70 consecutive adult critically ill patients who were intubated and mechanically ventilated for more than 24 hours in the adult ICU of Albert Einstein Hospital, São Paulo, Brazil. The study was approved by the ethics committee of Albert Einstein Hospital (CEP: 1506) and signed at “Plataforma Brasil” (SGPP 1260-10). Signed informed consent was obtained from each patient or the patient’s next of kin. The study was registered at ClinicalTrials.gov on 27 August 2013 (NCT02122016). The inclusion criteria were adult patients on mechanical ventilation for more than 24 hours who were able to initiate spontaneous inspiratory effort, presenting spontaneous eye opening and response to stimuli with minimal level of sedation, and in whom the cause that led or contributed to the need of mechanical ventilation was resolved. The patients had to have an FiO2 less than 0.4, peripheral oxygen saturation (SpO2) greater than 93 % and PEEP less than 10 cmH2O. They had to be hemodynamically stable as expressed by good tissue perfusion, independence from or low doses of vasopressors (dosage of vasoactive drug less than 0.05 μg/kg/min) and absence of decompensated coronary insufficiency or arrhythmias with hemodynamic repercussion. The exclusion criteria were presence of tracheostomy, neurologic damage with limited prognosis (after cardiac arrest or central neurologic damage) or Glasgow Coma Scale score less than 10. Subsequently, the patients screened as suitable for weaning were subjected to assessment of the following:Maximal inspiratory pressure (Pimax) measurement with a manometer (Commercial Medical Electronics, Tulsa, OK, USA) and a unidirectional valve (Hudson RCI; Teleflex, Morrisville, NC, USA) connected to the tube (Hudson RCI) and with use of a bacterial filter (Gibeck ISO-Gard; Teleflex) (Measurements were based on functional residual capacity.) Maximal expiratory pressure (Pemax) measurement with a manometer based on total lung capacity Spontaneous VT and FVC measurements with a spirometer (Wright spirometer, British model) plugged into the airway through a bacterial filter (ISO-Gard) (We measured initially minute ventilation in liters per minute and respiratory rate (f) for 1 minute to calculate VT. Next, to measure FVC, the respiratory physiotherapist ensured that the patient breathed in as deeply as possible and then asked the patient to forcibly blast the breath out as fast and as long as possible.) RSBI (We directly connected the Wright ventilometer [British model] to the endotracheal tube to measure the patient’s spontaneous VT and the respiratory rate (f) for a duration of 1 minute. The index was obtained by calculating f/VT [L].) After the measurements were performed, the patients were randomized by drawing folded slips of paper from a large, opaque envelope. Each slip of paper provided an identification number that assigned the patient to one of the two weaning modes (Fig. 1): (1) the respiratory physiotherapy–driven group or (2) the weaning with SmartCare™ group.Fig. 1Study flowchart. EtCO2, End-tidal carbon dioxide concentration; f/VT, Frequency of breaths (respiratory rate) divided by tidal volume; FiO2, Inspired oxygen fraction; FVC, Forced vital capacity; P0.1, Airway occlusion pressure at 0.1 second; PEEP, Positive end-expiratory pressure; Pemax, Maximal expiratory pressure; Pimax, Maximal inspiratory pressure; PSV, Pressure support ventilation; SpO2, Peripheral oxygen saturation; VT, Tidal volume BODY.RESPIRATORY PHYSIOTHERAPY–DRIVEN WEANING GROUP: The randomized patients assigned to the respiratory physiotherapy–driven weaning group were ventilated on continuous positive airway pressure (CPAP)/PSV mode (EVITA XL; Dräger) using PSV mode. All the patients were subjected to an SBT, which consists of an integrated assessment of the patients while they breathe spontaneously through PSV of 5–7 cmH2O and PEEP of 5 cmH2O. The SBT in the PSV mode is carried out for a minimum of 30 minutes and a maximum of 2 hours. Respiratory rate, level of PSV, VT, PEEP, FiO2, SpO2, EtCO2 and P0.1 were recorded at the beginning of the weaning process and before extubation. Patients who failed the SBT were returned to the PSV ventilatory mode that was titrated to a minimum level to ensure patient comfort: f <28 breaths/min and VT >5 ml/kg of predicted body weight. SBT failure was defined as tachycardia (heart rate >140 beats/min); increased respiratory rate (f >35 breaths/min); SpO2 <90 %; systolic blood pressure >180 mmHg or <90 mmHg; and signs or symptoms of agitation, sweating or altered level of consciousness. After the patients who failed an SBT were returned to the PSV ventilatory mode, they were then reevaluated to identify the causes of the failure. After 24 hours, the patients were reevaluated again, and, if they passed the daily assessment for eligibility to wean, a new SBT was performed. The duration of weaning (from randomization to extubation), the rate of extubation failure (need to return to invasive mechanical ventilation within 48 hours after extubation) and mechanical ventilation time (from intubation to extubation) were recorded. BODY.AUTOMATIC WEANING GROUP (SMARTCARE™ GROUP): The patients were ventilated on CPAP/PSV mode (EVITA XL) using PSV mode. The icon SmartCare™ was triggered, and a set of relevant information was adjusted: patient’s weight, type of tracheal prosthesis, whether the patient had neurological disease or chronic obstructive pulmonary disease and whether the mode was to work throughout the night. The apnea parameters were then set for apnea ventilation: f of 15 breaths per minute, VT of 6 ml/kg of predicted body weight and flow trigger sensitivity of 2 L/min. After these adjustments, the SmartCare™ mode was then initiated. The ventilator automatically adjusted pressure support at the minimum level while keeping the patient within a comfort zone. If the patient’s condition developed satisfactorily, the ventilator attempted an SBT and sounded an alarm when the patient was ready for extubation. As soon as the warning alarm illuminated and sounded, it was quickly detected by the bedside physiotherapy team, who immediately extubated the patient. Respiratory rate, level of PSV, VT, PEEP, FiO2, SpO2, EtCO2 and P0.1 were recorded at the beginning of the weaning process and before extubation. Weaning duration, extubation failure rate and mechanical ventilation duration were also recorded. BODY.STATISTICAL DATA ANALYSIS: Absolute frequencies and percentages of categorical variables were described for each group. Comparisons between groups were performed by χ2 or Fisher’s exact tests. Normality in the distribution of variables within each group was tested with the Shapiro-Wilk test, and homogeneity of variances was assessed with Levene’s test. Normally distributed variables were described by their mean and standard deviation and compared using Student’s t tests. Otherwise, medians and interquartile ranges (IQRs) were used, and the Mann–Whitney U test was used for comparisons. A Bonferroni correction for multiple comparisons was applied if necessary. Analyses were performed with IBM SPSS version 17.0 software (IBM, Armonk, NY, USA). p values less than 0.05 were considered statistically significant. BODY.RESULTS: BODY.PATIENTS’ CHARACTERISTICS: A total of 70 consecutive patients were enrolled, 35 in each weaning protocol group. Mean age, gender distribution and patient diagnoses were similar between the two groups (Table 1). Diagnoses at patient ICU admission were predominantly respiratory, hepatic, gastrointestinal and infectious. They did not differ between the two groups.Table 1Characteristics of ICU patients under the two weaning protocols: respiratory physiotherapy–driven weaning and automatic weaning (SmartCare™ mode) Weaning mode p ValueRespiratory physiotherapy–driven (n =35)SmartCare™ (n =35) n % n %Diagnosis at ICU admission Cardiac 2 5.7 % 1 2.9 % 0.722 Gastrointestinal 7 20.0 % 6 17.1 % Hepatic 7 20.0 % 6 17.1 % Infection 3 8.6 % 8 22.9 % Neurologic 2 5.7 % 2 5.7 % Oncologic 0 0.0 % 1 2,9 % Orthopedic 1 2.9 % 0 0.0 % Respiratory 13 37.1 % 11 31.4 % Total 35 100.0 % 35 100.0 % Age, yr 66 ± 18 (20–93) 62 ± 19 (33–97) 0.388 Sex Male 17 (49 %) 22 (63 %) 0.336 Female 18 (51 %) 13 (37 %) When we compared the two weaning groups, we found no significant difference in the Pimax (p =0.270) and Pemax (p =0.058) measured before the SBT. There was also no significant difference between the two groups concerning the FVC or the RSBI (Table 2).Table 2Maximum inspiratory and expiratory pressures, rapid shallow breathing capacity and forced vital capacity before weaning start among ICU patients Weaning mode p ValueRespiratory physiotherapy driven, median (IQR)Smart Care™, median (IQR) groupf/VT 40 (26–71) 35 (24–56) 0.404 FVC 975 (900–1550) 1200 (900–1900) 0.683 Pimax −40 (35–50) −45 (40–50) 0.270 Pemax 40 (22–45) 40 (30–60) 0.058 Abbreviations: f/VT respiratory rate divided by tidal volume in liters; FVC forced vital capacity in milliliters, P e max maximum expiratory pressure (cmH2O), P i max maximum inspiratory pressure (cmH2O) There was no difference in the need for reintubation between groups (two patients from each group required reintubation; p =1.00) or in the frequency of mechanical ventilator malfunctions between the ventilation modes (p =0.239). However, three technical hitches were observed in the automatic mode. Two difficulties in the calibration of EtCO2 coupled to the ventilator, and one difficulty during the decrease of PSV caused by psychomotor agitation of the patient led to tachypnea. There were more clinical complications during the weaning in the SmartCare™ group (p <0.001) (Table 3), which were not related to the ventilator mode itself. Of the two complications in the respiratory physiotherapy–driven weaning group, one was the result of a delay in performing a cuff leak test owing to psychomotor agitation and delirium, and the other was associated with a patient’s episode of arrhythmia during the weaning process, followed by medication before the process could continue. There were six clinical complications in the SmartCare™ group. One involved a positive cuff leak test that indicated upper-airway edema which needed to be treated with corticosteroids. One patient had temporary hemodynamic instability that had to be treated before extubation. One patient had gastrointestinal bleeding. One patient had nausea impairing the extubation. One was a case of difficult airway that the attending physician determined required extubation by bronchoscopy. One was a patient who needed insertion of a femoral catheter (for dialytic procedure) just after the weaning process was started.Table 3Reintubation rate, ventilator malfunction and clinical complications in the two weaning protocols Weaning mode p ValueRespiratory physiotherapy–driven weaningSmartCare™ group n % n %Reintubation rate 2 5.7 % 2 5.7 % 1.00 Ventilator related malfunction 0 0.0 % 3 8.6 % 0.239 Clinical complications not related to the ventilatory mode itself 2 5.7 % 6 22.9 % 0.001 BODY.SIMILAR VENTILATION TIME, BUT SHORTER WEANING DURATION, ASSOCIATED WITH RESPIRATORY PHYSIOTHERAPY–DRIVEN WEANING PROTOCOL: There was no difference between the two groups at the beginning of the weaning trial in FVC (p =0.683), f/VT (L) (p =0.404), PEEP (p =0.944), VT (p =0.509), FiO2 (p =0.499), SpO2 (p =0.774), f (p =0.947), EtCO2 (p =0.422) or P0.1 (p =0.201), except that PSV that was lower in the respiratory physiotherapy–driven weaning group (8 [IQR: 7–10] cmH2O vs. 10 [8–12] cmH2O; p =0.007). When the patients were ready for extubation, PSV was significantly higher in the respiratory physiotherapy–driven weaning group (8 [IQR: 7–8] cmH2O vs. 5 [IQR: 5–8] cmH2O; p =0.015), as was PEEP (8 [IQR: 7–8] cmH2O vs. 5 [IQR: 5–5] cmH2O; p <0.001), indicating that the decision to extubate was more rigidly controlled in the SmartCare™ automatic group (Table 4). Total duration of mechanical ventilation was similar in both groups (p =0.467). However, weaning duration and weaning time in the respiratory physiotherapy–driven weaning group were significantly shorter (60 [50–80] minutes vs. 110 [80–130] minutes; p <0.001) (Table 5).Table 4Respiratory parameters at the weaning start and end Start of weaningEnd of weaningWeaning modeRespiratory therapy–driven weaningSmartCare™ weaningRespiratory therapy–driven weaningSmartCare™ weaningMedian (IQR)Median (IQR) p ValueMedian (IQR)Median (IQR) p ValueVT (ml) 491 (350–611) 515 (413–612) 0.509 500 (420–600) 500 (450–630) 0.535 f (breaths/min) 20 (16–24) 20 (17–24) 0.947 17 (15–20) 18 (17–20) 0.354 PSV (cmH2O) 8 (7–10) 10 (8–12) 0.007 8 (7–8) 5 (5–8) 0.015a PEEP (cmH2O) 8 (8–10) 8 (8–10) 0.944 8 (7–8) 5 (5–5) b FiO2 (%) 0.30 (0.25–0.30) 0.30 (0.25–0.30) 0.499 0.30 (0.25–0.30) 0.30 (0.25–0.30) 0.572 SpO2 (%) 99 (98–100) 99 (98–100) 0.774 99 (98–100) 99 (98–99) 0.617 P0.1 (cmH2O) 2.5 (0.7–4.2) 1.3 (0.7–2.5) 0.201 2.8 (0.6–4.1) 1.6 (1.2–2.4) 0.201 EtCO2 Mean (SD) 35.7 (7.4) 33.6 (8.2) 0.422 35.4 (7.2) 34.9 (6.6) 0.826 Abbreviations: EtCO 2 end-tidal expiratory carbon dioxide, f respiratory rate, F i O 2 inspired fraction of oxygen, IQR interquartile range, P 0.1 airway occlusion pressure at the first millisecond, PEEP positive end-expiratory pressure, PSV pressure support ventilation, SD standard deviation, SpO 2 peripheral oxygen saturation, VT tidal volume a p <0.05 b p <0.01 Table 5Mechanical ventilation time and weaning duration in the two weaning protocols Weaning mode p ValueRespiratory physiotherapy–drivenSmartCare™ groupMechanical ventilation time (days), median (IQR) 3.5 (2.0–7.3) 4.1 (2.7–7.1) 0.467 Weaning duration (min), median (IQR) 60.0 (50.0–80.0) 110.0 (80.0–130) <0.001 Abbreviation: IQR interquartile range BODY.DISCUSSION: The results of this study demonstrate that weaning time using SmartCare™ was longer than that of the respiratory physiotherapy–driven protocol. A recent meta-analysis [7] involving 496 participants (7 trials of time from randomization to extubation) [11–17] showed that weaning time using SmartCare™ was decreased compared with non-automated weaning (MD −2.68 days; 95 % confidence interval [CI] −3.99 to −1.37; p value <0.0001) in the presence of substantial heterogeneity (I2 =68 %; p =0.005). One possible explanation for the unexpected result of this trial that showed an increased weaning time in the SmartCare™ group is that weaning time in the meta-analysis was defined as the time from randomization to extubation (as determined by the authors) [7]. Weaning time as defined by authors can vary among studies as follows: (1) from the initiation of spontaneous breathing (CPAP), (2) from the initiation of an assisted mode of ventilation (e.g., pressure support, volume-assisted, Proportional Assist Ventilation Plus [PAV+; Covidien, Boulder, CO, USA], adaptive support ventilation, Automatic tube compensation), or (3) as the patient is ready to undergo an SBT. In this study, we analyzed critically ill patients ventilated for more than 24 hours who successfully passed through a daily assessment of weaning evaluation, after which the SBT and extubation were quicker in the respiratory physiotherapy–driven protocol. This finding may be attributable to more efficient management of the intercurrences and individual variability, as there were human brains behind the decisions. Another explanation for the prolonged weaning with SmartCare™ is that more clinical complications not related to the ventilatory mode itself occurred in this group, which may have delayed the patients’ extubation procedures. If the start of a weaning trial is considered to be when the patient commences assisted ventilation, the weaning time would be increased compared with a patient who successfully passed a complete daily assessment for weaning evaluation. In terms of weaning duration, it took, on average, 60 and 110 minutes for the respiratory physiotherapy–driven weaning and SmartCare™ mode, respectively. Although this difference was significant, both values are within the ideal interval established in the literature for the performance of SBTs (30–120 [18-20] minutes). In a recent meta-analysis [7], the pooled results of 7 studies (496 patients) [11–17] showed that weaning time for SmartCare™ weaning groups was 4.57 (3.77) days vs. 7.56 (10.52) in non-automated groups. This much greater weaning time derived from the meta-analysis vs. minutes observed in the present study should represent a less sick ICU population or that our complete daily assessment for weaning is more complete and better selected the readiness for weaning and extubation. The low reintubation rate for this study (i.e., two cases in each arm [5.7 %]) reinforces the hypothesis that our patients recovered well from the respiratory failure and were in fact able to be extubated from the ventilator after the SBTs. The results of this study show that total invasive mechanical ventilation times were similar for both weaning modes; average mechanical ventilation duration was approximately 4 days in both weaning modes, a shorter time than previously reported values [8, 9]. In a recent meta-analysis, authors reported pooled data of 7 trials involving 520 participants and showed a significant reduction in total duration of mechanical ventilation of 1.8 days favoring SmartCare™ (MD: −1.68 days; 95 % CI: −3.33 to −0.03; p =0.05) with substantial heterogeneity (I2 =53 %; p =0.05) [11, 17–21]. The mean mechanical ventilation time (pooled data from 7 studies, 520 participants [11–17, 21]) was 8.34 (5.88) days for SmartCare™ versus 10.48 (8.25) days for non-automated weaning, showing that the condition of the patients in these seven polled studies was more severe than that of the patients in the present study. We believe that the efficacy of this respiratory physiotherapy–driven weaning protocol derives from the structure and composition of the ICU professionals in our institution. The adult ICU of Albert Einstein Hospital has physiotherapists (physiotherapists in Brazil also act as respiratory therapists) 24 hours per day, with one physiotherapist for every six to eight patients. These therapists are highly trained in weaning protocols of mechanical ventilation [8, 10, 22, 23]. The physiotherapist is able to watch patients closely, quickly identifying patients who are prepared for SBT through a daily assessment [8, 22, 24] in accordance with the Institute for Healthcare Improvement recommendations for the quality of care in ICUs. There are daily multidisciplinary rounds with clear goals for the assessment of the patients. Once all the patients’ goals have been met, there is a team discussion about each patient’s progress, which also speeds up the process of weaning. Therefore, it is possible that automatic modes could be faster in a service with fewer physiotherapists. It is worth noting the occurrence of no malfunction events related to the ventilator in the respiratory physiotherapy–driven weaning group, as well as three in the automatic mode, two of which were related to a malfunction of the capnography that prevented PSV from being decreased. After further inspection, these problems were found to be related to the EtCO2 cable that connects the ventilator to the cuvette where EtCO2 rate is measured. Following cable replacement, this problem was solved. Although previous studies with SmartCare™ have not reported this problem [3, 25–27], the observation of this problem in two of six units used indicates that manufacturers should ensure that EtCO2 cables are properly adapted. There was also one case where psychomotor agitation and tachypnea led to an unnecessary increase in PSV in the automatic weaning mode. A similar problem was previously reported [25] where a child was not weaned in automatic mode, owing to pain and tachypnea. Although the influence of respiratory rate for PSV adjustment was less frequent with SmartCare™ mode than with previously studied automatic systems [23], these findings indicate that automatic weaning may not be ideal for patients with tachypnea as a result of pain, fever or delirium, even when more parameters in addition to respiratory rate are monitored. We acknowledge that there are some limitations of this study. First, the study included only 70 patients (35 in each mode)—a small sample size, but sufficient for the detection of significant differences in weaning duration. Second, the study was conducted among adults; hence, it is not possible to generalize our findings with regard to other age groups. Further research should prove fruitful in this respect. Finally, we tested the SmartCare™ system after the patients successfully passed a complete daily assessment for weaning readiness, and this fact might have decreased the effectiveness of SmartCare™ performance during the weaning phase of invasive mechanical ventilation. BODY.CONCLUSIONS: A respiratory physiotherapy–driven weaning protocol can decrease weaning time compared with an automatic system, as it takes into account individual weaning difficulties. However, both modes have been shown to be safe in terms of the frequency of weaning failure and weaning completion, leading to the same invasive mechanical ventilation duration. BODY.KEY MESSAGES: A complete daily assessment of readiness for weaning in critically ill patients invasively mechanically ventilated for more than 24 hours can help clinicians choose a better modality for patients able to perform SBTs. A respiratory physiotherapy–driven weaning protocol can decrease weaning time compared with the SmartCare™ mode, as it takes into account individual weaning difficulties. SmartCare™-driven weaning compared with a respiratory physiotherapy–driven weaning led to similar mechanical ventilation duration, weaning completion and failure.

TITLE: Optimal site for facial nerve transection and neurorrhaphy: a randomized prospective animal study ABSTRACT.BACKGROUND: Since the first facial allograft transplantation was performed, several institutions have performed the procedure with the main objectives being restoration of the aesthetic appearance and expressive function of the face. The optimal location to transect the facial nerve during flap harvest in transplantation to preserve facial movement function is currently unknown. There are currently two primary methods to perform facial nerve neurorrhaphy between the donor and recipient-one protocol involves transection and repair of the facial nerve at the main trunk while the another protocol advocates for the neurorrhaphy to be performed distally at the main branches. The purpose of this study is to establish the optimal location for transection and repair of the facial nerve to optimize functional recovery of facial movement. ABSTRACT.METHODS: A prospective randomized controlled trial using a rat model was performed. Two groups of 12 rats underwent facial nerve transection and subsequent repair either at the main trunk of the nerve (group 1) or 2 cm distally, at the main bifurcation (group 2). Primary outcome of nerve functional recovery was measured using a previously validated laser curtain model, which measured amplitude of whisking at 2, 4, and 6 post-operatively. The deflection of the laser curtain sent a digital signal that was interpreted by central computer software. ABSTRACT.RESULTS: At week 2 post-nerve surgery, the average amplitude observed for group 1 and 2 was 4.4 and 10.8 degrees, respectively. At week 4, group 1 showed improvement with an average amplitude of 9.7 degrees, while group 2 displayed an average of 10.2 degrees. The week 6 results showed the greatest improvement from baseline for group 1. Group 1 and 2 had average amplitudes of 17.2 and 6.9 degrees, respectively. There was no statistically significant difference between the two groups at 2, 4, and 6 weeks after facial nerve surgery (p > 0.05). ABSTRACT.CONCLUSIONS: We found no statistical difference between these two locations of nerve repair using identical methods. Therefore, the authors recommend a single versus multiple nerve repair technique. This finding has potential implications for future facial allograft transplantations and at minimum necessitates further study with long-term follow-up data. BODY.INTRODUCTION: Since the first facial allograft transplantation was performed in Amiens, France, in 2005, several institutions have performed the procedure with the main objectives being restoration of the aesthetic appearance and expressive function of the face. An essential component of this procedure involves the neurorrhaphy of the donor facial nerve to the corresponding recipient patient’s facial nerve. The optimal location to transect the facial nerve during flap harvest in transplantation to preserve facial movement function is currently unknown. There are presently two primary methods to perform facial nerve neurorrhaphy between the donor and recipient-one protocol involves transection and repair of the facial nerve at the main trunk while another protocol advocates for the neurorrhaphy to be performed just distally to the main trunk at the main upper and lower branches. There are several known clinical factors that have an effect on peripheral nerve function recovery after nerve repair including time interval between trauma and repair, type of lesion and repair, and the age of the patient [1]. Furthermore, in order to optimize nerve function, there are certain techniques of nerve repair that have been shown to be vital for outcome. The basic requirement is to appose the cut ends of the nerve in such a fashion as to minimize scar formation and preserve the optimal blood supply [2]. In cases of sharp nerve division with minimal gap, as is the case with facial allograft transplantation, direct end-to-end nerve repair is indicated [3]. Furthermore, tension-free suture repair remains the preferred treatment option as tension will result in scaring and poor regeneration [2, 3]. Despite an abundance of knowledge regarding nerve regeneration physiology and nerve repair techniques, little is known about optimal sites of transection and repair along a peripheral nerve. Some literature has suggested that more proximal peripheral nerve injuries are associated with worse outcomes. In their 2009 study of upper extremity nerve injuries, Lohmeyer et al. found that increasing distance between nerve lesion and fingertip correlated significantly with decreasing fingertip sensibility [4]. The reason for this is complex and not fully understood but it is felt that the more proximal the nerve injury, the lower the chances for the axons to re-innervate adequate terminal receptors and organs because possible misdirection increase [4]. Also, in the time needed to reach the end organ, it is felt that multiple irreversible changes take place, which can negatively affect outcome [1]. In regards to peripheral nerve recovery, it is also well recognized that the functional outcome following repair of different individual nerves, in otherwise comparable circumstances, are not the same [1]. Although there is no widely accepted explanation, it is felt the intrinsic complexity of the function of the nerve plays a role [1]. Unfortunately, literature regarding optimal sites for transection and repair specifically of the facial nerve is exceedingly scarce. In their 2006 study, Liu et al. compared lesions of the central nervous system to those of the peripheral nervous system along the facial nerve. The authors found that axonal injuries of central facial motoneurons caused greater nerve damage than injuries along the axons of the peripheral facial nerve [5]. A recent study by Hadlock et al. did attempt to compare injuries along different lengths of the facial nerve [6]. The authors found no significant difference in recovery using similar repair techniques [6]. The technique of facial nerve transection and subsequent neurrorhaphy between donor and recipient during facial allograft transplantation proposed by the Amiens group specifies transecting the nerve at the main upper and lower bifurcation. That of the Cleveland group specifies transecting and repairing the nerve at the main trunk. There currently exists no literature comparing these two types of transection and repair. Our objective in completing this study was to directly compare these two methods in an established animal model to better predict the ideal location of facial nerve transection to optimize facial nerve regeneration and functional recovery following repair. BODY.METHODS: BODY.STUDY DESIGN: This was a prospective randomized control animal trial conducted at the Surgical Medical Research Institute (SMRI) at the University of Alberta. A previously validated rat facial nerve model was used. Ethics approval was obtained from the Animal Care and Use Committee (ACUC) overseen by the University Animal Policy and Welfare Committee (UAPWC) at the University of Alberta in Edmonton, Alberta [AUP00000785]. BODY.STUDY SUBJECTS: 24 female Wistar rats (Charles River Laboratories, Canada) weighing 200–220 g were used for this study. Sample size was based on the study by Heaton et al. which employed a similar outcome measure [7]. All rats were housed in pairs in cages at the Health Sciences Laboratory Animal Services (HSLAS) at the University of Alberta. Rats were weighed and handled daily 2 weeks prior to the commencement of the study to reduce animal stress during the study. The 24 rats were block randomized into two groups of 12. Each animal underwent unilateral facial nerve transection and repair at either the main trunk of nerve or at the main upper and lower bifurcation of the nerve. Facial nerve functional outcome assessment was collected at 2, 4, and 6 weeks post-operatively. BODY.FACIAL NERVE FUNCTIONAL OUTCOME ASSESSMENT: The facial nerve functional outcome assessment model we employed in this study was based on the model described and validated by Heaton et al. in their 2008 study [7]. This model employs a head fixation device, body restraint, and bilateral photoelectric sensors to detect precise whisker movements as an objective measure for facial nerve function. BODY.HEAD IMPLANT: In order to ensure proper head fixation during whisker movement measurement, an implantable head fixation device was required. In conjunction with the biomedical engineering department at the University of Alberta, we designed a unique head implant adequate for our purposes. The implant itself was composed of acrylic and long threaded screws. The exact procedure is described below in section 7 of the materials and methods. BODY.BODY RESTRAINT: Based on the design described by Heaten et al., we developed a custom body restraint device for the rat subjects in conjunction with the Metalworks Engineering Shop at the University of Alberta. Our body restraint apparatus consisted of a half-pipe (ABS-DWV IPEX Drainway) measuring 7.6 cm in diameter and 30 cm in length. Three Velcro® straps were then fastened across the top of the half-pipe for added restraint. A steel bar spanning across the half pipe provided a fixation point for the head implant as well as functioned to support the laser micrometers. Along the anterior portion of the half-pipe we added a circular platform to support the weight of the rat’s head while placed in the apparatus (Fig. 1).Fig. 1Custom built rat body restraint apparatus BODY.TRACKING WHISKER MOVEMENT: Two pairs of photoelectric sensors (Rx-Laser Micrometer, Metralight Inc., San Mateo, Ca) were placed along each side of the subject’s face in order to track whisker movement (Fig. 2). Thin tubing 1.5 mm in diameter was placed over a midline whisker on either side of the subject’s face to facilitate tracking by the laser micrometer. The laser micrometers were placed at exactly 17 degrees from the midline along each side of the face and this was considered parallel to the lateral surface of the face. The lasers were also positioned approximately 10 mm from the origin of the tracked whisker on each side of the face.Fig. 2Photoelectric sensors used in detecting rat whisking The laser micrometer itself was comprised of an emitter, which produced a 780 nm wavelength light curtain, and a detector composed of a 28 mm linear array of 4000 charge-coupled devices (CCD scanline). The emitter and detector were separated by a 5 cm vertical distance, producing a laser curtain. Movement detected within the laser curtain sent a digital signal that could then be recorded. The laser micrometers themselves were calibrated to not detect objects less than 1 mm in size to avoid tracing multiple whiskers. Instead the laser curtain detected only the marked whisker. BODY.DATA ACQUISITION: Whisker movement was elicited in each subject by providing a scented stimulus (chocolate milk). The laser micrometers themselves were connected to a 32-Channel Digital I/O Module (NI 9403, National Instruments, Dallas, Tx), which received digital output from the laser micrometers (Fig. 3). The I/O module was connected to a PC through a CompactDAQ chassis (cDAQ-9174, National Instruments, Dallas, Tx). The I/O module acquired the laser micrometer signal at a sampling rate of 1 kHz. LabVIEW (LabVIEW Full Development System, National Instruments, Dallas, Tx) software was used as the interface for data acquisition.Fig. 332-channel digital I/O module BODY.SURGICAL PROCEDURE: All subjects underwent both head implantation surgery as well as facial nerve surgery during the same anesthetic. All rats were first anesthetized with 3–4 % isoflurane. Subjects were then maintained under general anesthesia using 1.5 % isoflurane. Hair was then removed from the right side of the face and the top of the head using an electric shaver. BODY.FACIAL NERVE SURGERY: All facial nerve surgery was completed on the right side of the face on all subjects. A small incision was made just inferior to the right ear bony prominence. Under microscopic visualization, the parotid glad was visualized and everted and retracted out of the surgical field, without removing it completely. Subsequently, distal branches of the facial nerve were identified just inferior to the parotid bed. These were followed proximally until the main trunk of the facial nerve was identified. Once identified, the main trunk and upper and lower bifurcation of the facial nerve were carefully dissected. If the subject was randomized to group 1 (main trunk), a single transection of the main trunk of the facial nerve was made using straight microscopic scissors. If the subject was randomized to group 2 (bifurcation), two nerve transections were completed: one at the upper bifurcation and one at the lower bifurcation of the nerve. These transections were similarly completed using straight microscopic scissors. In both groups, the cut nerve ends were immediately repaired using a direct end-to-end technique. Using 9–0 sutures, four simple interrupted sutures were made within the proximal and distal epineural nerve endings. Care was taken to ensure proper nerve alignment. In group 1 subjects, only one nerve repair was necessary while group 2 subjects underwent two nerve repair techniques in this fashion. The parotid gland was then reflected back into the surgical field. Skin was approximated using 3–0 vicryl sutures. BODY.HEAD IMPLANT SURGERY: Following the facial nerve procedure, head implant surgery was then completed without reversing the general anesthetic. A small incision was made using a 15-blade scalpel from the anterior to posterior margin of the cranium. Blunt dissection was employed to fully expose the underlying bony cranium. Using an electric drill, 4 holes were made in each quadrant of the skull approximately 15 mm apart from each other. 1.6 mm screws were then placed within each drill site. (Fig. 4) Dry acrylic resin was then liquefied and placed onto the skull, covering the placed screws. Two larger 5 mm threaded screws were then inverted with the threads directed upwards into the acrylic before it solidified. Once the acrylic completely solidified, the skin was then re-approximated overtop of the acrylic with interrupted 3–0 vicryl sutures, leaving the two larger threaded screws exposed through the incision (Fig. 5 and Fig. 6).Fig. 4Rat cadaver depicting custom built head implantation device made from dry acrylic resin Fig. 5Study rat 1 week post-op from head implantation surgery Fig. 6Study rat 1 week post-op from head implantation surgery BODY.HEAD FIXATION AND BODY RESTRAINT: Two weeks prior to surgery, all animal subject were handled daily for conditioning. After surgery, all subjects were placed in body restraints daily for a week. At post-operative day 14, whisker measurements were started. Subjects were initially given an injection of low dose ketamine and transported to the body restraint apparatus described in Body restraint. Here they underwent head fixation with bolts applied across the exposed threaded screws (Fig. 7). Whisker markers were then placed on either side of the rat’s face as described in Tracking whisker movement.Fig. 7Rat cadaver depicting fixation bolts applied to head implant for rat head stability Once this was completed, a scented stimulus was introduced and recording started usually for a period of 5 min. The non-operative left side was used as the control for each subject. This procedure was completed for each rat at 2, 4, and 6 weeks post-operatively. BODY.RESULTS: All animals tolerated the surgical procedure very well. They exhibited normal cage behavior and did not lose weight. Three animals had problems with suture break down post-operatively. This occurred in all 3 animals within 5 days of the surgical procedure. For these animals, we re-anesthetized them with isoflurane and were able to re-approximate the incision edges with 3–0 vicryl sutures. No animals had to be removed from the study. All animals experienced complete ipsilateral loss of whisking amplitude post-operatively. At week 2 the average amplitude observed for group 1 was 4.4 degrees (Table 1). Similarly, the group 2 average was 10.8 degrees at 2 weeks post-operatively. At week 4, group 1 showed improvement having an average of 9.7 degrees, while group 2 remained relatively unchanged with an average of 10.2 degrees. The week 6 results showed the greatest improvement from baseline for group 1. Group 1 had an average amplitudes of 17.2 degrees at 6-weeks from surgery (Fig. 8). However, group 2 showed a slight decrease in amplitude with an average of 5.9 degrees. There was no statistically significant difference between the two groups at 2, 4 and 6 weeks after facial nerve surgery (p > 0.05).Table 1Post-operative whisking amplitudes at week 2, 4, and 6 Week 2 amplitude (degrees)Week 4 amplitude (degrees)Week 6 amplitude (degrees)MAIN TRUNK (group 1) Right side (operated) 4.4 9.7 17.2 MAIN TRUNK (group 1) Left side (control) 72.1 66.6 71.8 MAIN BIFURCATION (group 2) Right side (operated) 10.8 10.2 5.9 MAIN BIFURCATION (group 2) Left side (control) 74.9 70.9 67.5 Fig. 8Whisking amplitude in degrees at 2, 4, and 6 weeks postoperatively BODY.DISCUSSION: Since 2005, facial allograft transplantation has rapidly started becoming a more commonly employed surgical procedure, indicated for individuals disfigured from trauma, burns, and birth defects among other entities. As the procedure has become more commonly employed, knowing the exact location of where to transect and repair the facial nerve has become that much more vital. The most significant study attempting to answer the question of the location to transect and repair the facial nerve for optimal functional outcome was published by Hadlock et al. in 2010 [6]. The authors studied a variety of different types of facial nerve injuries and injury locations in the rat model. When comparing proximal facial nerve lesions of the main trunk to peripheral facial nerve lesions of distal branches, the authors found no statistically significant difference in whisking amplitude [6]. In our study, we specifically compared the two locations employed by the Cleveland and Amiens groups to transect and repair the facial nerve in facial allograft transplantation (main trunk and main nerve bifurcation, respectively). Our literature search found that these two methods had never been compared in a randomized study. Similar to Hadlock et al., we found that there was no statistically significant difference between injuries at the main trunk and more distal injuries, which in our study was specifically at the main bifurcation of the nerve. However, we did find a non-statistically significant improvement in whisking amplitude for group 1 (main trunk) as compared to group 2. The whisking amplitude of group 1 was consistently greater at week 6 postoperatively. Although the whisking amplitude difference is relatively small, it does raise the possibility that a greater follow-up time may reveal a larger, statistically significant difference between the two groups. This notion is further supported by the observation that the whisking amplitude difference between the two groups consistently became greater the further out from nerve surgery. However, given that our study showed only a minimal, non-statistically significant difference in whisking amplitude between the two groups, it seems logical with the given evidence to favor the Cleveland facial nerve protocol. The Cleveland protocol, as previously mentioned, entails only a single nerve transection and repair (group 1), minimizing operative time. Overall, facial nerve functional recovery remained fairly limited in both groups. This may be due to several reasons, including peripheral misrouting of axons and reduction of brainstem synaptic connection with facial motoneurons. A potential limitation of our study was our follow up time. A more protracted follow-up time may have elucidated a more significant difference between the two groups. Our study has important findings to guide future facial allograft transplantations. Given the minimal difference in whisking amplitude between the two groups, single nerve repair is more advisable as it has the added benefit of less required operative time and potential cost savings. BODY.CONCLUSION: Our study directly compared, in a rat model, the transection and subsequent neurorrhaphy of the facial nerve at two distinct locations commonly used during facial allograft transplantation; the main trunk (group 1) and main bifurcation (group 2). We found no statistical difference between these two locations of nerve repair using identical methods. Therefore, the authors recommend the protocol outlined by the Cleveland group, which requires only single nerve repair as opposed to that described by the Amiens group. This finding has potential implications for future facial allograft transplantations and at minimum necessitates further study with long-term follow-up data.

TITLE: Cardiovascular and renal protective role of angiotensin blockade in hypertension with advanced CKD: a subgroup analysis of ATTEMPT-CVD randomized trial The ATTEMPT-CVD study was prospective randomized active-controlled trial and the main findings had been reported. According to baseline GFR and albuminuria categories, we divided the patients of the ATTEMPT-CVD study into 2 subgroups: (Group 1) the patients with at least one of eGFR of <45 ml/min per 1.73 m2 and UACR of ≥300 mg/g creatinine, defined as G3b and/or A3; (Group 2) the patients except for Group 1, defined as the other patients. In patients with G3b and/or A3, the incidence of cardiovascular events was significantly less in ARB group than in non-ARB group (11 vs 22, respectively) (HR = 0.465: 95%CI = 0.224–0.965; P = 0.040). UACR was significantly less in ARB group than in non-ARB group during follow-up period in patients with G3b and/or A3 (P = 0.0003), while eGFR, plasma BNP levels, and blood pressure were comparable between ARB and non-ARB groups. Allocation to ARB therapy was a significant independent prognostic factor for cardiovascular events in patients with G3b and/or A3 (P = 0.0268). On the other hand, in the other patients, the occurrence of cardiovascular events was comparable between ARB and non-ARB groups. In patients with advanced CKD, ARB-based therapy may confer greater benefit in prevention of cardiovascular events than non-ARB therapy. BODY.INTRODUCTION: We performed a trial of telmisartan prevention of cardiovascular disease (ATEMPT-CVD)1,2 to compare the effects of angiotensin II receptor blocker (ARB)-based antihypertensive therapy and those of non-ARB antihypertensive therapy on biomarker level changes and the incidence of cardiovascular and renal events in Japanese hypertensive patients who had at least one of cardiovascular risk factors (type 2 diabetes, cerebrovascular, cardiac or peripheral arterial disease, or renal impairment). ATTEMPT-CVD study provided the evidence that ARB (telmisartan)-based antihypertensive therapy caused a greater decrease in urinary albumin/creatinine ratio (UACR) and a smaller increase in plasma BNP than non-ARB antihypertensive therapy, under similar blood pressure control1. However, in spite of more benefit in UACR and plasma BNP changes by ARB therapy than by non-ARB therapy, the incidence of cardiovascular and renal events did not differ between ARB group and non-ARB group1. Thus, the positive effects of ARB therapy on UACR and plasma BNP were not associated with cardiovascular outcome in overall hypertensive patients enrolled in the ATTEMPT-CVD study. Patients with chronic kidney disease (CKD) are at risk of not only end-stage kidney disease (ESKD) but also cardiovascular disease and death3. Glomerular filtration rate (GFR) level is most often used for definition and classification of CKD3–5, and most of previous clinical trials on CKD patients have defined CKD based on GFR category. GFR category is divided into 5 stages (≥90 [G1], 60–89 [G2], 30–59 [G3], 15–29 [G4], and <15 [G5] ml/min per 1.73 m2)3–5. Very importantly, stage G3 is subdivided into G3a (eGFR of 45–59 ml/min per 1.73 m2) and G3b (GFR of 30–44 ml/min per 1.73 m2)4,5. This subdivision of G3 stage by applying a cut-off value of 45 ml/min per 1.73 m2 is very critical for appropriate risk assessment of ESKD and cardiovascular disease, because the patients with GFR of <45 ml/min per 1.73 m2 have much higher risk for cardiovascular death, ESKD and all-cause death than those with GFR of ≥45 ml/min per 1.73 m2 3–5. However, there is no sufficient evidence from large-scale randomized trial addressing appropriate antihypertensive strategy in patients with GFR category of <45 ml/min per 1.73 m2 or worse stage. Besides GFR, the level of albuminuria is established to predict the prognosis of CKD progression and cardiovascular disease and death, independently of GFR6–11. KDIGO guideline highly recommend the use of both GFR and albuminuria categories for clinical decision making in CKD patients4,5,12. However, many previous clinical studies on CKD patients have paid attention to only GFR category but not to albuminuria category. Importantly, of albuminuria categories, UACR of ≥300 mg/g creatinine (macroalbuminuria), namely category A3, is established to be at much higher risk for ESKD and cardiovascular disease and death, regardless of GFR stage (even in the case of GFR stage G1 or G2)3,12,13. Based on these evidences, patients with at least one of eGFR of <45 ml/min per1.73 m2 (G3b cut-off value) and UACR of ≥300 mg/g creatinine (macroalbuminuria) have much higher risk for ESKD and cardiovascular morbidity and mortality than the patients with neither eGFR of <45 ml/min per 1.73 m2 nor macroalbuminuria3–5. However, it remains to be elucidated whether renin-angiotensin system blockers exert more benefit in prevention of cardiovascular and renal events than other antihypertensive drugs in hypertensive patients with at least one of eGFR of <45 ml/min per 1.73 m2 and UACR of ≥300 mg/g creatinine. Therefore, in the present subanalysis, we subdivided the hypertensive patients enrolled in the ATTMPT-CVD study into two groups; (1) the patients with at least one of eGFR of <45 ml/min per 1.73 m2 and UACR of ≥300 mg/g creatinine, defined as the patients with G3b and/or A3 and (2) the patients with neither eGFR of <45 ml/min per 1.73 m2 nor UACR of ≥300 mg/g creatinine, defined as the other patients. We examined the comparative effects of ARB therapy and non-ARB therapy on the incidence of cardiovascular and renal events and biomarker changes in the subgroup patients. BODY.RESULTS: BODY.CATEGORIZATION OF PATIENTS ACCORDING TO UACR AND EGFR CATEGORIES AT BASELINE: As 6 patients of 1,228 patients enrolled in the ATTEMPT-CVD study were excluded from the present subanalysis because of no availability of baseline eGFR data, 1,222 patients were included in the present subanalysis. Table 1 shows distribution of patients according to baseline eGFR and UACR categories. In patients enrolled in the ATTEMPT-CVD study, there was no patient with eGFR stage G5 and the proportion of patients with eGFR G4 stage was very small. The number of patients with at least one of eGFR of <45 ml/min per 1.73 m2 (G3b, G4 or G5) and UACR of ≥300 mg/g creatinine (A3 category) was 187, which were defined as patients with G3b and/or A3. The number of patients with both eGFR of ≥45 ml/min per 1.73 m2 (G1-G3a) and UACR of <300 mg/g creatinine (A1 or A2) was 1,035, which were defined as the other patients.Table 1Distribution of patients according to UACR and eGFR categories at baseline. GFR categorieseGFR (ml/min/1.73 m2)Albuminuria categoriesA1A2A3UACR of <30 mg/g creatinineUACR of 30–300 mg/g creatinineUACR of ≥300 mg/g creatinineG1 and G2 ≥60 510 (41.7%) 327 (26.8%) 81 (6.6%) G3a 45–59 109 (8.9%) 89 (7.3%) 29 (2.4%) G3b 30–44 25 (2.0%) 21 (1.7%) 20 (1.6%) G4 15–29 1 (0.1%) 3 (0.2%) 7 (0.6%) G5 <15 0 (0%) 0 (0%) 0 (0%) Categories in bold indicate patients with at least one of eGFR of <45 ml/min/1.73 m2 and UACR of ≥300 mg/g creatinine. Abbreviations: UACR, urinary albumin/creatinine ratio; eGFR, estimated glomerular filtration rate. Values are the number of patients belonging to each category. Number in parenthesis indicates percentage of patients to overall patients. BODY.DEMOGRAPHIC AND BASELINE CHARACTERISTICS OF PATIENTS WITH G3B AND/OR A3 AND THE OTHER PATIENTS: As shown in Table 2, median of UACR in patients with G3b and/or A3 was 490 mg/g creatinine and that in the other patients was 20.7 mg/g creatinine (P < 0.0001 between the groups). Median of eGFR in patients with G3b and/or A3 was 52.6 ml/min per 1.73 m2 and that in the other patients was 72.7 ml/min per 1.73 m2 (P < 0.0001 between the groups). Compared with the other patients, patients with G3b and/or A3 had higher plasma BNP (P < 0.0001), were older (P = 0.0063), and had higher serum creatinine (P < 0.0001), slightly higher serum potassium (P = 0.0329), higher blood sugar (P = 0.0002), higher hemoglobin A1c (P = 0.0007), lower hemoglobin (P = 0.0004) and higher uric acid (P < 0.0001). In patients with G3b and/or A3, baseline characteristics were well balanced between ARB group and non-ARB group, and there was no statistically significant difference regarding demographic and baseline characteristics listed in Table 2. In the other patients, baseline characteristics were also well balanced between the two treatment groups and there was no significant difference regarding baseline characteristics between ARB and non-ARB groups.Table 2Baseline characteristics of patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine and the other patients. Total patientsOverallG3b and/or A3The otherG3b and/or A3The otherP-valueARBNon-ARBARBNon-ARB(n = 1222)(n = 187)(n = 1035)(n = 96)(n = 91)(n = 516)(n = 519)UACR (mg/g creatinine) 26.1 (11.1–89.1) 490 (119–957) 20.7 (10.3–54.4) <0.0001 409 (102–914) 529 (300–974) 20.4 (10.5–50.6) 21.1 (10.1–61.6) eGFR (ml/min per 1.73 m2) 71.2 (60.0–84.6) 52.6 (40.2–74.0) 72.7 (62.4–85.3) <0.0001 51.0 (40.0–72.3) 55.3 (40.9–74.3) 72.9 (62.1–84.9) 72.6 (62.8–86.7) BNP (pg/mL) 18.0 (9.0–36.5) 23.3 (12.0–58.4) 17.4 (8.7–34.3) <0.0001 27.5 (13.8–70.1) 19.2 (9.6–46.6) 17.3 (8.1–34.1) 17.5 (9.1–34.8) Age (years) 66 ± 9 68 ± 9 66 ± 9 0.0063 68 ± 9 67 ± 9 66 ± 9 66 ± 10 Male, n (%) 712 (58.3) 113 (60.4) 599 (57.9) 0.5146 60 (62.5%) 53 (58.2%) 298 (57.8) 301 (58.0) BMI (kg/m2) 25.2 ± 3.8 25.4 ± 4.0 25.2 ± 3.8 0.5198 25.3 ± 3.9 25.6 ± 4.1 25.1 ± 3.8 25.3 ± 3.8 Systolic BP (mmHg) 150 ± 16 152 ± 16 150 ± 16 0.1393 151 ± 16 153 ± 15 151 ± 16 149 ± 15 Diastolic BP (mmHg) 84 ± 12 82 ± 12 84 ± 12 0.0201 81 ± 12 83 ± 13 85 ± 12 84 ± 12 Heart rate (b.p.m) 72 ± 11 74 ± 12 72 ± 11 0.0292 72 ± 11 75 ± 13 72 ± 11 72 ± 11 Diabetes mellitus, n (%) 817 (66.9) 131 (70.1) 686 (66.3) 0.3131 69 (71.9) 62 (68.1%) 340 (65.9) 346 (66.7) Hyperlipidemia, n (%) 704 (57.6) 106 (56.7) 598 (57.8) 0.7807 54 (56.3) 52 (57.1) 298 (57.8) 300 (57.8) Current smoker, n (%) 216 (17.7) 36 (19.3) 180 (17.4) 0.5394 17 (17.7) 19 (20.9) 90 (17.4) 90 (17.3) Serum or plasma values Creatinine (mg/dL) 0.8 ± 0.2 1.0 ± 0.4 0.7 ± 0.2 <0.0001 1.0 ± 0.4 1.0 ± 0.4 0.7 ± 0.2 0.7 ± 0.2 Potassium (mEq/L) 4.3 ± 0.5 4.4 ± 0.6 4.3 ± 0.5 0.0329 4.4 ± 0.7 4.3 ± 0.6 4.3 ± 0.5 4.3 ± 0.5 Total cholesterol (mg/dL) 196 ± 36 202 ± 48 195 ± 33 0.1420 199 ± 41 204 ± 54 195 ± 34 195 ± 32 LDL cholesterol (mg/dL) 112 ± 30 114 ± 33 112 ± 29 0.5415 110 ± 31 119 ± 35 112 ± 30 112 ± 28 HDL cholesterol (mg/dL) 56 ± 14 55 ± 14 56 ± 14 0.0736 56 ± 15 53 ± 13 57 ± 14 56 ± 14 Blood sugar (mg/dL) 135 ± 56 150 ± 63 132 ± 54 0.0002 148 ± 61 151 ± 66 130 ± 51 135 ± 57 Hemoglobin A1c (%) 6.4 ± 1.2 6.7 ± 1.4 6.3 ± 1.1 0.0007 6.5 ± 1.2 6.9 ± 1.6 6.3 ± 1.1 6.3 ± 1.2 Hemoglobin (g/dL) 13.9 ± 1.6 13.5 ± 1.8 14.0 ± 1.6 0.0004 13.4 ± 1.9 13.7 ± 1.7 14.0 ± 1.6 14.0 ± 1.5 Uric acid (mg/dL) 5.3 ± 1.3 5.7 ± 1.5 5.3 ± 1.3 <0.0001 5.8 ± 1.4 5.6 ± 1.5 5.2 ± 1.3 5.3 ± 1.3 Abbreviations: G3b and/or A3, patients with estimated glomerular filtration rate of <45 ml/min per 1.73 m2 and/or urinary albumin/creatinine ratio of ≥300 mg/g creatinine; The other, patients with both estimated glomerular filtration rate of ≥45 ml/min per 1.73 m2 and urinary albumin/creatinine ratio of <300 mg/g creatinine; UACR, urinary albumin/creatinine ratio; eGFR, estimated glomerular filtration rate; BNP, brain natriuretic peptide; BMI, body mass index; BP, blood pressure; LDL, low-density lipoprotein; HDL, high-density lipoprotein; ARB, antihypertensive treatment with angiotensin II receptor blocker; Non-ARB, treatment with antihypertensive drugs except for angiotensin II receptor blocker. UACR, eGFR, and BNP are expressed as median with interquartile range. Other data are mean ± s.d. for continuous values and number (%) for categorical variables. P-value was calculated using t-test or ManWhitney test for continuous variables and χ2 tests for categorical variables. Table 3 shows the proportion of baseline cardiovascular diseases of overall patients and patients with G3b and/or A3 or the other patients. Proportion of previous cardiovascular diseases was slightly less in patients with G3b and/or A3 than in the other patients (25.7% vs 33.7%; P = 0.0305), and this difference was mainly accounted for by less percentage of previous left ventricular hypertrophy in patients with G3b and/or A3 than in the other patients (8.6% vs 14.8%; P = 0.0232). The proportion of previous cerebrovascular disease and the proportion of previous peripheral artery disease were not different between patients with G3b and/or A3 and the other patients. In patients with G3b and/or A3, proportion of previous cardiovascular disease, previous cerebrovascular disease, and previous peripheral artery disease were similar between ARB and non-ARB groups. Also in the other patients, the proportion of previous cardiovascular diseases was comparable between the two treatments.Table 3Proportion of baseline cardiovascular disease of patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine and the other patients. Total patientsOverallG3b and/or A3The otherG3b and/or A3The otherP-valueARBNon-ARBARBNon-ARB(n = 1222)(n = 187)(n = 1035)(n = 96)(n = 91)(n = 516)(n = 519)Previous cardiac disease, n (%) 397 (32.5) 48 (25.7) 349 (33.7) 0.0305 27 (28.1) 21 (23.1) 173 (33.5) 176 (33.9) Myocardial infarction, n (%) 54 (4.4) 6 (3.2) 48 (4.6) 0.3815 5 (5.2) 1 (1.1) 24 (4.7) 24 (4.6) Angina pectoris, n (%) 114 (9.3) 18 (9.6) 96 (9.3) 0.8795 9 (9.4) 9 (9.9) 46 (8.9) 50 (9.6) Heart failure (NYHA I/II), n (%) 43 (3.5) 7 (3.7) 36 (3.5) 0.8563 4 (4.2) 3 (3.3) 19 (3.7) 17 (3.3) Left ventricular hypertrophy, n (%) 169 (13.8) 16 (8.6) 153 (14.8) 0.0232 9 (9.4) 7 (7.7) 81 (15.7) 72 (13.9) Atrial fibrillation, n (%) 87 (7.1) 13 (7.0) 74 (7.1) 0.9228 7 (7.3) 6 (6.6) 35 (6.8) 39 (7.5) Previous cerebrovascular disease, n (%) 165 (13.5) 32 (17.1) 133 (12.9) 0.1165 18 (18.8) 14 (15.4) 64 (12.4) 69 (13.3) Cerebral infarction, n (%) 97 (7.9) 19 (10.2) 78 (7.5) 0.2218 10 (10.4) 9 (9.9) 40 (7.8) 38 (7.3) Cerebral hemorrhage, n (%) 13 (1.1) 2 (1.1) 11 (1.1) 0.9934 2 (2.1) 0 (0) 4 (0.8) 7 (1.3) Subarachnoid hemorrhage, n (%) 8 (0.7) 0 (0) 8 (0.8) 0.2277 0 (0) 0 (0) 4 (0.8) 4 (0.8) Transient ischemic attack, n (%) 48 (3.9) 12 (6.4) 36 (3.5) 0.0569 7 (7.3) 5 (5.5) 16 (3.1) 20 (3.9) Previous peripheral artery disease, n (%) 6 (0.5) 2 (1.1) 4 (0.4) 0.2188 2 (2.1) 0 (0) 2 (0.4) 2 (0.4) Lower extremities bypass surgery or angioplasty, n (%) 3 (0.2) 1 (0.5) 2 (0.2) 0.3851 1 (1.0) 0 (0) 1 (0.2) 1 (0.2) Ankle-brachial index of <0.9 or with intermittent claudication, n(%) 4 (0.3) 2 (1.1) 2 (0.2) 0.0535 2 (2.1) 0 (0) 1 (0.2) 1 (0.2) Abbreviations used are the same as in Table 2. Data are number (%). P-value was calculated using χ2 tests. BODY.INCIDENCE OF CARDIOVASCULAR AND RENAL EVENTS IN PATIENTS WITH G3B AND/OR A3 AND THE OTHER PATIENTS: 1 shows the incidence of cardiovascular and renal events in ARB group and in non-ARB group of patients with G3b and/or A3 (Fig. 1(a)) and of the other patients (Fig. 1(b)). The incidence of cardiovascular and renal events was much greater in patients with G3b and/or A3 than in the other patients. In patients with G3b and/or A3, the incidence of cardiovascular and renal events was significantly less in ARB group than in non-ARB group (HR = 0.465: 95%CI = 0.224–0.965; P = 0.040) (Fig. 1(a)). On the other hand, in the other patients, there was no significant difference regarding incidence of cardiovascular and renal events between ARB and non-ARB groups (HR = 0.913: 95%CI = 0.538–1.551; P = 0.737) (Fig. 1(b)). The P value for the treatment-subgroup interaction was 0.1397. Table 4 indicates the detail of cardiovascular and renal events occurred in ARB and non-ARB groups in patients with G3b and/or A3 or the other patients.Figure 1Kaplan-Meier curves for composite cardiovascular and renal events during the follow-up period in patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine (a) and the other patients (b). In (a), the numbers of patients in ARB and non-ARB groups were 96 and 91, respectively, and the number of occurrence of endpoints was 11 and 22 in ARB group and non-ARB group, respectively. In (b), the number of endpoints was 27 in 516 patients assigned ARB group and 29 in 518 patients assigned non-ARB group. Abbreviations: G3b and/or A3, patients with estimated glomerular filtration rate of <45 ml/min per 1.73 m2 and/or urinary albumin/creatinine ratio of ≥300 mg/g creatinine; The other, patients with both estimated glomerular filtration rate of ≥45 ml/min per 1.73 m2 and urinary albumin/creatinine ratio of <300 mg/g creatinine; ARB, antihypertensive treatment with angiotensin II receptor blocker; Non-ARB, treatment with antihypertensive drugs except for angiotensin II receptor blocker; HR, hazard ratio; 95%CI, 95% confidence interval. Table 4Comparison of composite cardiovascular events between ARB and non-ARB groups in patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine and the other patients. EventG3b and/or A3The otherARB (n = 96)Non-ARB (n = 91)ARB (n = 516)Non-ARB (n = 519)Total cardiovascular and renal events, n 11 22 27 29  Stroke, n 1 3 7 8  Transient ischemic attack, n 0 1 1 1  Sudden death, n 1 0 1 2  Acute myocardial infarction, n 2 0 3 1  Angina pectoris, n 0 1 1 4  Heart failure, n 2 4 1 2  Aortic aneurysm 0 0 1 2  Aortic dissection, n 0 1 0 0  Peripheral artery disease, n 1 2 4 3  Diabetic nephropathy, n 0 0 1 1  Diabetic retinopathy, n 1 2 5 5  Doubling of serum creatinine, n 2 8 2 0  End stage renal disease, n 1 0 0 0 Abbreviations used are the same as in Table 2. BODY.TIME COURSE OF CHANGES IN UACR DURING THE FOLLOW-UP PERIOD: Figure 2 shows the time course of changes in UACR in the patients with G3b and/or A3 and the other patients. As shown in Fig. 2(a), UACR in ARB group was significantly less than in non-ARB group during follow-up period (P = 0.0003). On the other hand, there was no significant difference in time course of UACR changes between the ARB and non-ARB groups in the other patients (P = 0.4018) (Fig. 2(b)).Figure 2Time course of % changes in urinary albumin/creatinine ratio (UACR) in ARB and non-ARB groups in patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine (a) and the other patients (b). Abbreviations used are the same as in Fig. 1. Values are mean ± SD. BODY.TIME COURSE OF EGFR DURING THE FOLLOW-UP PERIOD: As shown in Fig. 3(a), in patients with G3b and/or A3, no significant difference was noted regarding eGFR between ARB and non-ARB groups (p = 0.1434). On the contrary, eGFR in ARB group was significantly less than that in non-ARB group in the other patients (P < 0.0001) (Fig. 3(b)).Figure 3Time course of changes in estimated glomerular filtration rate (eGFR) in ARB and non-ARB groups in patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine (a) and the other patients (b). Abbreviations used are the same as in Fig. 1. Values are mean ± SD. BODY.TIME COURSE OF CHANGE IN PLASMA BNP DURING THE FOLLOW-UP PERIOD: In both patients with G3b and/or A3 (Fig. 4(a)) and the other patients (Fig. 4(b)), plasma BNP levels were similar between ARB and non-ARB groups throughout the follow-up period.Figure 4Time course of % changes in plasma brain natriuretic peptide (BNP) in ARB and non-ARB groups in patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine (a) and the other patients (b). Abbreviations used are the same as in Fig. 1. Values are mean ± SD. BODY.TIME COURSE OF BLOOD PRESSURE DURING THE FOLLOW-UP PERIOD: Figure 5 and Table 5 indicate time course of blood pressure in each group of patients. In patients with G3b and/or A3, there was the significant difference between ARB and non-ARB groups regarding time course of systolic BP (P = 0.0001) and diastolic BP (P = 0.0306). At 3, 6, and 12 months, systolic BP in ARB group tended to be higher than that in non-ARB group. However, at all time points examined, the difference did not reach statistical significance regarding systolic or diastolic BP between ARB and non-ARB groups in patients with G3b and/or A3. Proportion of the patients achieved BP of <140/90 mmHg was 63.8% in ARB group and 69.3% in non-ARB group of patients with G3b and/or A3, and there was no significant difference between the groups (P = 0.4331).Figure 5Time course of systolic and diastolic blood pressure (BP) in ARB and non-ARB groups in patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine (a) and the other patients (b). Abbreviations used are the same as in Fig. 1. Values are mean ± SD. Table 5Time course of systolic and diastolic BP in patients with eGFR of <45 ml/min per 1.73 m2 and/or UACR of ≥300 mg/g creatinine or the other patients 0 months3 months6 months12 months24 months36 monthsG3b and/or A3   Systolic BP (mmHg)     ARB group 151.0 ± 16.2 139.3 ± 16.6 136.2 ± 15.4 137.7 ± 15.1 133.5 ± 13.4 129.6 ± 16.8     Non-ARB group 152.9 ± 15.1 134.1 ± 14.5 132.8 ± 13.3 134.4 ± 13.4 136.1 ± 14.8 136.8 ± 16.6   Diastolic BP (mmHg)     ARB group 81.2 ± 11.6 75.6 ± 11.8 75.8 ± 10.5 74.1 ± 9.9 72.3 ± 9.2 70.7 ± 11.3     Non-ARB group 83.4 ± 12.6 75.0 ± 11.1 74.5 ± 10.2 75.2 ± 10.4 75.2 ± 10.2 75.0 ± 10.8 The other Systolic BP (mmHg)     ARB group 151.0 ± 16.3 134.7 ± 15.1 132.8 ± 14.1 132.7 ± 13.8 131.8 ± 13.5 131.0 ± 14.4     Non-ARB group 149.4 ± 15.3 133.0 ± 13.1 132.3 ± 13.5 132.4 ± 13.3 132.3 ± 13.5 131.4 ± 15.4 Diastolic BP (mmHg)     ARB group 84.7 ± 11.6 77.1 ± 10.6 76.6 ± 10.6 76.1 ± 10.1 75.0 ± 10.1 74.3 ± 10.7     Non-ARB group 84.2 ± 11.9 75.5 ± 10.0 75.5 ± 10.2 75.6 ± 10.5 74.6 ± 10.3 73.3 ± 9.9 Abbreviations used are the same as in Table 2. Values are mean ± SD. In the other patients, time course of systolic (P = 0.2822) and diastolic (P = 0.6205) BP was comparable between ARB and non-ARB groups during follow-up period. Proportion of the patients achieved BP of <140/90 mmHg was similar between ARB group and non-ARB group (76.5% vs 77.5%; P = 0.7061). BODY.ASSOCIATION OF PROGNOSTIC FACTORS WITH CARDIOVASCULAR AND RENAL EVENTS: Table 6 shows the results of multivariable Cox regression analysis for overall patients, patients with G3b and/or A3, and the other patients. In overall patients, ARB allocation was not significantly associated with cardiovascular and renal events (P = 0.1520), while gender (P = 0.0353), previous CV disease (P < 0.0001), and previous diabetes (P < 0.0001) were significantly associated with cardiovascular and renal events. In patients with G3b and/or A3, ARB allocation (P = 0.0268) was significantly associated with cardiovascular and renal events, and previous diabetes (P = 0.0126) was also significantly associated with cardiovascular and renal events. In the other patients, ARB allocation (P = 08604) was not associated with cardiovascular and renal events, whereas age ≥ 65 years (P = 0.0165), previous CV disease (P = 0.0218), and previous diabetes (P = 0.0083) were significantly associated with cardiovascular and renal events.Table 6Adjusted hazard ratios of prognostic factor for cardiovascular and renal events. Overall patientsG3b and/or A3The otherHR (95%CI)P-valueHR (95%CI)P-valueHR (95%CI)P-valueARB (+) 0.735 (0.483–1.120) 0.1520 0.437 (0.210–0.909) 0.0268 0.954 (0.565–1.612) 0.8604 Male gender 1.623 (1.034–2.548) 0.0353 2.097 (0.915–4.802) 0.0799 1.414 (0.817–2.449) 0.2159 Age ≧65 years 1.479 (0.929–2.355) 0.0990 0.847 (0.409–1.753) 0.6546 2.160 (1.151–4.055) 0.0165 Previous CV disease 3.235 (1.996–5.242) <0.0001 3.221 (0.966–10.736) 0.0569 2.034 (1.109–3.730) 0.0218 Previous diabetes 3.678 (2.121–6.378) <0.0001 3.433 (1.303–9.041) 0.0126 2.594 (1.279–5.263) 0.0083 Abbreviations: HR, hazard ratio; 95%CI, 95% confidence interval; CV, cardiovascular. Other abbreviations used are the same as in Table 2. BODY.DISCUSSION: The present post-hoc analysis of the ATTEMP-CVD study was performed to examine the comparative effect of ARB (telmisartan)-based antihypertensive therapy and non-ARB antihypertensive therapy on the incidences of composite cardiovascular and renal events and biomarker changes in hypertensive patients with G3b and/or A3 (with at least one of eGFR of <45 ml/min per 1.73 m2 and UACR of ≥300 mg/g creatinine) or the other patients. The major findings of our subanalysis were as follows: (1)ARB-based antihypertensive therapy more reduced the incidence of composite cardiovascular and renal events than non-ARB therapy in patients with G3b and/or A3, while there was no significant difference in the incidence of cardiovascular and renal events between the two therapies in the other patients; (2) More benefit of ARB-based therapy over non-ARB therapy in prevention of cardiovascular and renal events in patients with G3b and/or A3 was associated with the significant decrease in UACR but not with blood pressure, eGFR or plasma BNP. The present subanalysis provided a new insight into antihypertensive therapeutic strategy in patients with G3b and/or A3 in terms of prevention of cardiovascular morbidity and mortality. Solid evidence indicates that CKD is a risk factor for not only ESKD but also cardiovascular disease and death3,4. KDIGO guideline4 highly recommends that the definition and classification of CKD is determined by the level of both GFR and albuminuria, because decreased GFR and increased albuminuria levels both are significant risk factors for ESKD and cardiovascular disease and death independently of one another6,8,9,11,12. However, in most of previous clinical studies on CKD patients, the definition and classification of CKD patients have been determined based on only GFR category, and there is no sufficient evidence on randomized trials of CKD patients based on both GFR and albuminuria categories. The patients with eGFR of <45 ml/min per 1.73 m2 (G3b cut-off value) are at much higher risk for cardiovascular morbidity and mortality as well as for ESKD than the patients with eGFR of ≥45 ml/min per 1.73 m2 3–5. Furthermore, regardless of GFR levels, patients with UACR of ≥300 mg/g creatinine (macroalbuminuria) have much higher risk for renal and cardiovascular diseases and death than patients without macroalbuminuria3–5. Therefore, CKD patients with at least one of eGFR of <45 ml/min per 1.73 m2 and macroalbuminuria (defined as G3b and/or A3, in the present study) are at higher risk regarding the prognosis than the other CKD patients, and randomized trial on such patients at higher risk should be more encouraged to develop more appropriate antihypertensive therapy for ESKD and cardiovascular outcomes. However, there is insufficient evidence for antihypertensive strategy in patients with G3b and/or A3. These findings encouraged us to examine the subgroup of the hypertensive patients enrolled in ATTEMPT-CVD study with at least one of eGFR of <45 ml/min per 1.73 m2 and UACR of ≥300 mg/g creatinine. In the present study, patients with G3b and/or A3 had much greater incidence of cardiovascular and renal events than the other subgroup, being in good agreement with the established evidence3–5. The renoprotective effects beyond BP control of RAS blockers including ARBs and ACE inhibitors have been well established in CKD patients14–21 (particularly the patients with diabetic nephropathy). However, it remains to be elucidated whether RAS blockers are superior to other classes of antihypertensive drugs in CKD patients with G3b and/or A3 regarding prevention of cardiovascular events. The present analysis demonstrated that in hypertensive patients with G3b and/or A3, ARB therapy had less incidence of cardiovascular and renal events than non-ARB therapy, and allocation to ARB therapy was a significant independent prognostic factor of cardiovascular and renal events in such patients. Moreover, the incidence of doubling of serum creatinine was less in ARB group than in non-ARB group, further supporting the renoprotective effect of RAS blockade beyond BP control. Therefore, our study provided the evidence suggesting that ARB therapy may be more beneficial than non-ARB therapy in terms of prevention of cardiovascular and renal events in patients with G3b and/or A3. It is well established that BP reduction itself causes the prevention of CKD progression and cardiovascular disease and death4,22–24. However, in the present study, in patients with G3b and/or A3, BP during follow-up period tended to be higher in ARB group than in non-ARB group, although the difference did not reach statistical significance between ARB and non-ARB groups. Furthermore, there was no difference between ARB and non-ARB groups in the proportion of patients achieved BP of <140/90 mmHg. Therefore, it is unlikely that more benefit of ARB therapy over non-ARB therapy regarding prevention of cardiovascular and renal events in the patients with G3b and/or A3 might be attributed to blood pressure. Thus, BP-independent effects of ARB-based therapy seem to be responsible for less incidence of cardiovascular and renal events in ARB group than in non-ARB group of patients with G3b and/or A3. We have previously reported the findings of the OSCAR study25,26, which is a prospective, randomized trial to investigate comparative effect of ARB (olmesartan 20 mg/day) plus CCB combination therapy versus high-dose ARB (olmesartan 40 mg/day) therapy in Japanese elderly hypertensive patients who had at least one of cardiovascular diseases or type 2 diabetes at baseline. In CKD subgroup analysis of the OSCAR study25, we defined CKD patients as those with eGFR of <60 ml/min per 1.73 m2 but did not measure UACR, and found that ARB plus CCB combination reduced the incidence of cardiovascular events more than high-dose ARB therapy in elderly hypertensive patients with CKD (with eGFR of <60 ml/min per 1.73 m2), and more benefit of ARB plus CCB combination in prevention of cardiovascular events was associated with more reduction of BP25. Therefore, our CKD subanalysis of the OSCAR study showed that the combination of ARB with CCB is superior to uptitration of ARB (high-dose ARB) in prevention of cardiovascular events in elderly hypertensive patients with eGFR of <60 ml/min per 1.73 m2, probably because of greater BP-lowering effect of ARB plus CCB combination. Of note, as previously described1, in the ARB group enrolled in the ATTEMPT-CVD, the percentage of patients prescribed CCB was only 28% at baseline and 37% at 36 months (the end of the study) and the percentage of patients prescribed β-blockers and diuretics was 13% and 14%, respectively, at 36 months. On the other hand, in non-ARB group of the ATTEMPT-CVD, the percentage of the patients prescribed CCB was 99% at baseline and 100% at 36 months, and other main prescribed antihypertensive drugs were β-blockers (25%), diuretics (20%),and ACE inhibitors (13%) at 36 months1. Therefore, differing from the findings of the CKD subanalysis of OSCAR study25, in the present study, ARB monotherapy itself seems to have more benefit in prevention of cardiovascular and renal events than non-RAS antihypertensive agents in hypertensive patients with G3b and/or A3. Future large-scale randomized trial on CKD patients with G3b and/or A3 addressing this issue is needed to elucidate our proposal. It is suggested that UACR16,27–30 and eGFR changes are potentially useful for predicting future progression or prognosis of cardiovascular and renal diseases. In the present subanalysis, we also compared between ARB and non-ARB groups regarding the change of UACR and eGFR. Interestingly, in patients with G3b and/or A3, UACR during follow-up period was significantly less in ARB group than in non-ARB group, while ARB and non-ARB groups had similar eGFR changes. The improvement of UACR by ARB therapy, but not eGFR, was associated with less incidence of cardiovascular and renal events in patients with G3b and/or A3. On the other hand, in the other patients, no difference was noted between ARB and non-ARB groups regarding UACR, while ARB group has less eGFR than non-ARB group. Therefore, monitoring of albuminuria change may be more useful than that of GFR change in patients with G3b and/or A3. Plasma BNP is also proposed as a potential biomarker predicting the prognosis of cardiovascular and renal diseases31–33. Importantly, in patients with G3b and/or A3, plasma BNP levels were significantly higher than in the other patients. However, the change of plasma BNP levels was comparable between ARB and non-ARB groups in patients with G3b and/or A3 or in the other patients. Therefore, the present study provided no evidence for the significance of plasma BNP as a marker predicting the prognosis of cardiovascular and renal events in CKD with G3b and/or A3. ARB used in the present study was telmisartan. There are abundant experimental evidences indicating that differing from other ARBs, telmisartan is an ARB with partial PPAR-γ activity34–37. Therefore, it is an important question whether the benefit of telmisartan in prevention of cardiovascular events observed in this study was partially attributed to its PPAR-γ activity. However, in contrast to much experimental evidences, there is no clinical evidence indicating the significant role of PPAR-γ activity in telmisartan-induced organ protection. Furthermore, previous randomized double-blind placebo-controlled study38, which investigated the effect of telmisartan on PPAR-γ target genes CD36 and CD163 in patients with metabolic syndrome, showed that the activation of PPAR-γ target gene was not demonstrated by telmisartan at 80 mg (the highest dose used in our study) and partial activation of PPAR-γ target gene was observed only in 160 mg of telmisartan. Taken together, it is likely that cardiovascular and renal protective effect of telmisartan in this subanalysis was mediated by AT1 receptor blockade rather than PPAR-γ activity. Accumulating evidence36,39 supports the notion that AT1 receptor inhibition causes pleiotropic effects such as the amelioration of tissue oxidative stress and inflammation, glomerular hypertension, and cardiovascular remodelling. Therefore, the potential mechanisms underlying the benefit of telmisartan demonstrated in this subanalysis seem to be derived from the above mentioned pleiotropic effects36,39 induced by AT1 receptor inhibition. Future randomized study is required to elucidate the precise mechanism of cardiovascular protection by ARBs. BODY.STUDY LIMITATION: There are several study limitations in our subanalysis. First, the number of patients with G3b and/or A3 was small and our present findings are hypothesis generating. Such insufficient sample size seems to account for the finding that the treatment-subgroup interaction did not reach statistical significance. Large-scale prospective randomized trial on patients with G3b and/or A3 is required to define our present findings. Second, our present subanalysis is post-hoc analysis. However, we believe that our present proposal of categorization of the patients based on G3b cut-off value (eGFR of 45 ml/min per 1.73 m2) and A3 cut-off value (UACR of 300 mg/g) has the significant clinical implication, because the patients with G3b and/or A3 are at higher risk regarding prognosis. Furthermore, it provides important information for clinical decision making in terms of the therapeutic strategy for renal and cardiovascular outcomes. Finally, the present subanalysis did not allow us to determine whether more reduction of UACR by ARB therapy in patients with G3b and/or A3 was directly linked to fewer incidences of cardiovascular and renal events by ARB therapy. Future study demonstrating the direct association of UACR change with incidence of cardiovascular and renal events should be designed. In conclusion, our present subanalysis provided the evidence suggesting that ARB-based antihypertensive therapy may have the benefit in prevention of cardiovascular and renal events in hypertensive patients with G3b and/or A3 (with either eGFR of <45 ml/min per 1.73 m2 or UACR of ≥300 mg/g creatinine, or both). Our subanalysis provides a new insight into antihypertensive strategies for CKD. However, large-scale randomized trial is required to define our hypothesis generating findings. BODY.METHODS: BODY.STUDY DESIGN AND TREATMENT PROTOCOL: The detail of study design and treatment protocol of the ATTMEPT-CVD study has been previously reported2. This trial was registered with ClinicalTrials.gov number NCT01075698. In brief, ATTEMPT-CVD study is a multicentre, prospective, randomised, open-label, active-controlled trial with blinded end-point assessment of 1,228 hypertensive patients aged 40 to 80 years who had at least one of cardiovascular risk factors (type 2 diabetes, renal dysfunction, cerebrovascular disease, cardiac disease or peripheral artery disease). Full inclusion and exclusion criteria are described in our previous papers1,2. The study protocol was in agreement with the ethics committee guidelines of our institution and complied with the Declaration of Helsinki. The institutional review board of each participating hospital approved this trial, and written informed consent was obtained from each patient. The study protocol was approv The eligible patients were randomly assigned in a 1:1 ratio by computer-generated stratified randomization sequence were stratified for age, sex, history of cardiovascular events, history of diabetes mellitus, and usage of an ACE inhibitor and was allocated (1) treatment with telmisartan, an ARB (ARB group) or (2) treatment with antihypertensive drugs except for ARB (non-ARB group). Patients and study investigators were not masked for treatment allocation. After completion of registration and allocation, administration of telmisartan at the indicated low (20 mg/day) or middle (40 mg/day) dose was started in the ARB group. The dose level of telmisartan could be increased to the middle or highest dose (80 mg/day) by the physician’s decision in patients who did not achieve the target blood pressure. In the non-ARB group, an antihypertensive drug except for ARB was started. At study registration and after 3, 6, 12, 24 and 36 months, a physician examined the survey items including discontinuation/dropout, occurrence of any cardiovascular events, and occurrence of any adverse events. At 6, 12, 24 and 36 months, drug compliance, concomitant drugs, concurrent therapies, physical findings, and laboratory tests and biomarkers including urinary albumin/creatinine ratio (UACR), plasma BNP levels, serum high-sensitivity C-reactive protein (hsCRP) levels, urinary 8-hydroxy-deoxy-guanosine (8-OHdG), serum adiponectin and high-molecular weight adiponectin levels and estimated glomerular filtration rate (eGFR) were examined. BODY.ENDPOINTS: The primary endpoints of ATTMEPT-CVD study were changes in UACR and in plasma BNP levels from baseline2. The secondary endpoint was the time to the first occurrence of composite cardiovascular and renal events consisting of cerebrovascular events, cardiac events, peripheral arterial events, complication of diabetes, and aggravation of renal function2. In addition, changes in eGFR, hsCRP, urinary 8-OHdG, serum adiponectin, and serum high-molecular weight adiponectin were also the secondary endpoints. BODY.SUBGROUP ANALYSIS ACCORDING TO EGFR AND UACR CATEGORIES AT BASELINE: In the present analysis, the patients enrolled in the ATTEMPT-CVD study were divided into two subgroups by applying G3b cut-off value (GFR of 45 ml/min per 1.73 m2) and albuminuria A3 cut-off value (UACR of 300 mg/g creatinine). One subgroup was the patients with at least one of eGFR of <45 ml/min per 1.73 m2 and UACR of ≥300 mg/g creatinine (macroalbuminuria) at baseline, which were defined as patients with G3b and/or A3 (see the category enclosed by a thick line in Table 1). The other subgroup was patients with both eGFR of ≥45 ml/min per 1.73 m2 and UACR of <300 mg/g creatinine at baseline, which were defined as the other patients. BODY.STATISTICAL ANALYSIS: Sample size and power of the study were estimated as previously described1,2. All analyses were performed on the intention-to-treat population. All randomized patients with at least one on-treatment observation of laboratory data and safety information at that point were included in efficacy and safety analyses. Subjects who withdrew consent were excluded. As for cardiovascular and renal events, time to first event curves were estimated by the Kaplan-Meier method and the log-rank test was used to show the differences between ARB and non-ARB groups. Using Cox proportional hazard model, the hazard ratio (HR) of the ARB group to the non-ARB group and its 95% confidence interval (CI) were calculated. To estimate the heterogeneity of the HR for the subgroup according to baseline eGFR and UACR, the interaction between treatment groups and the subgroup according to eGFR and UACR was assessed using the interaction terms in a stratified Cox proportional-hazards model. Repeated measures analysis of variance was used to compare between ARB and non-ARB groups for time course of blood pressure during the follow-up period, and compared using the unpaired t-test adjusted by Holm’s method to avoid multiplicity at multiple time points. Multivariable Cox proportional hazards analysis was performed to determine the association of each prognostic factor with the incidence of cardiovascular and renal events adjusted for the following covariate: sex, age, treatment group, baseline cardiovascular diseases, baseline diabetes. The changes in UACR, plasma BNP, or eGFR were compared between ARB and non-ARB groups and analysed by repeated-measure analysis of variance (ANOVA) with measurement time points as repetition. Windows SAS Version 9.2 and subsequent versions were used as the statistical analysis software. P-values of less than 0.05 were considered statistically significant. BODY.DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary Information

TITLE: A randomized double blind placebo controlled trial assessing the efficacy and safety of praziquantel for the treatment of human schistosomiasis during pregnancy ABSTRACT.SUMMARY: ABSTRACT.BACKGROUND: Despite WHO recommendations to offer pregnant women treatment with praziquantel, many nations continue to withhold treatment, awaiting data from controlled trials addressing safety and efficacy. The objectives of the study were to 1) assess whether treatment of pregnant women with schistosomiasis at 12–16 weeks gestation leads to improved maternal and newborn outcomes and 2) collect maternal and newborn safety data. ABSTRACT.METHODS: Women who were otherwise healthy and infected with S. japonicum (N=370) were enrolled and randomized 1:1 to receive either over-encapsulated praziquantel (60 mg/kg in split dose) or placebo. The following efficacy outcomes were ascertained: maternal hemoglobin, iron status, and gestational weight gain, birth weight (primary outcome), newborn hemoglobin and iron status. Safety data were collected including immediate reactogenicity, post dosing toxicology ascertained 24 hours after study agent administration, and maternal and newborn serious adverse events. ABSTRACT.FINDINGS: Most women harbored low intensity infections (90.9%). Treatment with praziquantel did not have a significant impact on birth weight (2.85 kg in both groups, Beta −0.002, [0.88, 0.083]) or the incidence of low birth weight (OR 1.319 [0.729, 2.387]. Lack of treatment success may be due to the lack of difference in measures of maternal inflammation at 32 weeks gestation. Treatment with praziquantel resulted in a higher likelihood of treatment success (OR 5.815, [3.52, 9.61], P < 0.0001). Treatment was well tolerated with reactogenicity rates similar to that observed in non-pregnant subjects. There were no significant differences in key safety outcomes including abortion, fetal death in utero and congenital anomalies. ABSTRACT.INTERPRETATION: Results from this study provide important data from a controlled trial in support of the expansion of treatment policies to include pregnant women as recommended by WHO. ABSTRACT.FUNDING: The trial was funded by the United States National Institutes of Health, National Institute of Allergy and Infectious Diseases (U01AI066050). BODY.INTRODUCTION: Over 200 million individuals are infected with one of three species of schistosomes globally,1 including an estimated 40 million women of reproductive age. Schistosomiasis remains a significant cause of morbidity and mortality in low and middle income countries, despite the availability of effective pharmacologic therapy with praziquantel.2 Praziquantel was released in 1979, but was never studied in pregnant or lactating women and remains a United States Federal Drug Administration pregnancy Class B drug. Its Class B designation is based on numerous animal studies supporting its safety,3, 4 but a lack of well-controlled trials during human pregnancy. In 2002, a World Health Organization (WHO) report recommended that all schistosomiasis infected pregnant and lactating women be considered high-risk groups and be offered treatment with praziquantel individually or during treatment campaigns.5–7 This recommendation was reissued in 2006 as part of the WHO’s Guidelines for Preventative Chemotherapy for Helminthiasis,2 in which it was recommended that pregnant and lactating women be included in mass drug administration (MDA) campaigns. These recommendations were largely motivated by 1) the expected progression of both end-organ morbidity and anemia if women remained untreated during repeated cycles of pregnancy and lactation, 2) demonstrated safety in animal models of pregnancy, and 3) expected safety in humans based on inadvertent exposures during human pregnancy. Although some nations, particularly in Africa, have adopted this policy, many others, including The Philippines, have not, awaiting safety data from well-controlled trials in humans. Millions of women of reproductive age, therefore, are not treated for many years during repeated cycles of pregnancy and lactation. In addition to the lack of safety data in humans, the specific impact of human schistosomiasis on pregnancy outcomes remains understudied. Schistosomiasis has been implicated as a contributor to undernutrition8–14 among non-pregnant subjects. In particular, it is thought that schistosomiasis culminates in undernutrition through effects on appetite15 (anorexia and symptomatology) and inflammation-mediated cachexia.16 Schistosomiasis also contributes to the global burden of anemia, largely through anemia of inflammation.10, 12, 17, 18 In addition, studies support the role of schistosomiasis in iron deficiency anemia at higher intensities of infection, as individuals experience occult blood loss in urine and stool.19–22 Given demonstrated morbidity among non-pregnant subjects, a few studies have sought to address the impact of schistosomiasis infection in human pregnancy.23, 24 One randomized, controlled trial conducted in Uganda, examined the impact of praziquantel given to pregnant women during the second or third trimester (mean gestational age 26.6 weeks).24 That study differed from this trial as women who were and were not infected with schistosomiasis were included in the randomized sample. That trial did not demonstrate a significant impact of praziquantel on maternal anemia or birth weight, even among the approximately 18% of women who were infected with S. mansoni. No studies have examined whether treatment earlier in gestation improves pregnancy outcomes, none have examined the impact of treatment for S. japonicum, and none have examined use of the higher dose of praziquantel recommended for S. japonicum. The objectives of the study were to 1) assess whether treatment of pregnant women with schistosomiasis at 12–16 weeks gestation leads to improved maternal and newborn outcomes including birth weight (primary endpoint) and both maternal and newborn anemia and iron status and 2) collect safety data addressing immediate reactogenicity, adverse events during pregnancy, and adverse newborn outcomes such as congenital anomalies. We hypothesized that treatment of S. japonicum during pregnancy would lead to higher newborn birth weight by improving maternal appetite and nutritional status, higher maternal hemoglobin and bio-available iron through decreasing the risk for anemia of inflammation during pregnancy, and improved newborn iron stores through greater iron bio-availability to the developing fetus. BODY.METHODS: BODY.STUDY DESIGN AND PARTICIPANTS: This study was a Phase 2, double blind, placebo controlled trial addressing the effects of praziquantel given at 12–16 weeks gestation on maternal and newborn birth outcomes (efficacy) and initial safety and toxicology. The initial target sample size was 500 pregnant women, with a plan to enroll equal numbers of women with low intensity and moderate/high intensity infections with S. japonicum. The protocol was modified to omit this requirement when it was noted that the prevalence of moderate and high intensity infections was much lower than expected. It was later modified to decrease the target enrollment to N = 370 given a concern regarding study duration as the overall prevalence of S. japonicum infection was lower than originally anticipated. This approach was tenable as attrition rates were significantly lower than originally anticipated such that the primary outcome was still captured on more than the planned number of subjects (162 in each arm) needed to detect a difference in birth weight of 125 grams, with a type one error of 0.05, and a power of 0.80. The study was conducted in approximately 50 baranguays (villages) serviced by six Municipal Health Centers (MHCs), in a schistosomiasis endemic region of northeastern Leyte, The Philippines. At this study site, soil-transmitted helminths (STHs), but not malaria, are endemic and the prevalence of HIV is <0.1%.25 Informed consent for screening procedures was conducted by one of 12 midwives servicing the six MHCs who were members of the study staff. Initial eligibility screening included a urine pregnancy test and three stool samples collected on different days for the quantification of S. japonicum and STH eggs using the Kato-Katz method. Kato-Katz was performed by trained medical technologists at the study laboratory in Palo. Two Kato-Katz slides were examined for each sample within 30 minutes for hookworm and up to 24 hours later for other helminth infections including schistosomiasis. Each stool sample was quantified in duplicate and mean determined. Intensity of infection was determined as the mean of the three samples. S. japonicum infection intensity was categorized as low (0–99 eggs per gram (epg) of stool, moderate (100–399 epg of stool) or high (400 + epg of stool). The second phase of screening was conducted at Remedios Trinidad Romualdez (RTR) Hospital in Tacloban, Leyte. The study physician performed a history and physical examination and a trans-abdominal ultrasound to assess fetal viability and estimate gestational age. In addition, a blood sample was obtained prior to administration of the study agent to measure serum chemistries and a complete blood count (CBC). Weight, height and other anthropometric measures were made including thigh skin fold thickness as described.26, 27 Women were eligible for the trial if they provided informed consent and were infected with S. japonicum, age 18 or older, otherwise healthy as determined by physician history, physical examination and laboratory studies, and pregnant at 12–16 weeks gestation with a live, singleton, intrauterine fetus. Pregnancies with estimated gestation less than 12 weeks were scheduled to return, as were women with an acute medical condition that could potentially be addressed prior to 12–16 weeks gestation. All women were offered treatment for schistosomiasis and soil-transmitted helminths at the conclusion of lactation as it was the Philippines Department of Health policy to defer treatment until cessation of breast feeding. Infants who had a medical disorder or malnutrition diagnosed during the newborn period were referred for care. The study was separately approved by both the Rhode Island Hospital Institutional Review Board in Providence, RI and the Ethics Review Board of the Research Institute of Tropical Medicine in Manila, The Philippines. This trial was registered with ClinicalTrials.gov, number NCT00486863. BODY.RANDOMIZATION AND MASKING: Women who met eligibility criteria were randomly assigned (1:1) to receive either over-encapsulated praziquantel (30 mg/kg×2) or over-encapsulated placebo (dextrose), as a split dose over three hours in a double blind fashion. The study agent was compounded by The Rhode Island Hospital Pharmacy, using praziquantel tablets obtained from Bayer’s United States distributor, Shering Plough. Dextrose and gelatin capsules were provided by Gallipot. Both provided certificates of analysis for these compounds prior to study initiation. Two capsule doses were made which were differentiated by color, containing 300 or 150 mg to allow for optimal dosing by weight. Statisticians at the EMMES Corporation, the Statistical and Data Coordinating Center for the study, randomly allocated study numbers in blocks of six. Study drug was administered at RTR Hospital. BODY.PROCEDURES: Following each of the split doses, subjects were actively assessed for adverse drug reactions and then observed in the hospital for 24 hours. Prior to discharge, a repeat blood sample was drawn for serum chemistries and CBC. In addition, after approximately N=100 subjects were recruited, the protocol was amended to include two additional blood samples collected at varying times post-dosing for population pharmacokinetics, the results of which will be reported elsewhere. Approximately 10–14 days after discharge, subjects were visited at their home by study staff to elicit any symptoms experienced since hospital discharge. Subjects had two scheduled follow up visits prior to delivery at 22 +/− 2 weeks and 32 +/− 2 weeks gestation, both at RTR Hospital. Stool was collected at the 22 week visit for assessment of parasitologic cure. In addition, women were seen by the study obstetrician and a detailed history and physical examination were performed, including measures of nutritional status as described above. At the 32 week visit, the history, physical exam, and nutritional measures were repeated, a blood sample was collected for CBC as well as analytes capturing iron (ferritin, serum transferrin receptor (sTfR)) and inflammatory (C-reactive protein (CRP), hepcidin, TNF-α, IFN-λ, IL-6, and IL-1) markers. Serum samples were aliquoted and stored at −80°C. Bioactive serum hepcidin (DRG® Hepcidin 25 bioactive ELISA (EIA-5258)) was measured according to manufacturer's instructions. Other analytes were quantified using a multiplex bead-based platform (Bio-Rad, Hercules, CA) as described previously.8 A urine sample was collected to screen for pre-eclampsia and urinary tract infections. Women were scheduled for additional visits as needed based on obstetrician-identified diagnoses and were also asked to come to RTR for unscheduled visits for other concerns that arose. Mothers gave birth in one of the six MHCs or were referred to RTR Hospital if indicated. Gestational age at birth was assessed by a modified Dubowitz scoring system developed by Ballard.28 For deliveries that occurred at home based on maternal preference or inability to get to an MHC, mothers contacted the study midwife who visited the mother within 48 hours of delivery. Both mother and newborn returned to RTR hospital at 2–6 days of life for follow-up. The study pediatrician performed a history and physical examination and a heel stick blood sample was obtained for newborn screening, CBC, and the aforementioned iron and inflammatory analytes. Study close out occurred at RTR hospital at 28 days of life, at which point the pediatrician again performed a history and physical examination and referred the newborn for any identified concerns. BODY.OUTCOMES: The primary outcome was birth weight. Newborns were weighed within 48 hours of delivery on a Tanita model BD-585 portable scale (Arlington Heights, MD) to 10 grams. Birth weights taken 24–48h hours after delivery were corrected by a factor of +2%, to obtain the estimated weight at birth. Secondary outcomes included low birth weight, defined as weight < 2.5 kg. Small for gestational age (SGA), used as an indicator for possible intra-uterine growth restriction (IUGR), was defined based on gestational age determined at the 12–16 week ultrasound. SGA was defined as gestational age adjusted birthweight that was <10th percentile based on the multi-racial Williams curves.29 Other secondary efficacy outcomes included change in maternal nutritional status, maternal anemia (hemoglobin < 11.0 g/dL), hemoglobin, and iron status at 32 weeks gestation, newborn hemoglobin and iron status measured on a heel stick blood sample at 2–6 days of life, prematurity defined as gestation < 37 weeks, and parasitologic “cure” as defined by a >90% reduction in S. japonicum egg count from screening to 22 weeks gestation. Safety outcomes included toxicity to maternal bone marrow, kidney, and liver as measured by laboratory parameters collected just before, and 24 hours after dosing, maternal seizures, immediate toxicity to the fetus as assessed by abortion (fetal demise before 20 weeks gestation), live birth rate, and newborn congenital anomalies. BODY.ASSESSMENT OF POTENTIAL CONFOUNDERS AND MODIFYING COVARIATES: Household socio-economic status was captured using a questionnaire developed previously for use in this population as described.30 A summary score reflected numeric weights assigned to SES questionnaire items as described by Filmer,31 using the FACTOR procedure in SAS 9.3 (Cary, NC). Summary SES scores were categorized by quartile and compared by treatment group. The following other pre-specified covariates were evaluated as potential confounders: maternal age, maternal weight and height at enrollment, smoking status at enrollment (yes/no), reported alcohol intake at enrollment (1–2, 3–5, 6–8, 9–10 and more than 10 glasses of alcohol consumed per week), newborn sex, reported compliance with iron supplementation, intensity of infection with S. japonicum at enrollment (low, medium, high as described above) and treated as an ordinal variable in analyses, obstetrical history (defined as having one or more occurrences of a miscarriage, abortion or stillbirth) treated as a dichotomous variable in analyses, and intensity of infection with geohelminths at enrollment defined using World Health Organization (WHO) criteria. BODY.STATISTICAL ANALYSIS: Outcomes and potential confounders were compared between praziquanel and placebo groups at enrollment using Chi-square and Wilcoxon two-sample or t-tests. Potential confounders were considered for inclusion in final models if associated with treatment at the alpha level of 0.10. Of note, none met this criteria such that outcomes are presented by treatment group without adjustment for potential confounders. Primary and secondary efficacy analyses were performed using hierarchical linear models to account for potential clustering of observations within villages and municipalities. Models of each outcome included a main effect for treatment and random intercepts for villages and health centers. Intraclass correlations were estimated from the covariance parameters for each random effect to evaluate the proportion of the total variance attributable to village or health center. Covariance parameter estimates were evaluated for significance with a Wald test. Random effects whose intercept variance estimates were not significantly different from zero at the alpha level of 0.05 were removed from the final model. Standard model diagnostics were used to evaluate model assumptions of linearity, normality and constant variance of residuals. Outcomes were log transformed if necessary to avoid violation of regression assumptions. Analyses were performed using the MIXED procedure for continuous outcomes and NLMIXED procedure for binary outcomes in SAS 9.3. Primary efficacy models additionally evaluated the interaction of infection intensity with S. japonicum (epg) at enrollment with treatment allocation, to determine whether baseline intensity of infection modified the relationship between treatment and birth weight. Analyses were performed based on the intention to treat population, from which there were no deviations in product received. BODY.ROLE OF THE FUNDING SOURCE: The study funder (United States National Institutes of Health, National Institute of Allergy and Infectious Diseases) supported protocol development in collaboration with the investigators and engaged a data management and statistical analyses partner, EMMES Corporation, responsible for design of data bases, summary reports for Data Safety and Monitoring Board meetings, and final statistical analyses for this report. The funder had no role in generation of hypotheses, data collection, data analysis, data interpretation, or writing of the report. The authors, with the exception of Dr. Watson, did not have access to study data until all databases were locked and the analyses reported in the manuscript were complete. The corresponding author had access to the locked data set and had final responsibility for the decision to submit for publication. BODY.RESULTS: Of 2637 women screened for eligibility, the primary reasons for exclusion were 1) not infected with S. japonicum (N=2046) and 2) ultrasound not consistent with viable intrauterine, singleton pregnancy at 12–16 weeks gestation (N=139). Of 370 pregnant women enrolled and randomized, there were five fetal deaths in utero. Birth weight was ascertained for 361 of 365 live births (Figure 1). The randomized cohort included 334 women, most with low intensity infection (90.3%). Table 1 presents baseline socio-demographic, obstetrical, nutritional, and clinical covariates by treatment allocation. These variables were equally distributed between treatment groups. Treatment with praziquantel did not have a significant impact on the primary outcome, birth weight (2.85 kg in both praziquantel and placebo groups, P = 0.99) or secondary efficacy outcomes including prevalence of low birth weight and SGA newborns. (Table 2) The estimated association of treatment with birth weight (kg) from the mixed effects model was Beta = −0.002 ([95% CI −.088, 0.083]; P = 0.962). In mixed models of outcomes other than birth weight, covariance parameter estimates for the village and municipality random effects were not significantly different from zero, indicating that reported bivariate comparisons in Table 2 are sufficient for these data. With respect to maternal nutritional status, treatment with praziquantel did not impact maternal weight gain or change in thigh circumference (Table 2). At 32 weeks gestation, the group that received praziquantel had significantly higher ferritin, however, there was no significant difference in maternal hemoglobin at 32 weeks gestation. There was no significant difference between groups in hepcidin, the principal regulator of systemic iron homeostasis. (Table 2) Of note, there were no significant differences between groups with respect to inflammatory markers such as CRP, TNF-alpha, and IL-6. In both groups, all women (100%) reported taking pre-natal vitamins with iron as directed. Treatment was effective at reducing maternal intensity of infection as evidenced by a significantly higher proportion of women with a >90% reduction in egg (85.3 versus 50.0%, P < 0.0001). Praziquantel had a significant impact on cure rate as defined by the percentage of women who were not infected with S. japonicum at 22 weeks gestation (83.7% versus 46.2%, P < 0.0001). Praziquantel treatment had no effect on the prevalence or intensity of the STH infections, in particular hookworm. There was also a difference between groups in the percent change in S. japonicum (epg) from screening to 22 weeks gestation, though this did not reach statistical significance (−53.11 versus 128.7; P = 0.054). With respect to other secondary efficacy outcomes for newborns, maternal treatment did not influence newborn hemoglobin level or ferritin. (Table 2) Non-significant differences were noted for newborn sTfR and sTfR:ferritin ratios; newborns of mothers treated with praziquantel had slightly lower levels for both bio-markers, potentially indicating less cellular iron thirst and greater total body iron, respectively, among newborns of treated versus untreated mothers. (Table 2) There were no significant changes in serum chemistries or hematologic parameters in mothers before and 24 hours after dosing, with the following exceptions. There was a significant difference in the change in white blood cell and lymphocyte counts with women in the placebo group experiencing a greater increase from pre- to post-treatment. (Table 3) Women in the praziquantel group experienced a significantly higher change in creatinine from pre to post-dosing compared to the placebo group (0.07 versus 0.01 mg/dL, respectively; P = 0.004), however, these were all mild (grade I) elevations. Overall 18 subjects in the praziquanel group and 16 in the placebo group experienced a serious adverse event (SAE) with a wide range of event classifications that were largely expected during pregnancy in a high-risk population. None occurred in the immediate post-treatment period. There were no significant differences in the rate of fetal death in utero, the live birth rate or the number of congenital anomalies. (Table 3) Congenital anomalies included two newborns (one in each treatment group) with talipes equinovarus (club feet) and three infants with cleft lip/palate (two in praziquantel group and 1 in the placebo group). With respect to immediate reactogenicity, 323 subjects, 169 of whom received praziquanel, reported at least one side effect of treatment that was of mild or greater severity. Reactions graded as severe occurred in five subjects in the praziquanel group and two in the placebo and included headache, fever and malaise. All severe reactions were attributed to study product administration and resolved during the 14-day reactogenicity period. BODY.DISCUSSION: This is the first clinical trial to investigate the efficacy and safety of treating pregnant women with S. japonicum infection, at the higher recommended dose of praziquantel (60 mg/kg) recommended for Asian Schistosomiasis. The study was a phase two trial designed to assess impact on pregnancy outcomes and collect initial toxicology data for both mother and newborn. Treatment did not have a significant impact on the primary outcome, birthweight, or the rate of low birth weight newborns. Treatment may have led to improved iron status in both the mother at 32 weeks gestation and newborn. Importantly, praziquantel was well-tolerated with rates of immediate reactogenicity similar in this cohort of pregnant women to those observed in non-pregnant subjects.13 In addition, there were no significant differences in the rates of serious adverse events among mothers or newborns, comparing the treated versus untreated groups. This trial differs from another important, four arm RCT conducted in an S. mansoni endemic region of Uganda. In that study women were randomized to receive either albendazole + praziquantel, albendazole alone, praziquantel alone, or placebo. All women were randomized to one of the four groups, whether or not they harbored specific baseline infections with STH and/or S. mansoni.24 The mean gestational age at enrollment was 26.6 weeks. Neither the Ugandan study, which treated women later in gestation, nor our trial, which randomized women early in the second trimester, demonstrated a significant impact on birth weight. In the Ugandan study, this was true even when analyses were restricted to women found to be infected with S. mansoni. In both studies, of the women who were infected, most had low intensity infections. In the present study, interactions between baseline intensity of infection and treatment effect on birth weight were also not significant, suggesting that there were not differences in treatment efficacy among women with moderate or high intensity infections. This should be interpreted with caution, however, due to the small number of women with moderate and high intensity infections enrolled (9.7% at baseline). Other explanations for lack of impact on birth weight include the fact that treatment did not significantly impact maternal weight gain, a hypothesized potential mechanism. This may be due, in part, to a lack of effect on maternal inflammation even by 32 weeks gestation. In previous observational studies conducted by our group, women infected with S. japonicum had significantly higher TNF-α levels at 32 weeks gestation (mean 2.11 pg/ml) than uninfected women (mean 0.34 pg/ml).32 In the trial, these values at 32 weeks gestation were 1.6 pg/ml and 1.3 pg/ml for the placebo and praziquantel group, respectively, suggesting that treatment did not resolve inflammation. Another hypothesized mechanism through which praziquantel could lead to improved birth weight was through effects on placental health. Observational studies from our group suggested that infection with S. japonicum culminates in a pro-inflammatory signature at the maternal-fetal interface and this is associated with lower birth weight.32 Further, schistosome antigens elicit a pro-inflammatory immune response from primary human trophoblast cells from healthy placentas.33 It is possible, however, that praziquantel given during gestation does not sufficiently disrupt the delivery of immunogenic schistosome antigens to the placenta, given treatment results in a period of increased circulating antigens with a prolonged inflammatory immune response.8, 34 It remains possible that schistosome infections adversely affect placental health, but treatment after the first trimester may not mitigate this risk. In addition, neither our study nor the Ugandan trial demonstrated a significant impact on maternal hemoglobin or prevalence of maternal anemia. In studies conducted by our group among non-pregnant subjects, we have demonstrated a significant time lag after treatment before improvements in hemoglobin are noted, with no significant difference noted three months following therapy, but a significant increase six months following treatment.8 We did find that women treated with praziquantel had significantly higher ferritin levels at 32 weeks gestation. In addition, there was a trend toward increased newborn iron endowment as captured by sTfR:ferritin ratio, a marker for total body iron, and sTfR, a marker of cellular iron thirst among their newborns. It is likely that treatment began to modify the risk of maternal anemia of inflammation, which would be expected to increase iron absorption from the gut and increase the pool of maternal bioavailable iron before an impact on maternal hemoglobin could be observed.35 With respect to safety outcomes, both the Ugandan and our study did not observe significant differences in the rate of congenital anomalies or other serious adverse events comparing the praziquantel and placebo groups. Our study additionally captured abortion, defined as fetal loss occurring before 20 weeks gestation, which in this case would have been within 4–8 weeks of praziquantel dosing. There were no differences between groups in either abortion or rates of fetal death in utero at any point in gestation. Taken together, these studies contribute key toxicology data regarding the use of praziquantel during human pregnancy, supporting its safety at different timepoints during pregnancy after 12 weeks gestation. In WHO guidelines from 2002 and 2006, it was recommended that all schistosomiasis infected pregnant and lactating women be considered high-risk groups and be offered treatment with praziquantel individually or during treatment campaigns.5–7 These recommendations were based on safety from animal studies, as well as inadvertent exposures during human pregnancy, rather than data from controlled trials in humans. As such, the recommendations did not provide conclusive data regarding safety and, due in part to this constraint, many schistosomiasis endemic regions continue to exclude pregnant and lactation women from both MDA and individualized treatment programs. Results from this study, together with the Ugandan study, should provide necessary reassurance that praziquantel given after 12 weeks gestation is safe, effectively treats schistosomiasis, and may improve both maternal and newborn iron status. Study limitations include the low number of women who harbored moderate or high intensity infections, who may have been more likely to experience schistosomiasis-associated morbidity and benefit from treatment. This limits our ability to understand the impact of treatment in the context of higher intensity infections. We were also surprised to find that 46.2% of women in the placebo group were “cured” by 22 weeks gestation. It is unlikely that this is due to treatment outside of the study given it was the strict policy of the Philippines Department of Health to withhold treatment from pregnant women and almost all treatment occurs during MDA campaigns. Further, all women in both groups provided three stools at follow up for evaluation. It is more likely that many women in the placebo group with light infections at baseline may have been missed at 22 weeks gestation, contributing to an apparently high rate of “cure.” In addition, by design, our study sample consisted of otherwise healthy, adult women, limiting generalizability of findings somewhat. In addition, in many parts of the world, MDA is the current approach to schistosomiasis control, whereas women in this study had to be infected with S. japonicum to participate. In addition, given the use of single dosing in MDA campaigns as opposed to the split dose employed, reactogenicity in practice may be higher than observed in this trial. Finally, in the context of inflammation, ferritin is elevated, thus complicating its interpretation as a measure of stored iron. Therefore, it is possible that treated women had higher levels of ferritin at 32 weeks gestation due to prolonged inflammation following treatment. This is unlikely given the expectation that acute phase responses such as elevated ferritin would resolve 4–5 months after treatment and is further evidenced by a lack of difference in other acute phase proteins such as CRP. Results from this trial provide important data from a controlled trial in support of the expansion of treatment policies to include pregnant women as recommended by WHO. Though this study was not powered to detect differences in rates of rare outcomes such as congenital anomalies, these results, together with results from the Ugandan study, post-marketing surveillance data including experience treating pregnant women in MDA campaigns in some African nations, and data from animal studies support ending the exclusion of pregnant women from MDA campaigns and individualized treatment. Though we did not observe an impact on birth weight, it remains possible, based on animal models, as well as human observational and mechanistic studies, that schistosomiasis adversely affects pregnancy outcomes, however treatment during gestation is too late to be impactful. If this is the case, including pregnant women in MDA campaigns will increase the likelihood that they enter subsequent pregnancies free of schistosomiaisis infection.

TITLE: Supplementation with a highly concentrated docosahexaenoic acid plus xanthophyll carotenoid multivitamin in nonproliferative diabetic retinopathy: prospective controlled study of macular function by fundus microperimetry ABSTRACT.OBJECTIVE: There is little evidence of real-life outcomes of dietary supplementation with high-dose docosahexaenoic acid (DHA) and carotenoids in patients with diabetic retinopathy (DR). We assessed the effect of supplementation with DHA triglyceride (1,050 mg/d) + xanthophyll carotenoid multivitamin on macular function in nonproliferative DR. ABSTRACT.METHODS: Asymptomatic patients with nonproliferative DR were included in a prospective controlled study and assigned (1:1) to the DHA supplementation group or the control group. Macular sensitivity and macular integrity area were the main outcome measures. Functional vision measures (macular function [MAIA™ CenterVue], best-corrected visual acuity), structural retinal measures (central subfield macular thickness), and biochemical parameters (plasma total antioxidant capacity, DHA content of the erythrocyte membrane, and plasma IL-6) were evaluated at baseline and after 45 and 90 days of DHA supplementation. ABSTRACT.RESULTS: The study included 24 patients (48 eyes) (12 patients, 24 eyes in each group). Baseline clinical characteristics of patients in both groups were similar. Macular sensitivity increased from a mean (SD) of 25.9 (2.4) dB at baseline to 27.3 (2.3) dB at 90 days (P=0.030) in the DHA group only (between-group differences P<0.19). The macular integrity index decreased from 71.2 (33.2) at baseline to 63.5 (36.4) at 45 days and to 51.6 (35.9) at 90 days (P=0.002) in the DHA group only (between-group differences P<0.05). Best-corrected visual acuity and central subfield macular thickness did not vary significantly in any of the comparisons and in none of the groups. DHA content of erythrocyte membrane and total antioxidant capacity levels increased significantly only in the DHA group. Plasma IL-6 levels decreased significantly only in the DHA group. ABSTRACT.CONCLUSION: In an early stage of DR, supplementation with high-dose DHA plus xanthophyll carotenoid multivitamin during 90 days was associated with a progressive and significant improvement of macular function measured by microperimetry. Biochemical changes supported the effect of DHA. BODY.INTRODUCTION: Diabetic retinopathy (DR) is a microangiopathic complication of diabetes and the leading cause of vision loss in working-age adults.1 Approximately 1 in 3 people living with diabetes have some degree of DR, and 1 in 10 will develop a vision-threatening form of the disease. Diabetes mellitus is becoming a global epidemic, with 415 million people having diabetes, and the number is expected to increase to 642 million by 2040.2 Approximately 75% of the global diabetes burden occurs in low- and middle-income countries, and 46.5% of adults with the disease are underdiagnosed.2 These alarming statistics urgently require effective strategies for prevention, early diagnosis, and appropriate treatment. Good glycemic control and treatment of modifiable risk factors have been extensively recognized as essential factors for the successful ophthalmic care of patients with diabetes.3–5 The pathogenesis of DR is multifactorial, and a number of interconnecting events including capillary endothelial vascular dysfunction and retinal pericyte loss as a result of hyperglycemia; local inflammatory activity with upregulation of proinflammatory mediators, interleukins, and growth factors; cellular hypoxia; oxidative stress; breakdown of the blood–retinal barrier; and retinal neurodegeneration have been reported.6–11 Full understanding and the sequence of these underlying mechanisms remains unclear. The pleotropic mechanisms of action of long-chain polyunsaturated fatty acids (ω-3 PUFAs), particularly docosahexaenoic acid (DHA),12–22 as well as the xanthophylls, lutein and zeaxanthin,23–25 are involved in the molecular pathways implicated in DR (Figure 1)15,17,23–34 and support the rationale of dietary supplementation with DHA and xanthophyll carotenoids in DR. However, there is little evidence of real-life outcomes of dietary supplementation with DHA in patients with DR. In a randomized controlled study of patients with diabetic macular edema (DME) treated with intravitreal ranibizumab, the addition of a dietary supplement rich in DHA reduced macular thickness after 2 years of follow-up as compared with ranibizumab alone.35 This anatomical improvement was accompanied by a trend for an amelioration of visual acuity.35 In patients with DR and well-controlled diabetes, increasing PUFA intake was associated with a reduced likelihood of the presence and severity of DR.36 Interestingly, in a study of lipidomic analyses on red blood cell membranes from controls and type 2 diabetes patients, a significant decrease in levels of DHA and arachidonic acid in erythrocytes of diabetic patients with and without retinopathy was observed.37 This observations provide a good rationale to supplement the diet with DHA, although DHA is mainly used for its anti-inflammatory, antioxidant, and antiangiogenic effects.14–17,22 The possibility that DHA supplementation could prevent the progression of DR acting on an early stage of the disease is an appealing hypothesis. To this end, a prospective controlled study was designed to assess the effectiveness of dietary supplementation with a high rich DHA oral nutraceutical formulation in patients with non-proliferative DR (NPDR). Microperimetry, a relatively new and extremely sensitive method for studying retinal and optic nerve diseases, was used to assess macular function. Evaluation of subtle changes in macular function with an objective method, such as microperimetry, may support the advantage of early DHA supplementation in diabetic subjects with incipient DR. To our knowledge, studies on macular function using microperimetry in asymptomatic patients with type 2 diabetes and NPDR have not been previously reported. BODY.METHODS: This study was approved by the Ethics Committee of Hospital Universitario Virgen de la Arrixaca (Murcia, Spain) and adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent. The study was registered in the European Clinical Trials Database (EudraCT) (EudraCT trial number 2017-000856-25 for the Sponsor’s Protocol code number DHA/RDNP). BODY.STUDY DESIGN AND PARTICIPANTS: A prospective controlled study was conducted at the outpatient clinics of the Service of Ophthalmology of an 860-bed acute care teaching hospital in Murcia, Spain. Patients of both sexes aged >18 years were invited to participate in the study during an ophthalmologic appointment at the study center. All patients had type 2 diabetes mellitus and were asymptomatic at the time of enrollment. They had been diagnosed with NPDR elsewhere and were referred to our service for a full vision evaluation between June 2014 and June 2016. The diagnosis of NPDR in all referred asymptomatic type 2 diabetes patients was confirmed, with fundus examination showing the presence of microaneurysms and some isolated hard exudate. Clinically significant macular edema was absent. Patients unable to participate in the study according to the criteria of the ophthalmologist and those who refused to sign the written consent were excluded from the study as were patients using vitamin/mineral or fatty acids supplements and those with hypersensitivity to these compounds. BODY.STUDY INTERVENTION: Each participant contributed 2 study eyes to the protocol. The diagnosis of NPDR according to criteria of the Early Treatment Diabetic Retinopathy Study (ETDRS)38 was confirmed before enrollment. The diagnosis was based on clinical examination and retinography and was made by 2 specialized ophthalmologists. Study patients were consecutively assigned with a 1:1 sequential allocation to the DHA supplementation (experimental) group or to the control group. The inclusion criteria were identical for all study patients. Subjects in the control group received no treatment at all and were masked regarding the existence of the experimental group. Patients in the DHA group received a high rich DHA (1,050 mg/d) nutraceutical formulation (Brudyretina 1.5 g; Brudy Lab, S.L., Barcelona, Spain). This is a concentrated DHA triglyceride having a high antioxidant activity patented to prevent cellular oxidative damage.39,40 Table 1 shows the composition of the nutraceutical formulation, which includes a high dose of DHA (1 g), eicosapentaenoic acid, a mixture of B vitamins, vitamins C and E, lutein, zeaxanthin, and minerals. All fatty acids were present in the form of triglycerides (>95%) or ethyl esters (<5%). Patients were instructed to take 3 capsules of Brudyretina 1.5 g once daily. The treatment evaluator (M.E.R.G-H.) was masked of which subjects were receiving DHA supplementation. BODY.STUDY PROCEDURES AND OUTCOME: The duration of the study was 90 days. All patients were evaluated at baseline and at 45 and 90 days thereafter. At each visit, best-corrected visual acuity (BCVA) and measurement of central subfield macular thickness (CSMT) by spectral-domain optical coherence tomography (Cirrus HD-OCT, Carl Zeiss Meditec, Dublin, CA, USA) were assessed. BCVA was assessed using the ETDRS optotype at 2 m distance from the observer. Other investigations included retinography (VISUCAM®PRO NM, Zeiss) and assessment of macular function by microperimetry (MAIA 1™ CenterVue, Topcon España, S.A., Sant Just Desvern, Barcelona, Spain). Macular function included assessment of retinal sensitivity (10° diameter area) and macular integrity index. The MAIA sensitivity scale is 0–36 dB. The macular integrity index is a numerical value that describes the likelihood that a patient’s responses are normal, suspect, or abnormal when compared to age-adjusted normative data. Higher numbers suggest a greater likelihood of abnormal findings, while lower values suggest a greater likelihood of normal findings. At the beginning of the study and at 90 days, the following were measured: erythrocyte membrane DHA content, plasma total antioxidant capacity (TAC), and plasma levels of IL-6. The National Eye Institute Visual Function Questionnaire-25 (NEI-VFQ-25)41 was also evaluated at baseline and at 45 and 90 days. Total score of the NEI-VFQ-25 ranges between 0 and 100 (a high score represents better functioning). At each visit, the nutraceutical formulation was delivered to the patient for 45-day treatment. Compliance with DHA supplementation was assessed at the study visits by return of supplementation tablet counts and analytical data, especially erythrocyte membrane DHA content. Ophthalmologists paid special care to insist on the importance of compliance with the dietary supplement and the benefit that the patient may receive from the supplement. TAC in plasma samples was measured using the OxiSelect Total Capacity Assay kit (STA-360, Cell Biolabs Inc., San Diego, CA, USA) following the manufacturer’s instructions. High values in the TAC assay reflect high antioxidant capacity (ie, greater protection). Uric acid equivalent was used to calculate copper-reducing equivalent values (µM copper-reducing equivalent). The composition of fatty acids on the erythrocyte membrane (ω-3 DHA) was determined using the method described by Lepage and Roy,42 analyzed by gas chromatography–mass spectrometry and identified by comparing the elution pattern and relative retention times of fatty acid (FA) methyl esters with a reference FA methyl esters mixture (GLC-744 Nu-Chek Prep. Inc., Elysian, MN, USA). The results were expressed in relative amounts (% of total FA). Plasma levels of IL-6 were measured by a solid-phase sandwich enzyme-linked immunosorbent assay with a commercial kit (Human IL-6 ELISA Kit, Cat. No 950.030 purchased from Diaclone SAS, Besancon Cedex, France). This assay recognizes both natural and recombinant human IL-6. The instructions provided by the manufacturer were followed for the qualitative and quantitative determination of IL-6. The samples or standards were added in wells of the microtiter strip plate coated with anti-IL-6 monoclonal. Then, a biotin-conjugated monoclonal anti-IL-6 antibody was added and bonded to IL-6 captured by the first antibody. Later enzyme-linked secondary antibody (Streptavidin-HRP) was added, and bonded to detecting antibody. Ultimately, a chromogen substrate was added and enzymatically converted to detectable form by absorbance and measured at a wavelength of 450 nm with a microplate reader Synergy H1 Hybrid Multimode (BioTek Instruments, Winooski, VT, USA). The primary outcome of the study was the changes in macular sensitivity and macular integrity index during the study period. Secondary outcome variables were BCVA, CSMT, plasma TAC, erythrocyte membrane DHA content, plasma IL-6 levels, and the NEI-VFQ-25 score. BODY.STATISTICAL ANALYSIS: The sample size calculation was based macular sensitivity as the main variable of the study. Considering a standard deviation of 2.3 dB as reported by Roisman et al43 with a precision of 1.7 dB, an α risk of 5% and a power of 80%, the sample size per group was 23 eyes. Assuming 5% losses at follow-up, 24 eyes per group were needed (total 48 eyes). Analysis of visual function tests, including microperimetry, BCVA, and CSMT, was based on 48 eyes (24 eyes in each study group), whereas analysis of laboratory tests was based on 24 patients (12 patients in each study group). Categorical data are expressed as frequencies and percentages and continuous data as mean and standard deviation (±SD). Mixed linear model analysis was used to assess differences in the study variables between DHA supplementation and control groups over the 90-day study period (covariate: baseline data of variables; random factor: patients). Statistical significance was set at P<0.05. Statistical analyses were performed with the Statistical Package for the Social Sciences, version 11.0 software (SPSS Inc., Chicago, IL, USA). BODY.RESULTS: A total of 62 eyes from 31 patients diagnosed with NPDR were referred for a full vision evaluation during the study period, but 7 patients (14 eyes) were excluded because of the presence of neovessels, high risk for proliferative retinopathy, or doubts regarding adherence to the study protocol. No drop outs were recorded, and results were based on analysis of the per-protocol data set. The study population included 24 patients (48 eyes) with type 2 diabetes. There were 17 men and 7 women, with a mean age of 60.6 years (range 36–79 years). As shown in Table 2, the clinical characteristics of patients were similar in the 2 study groups. The mean ± SD values of all study variables at baseline and at 45 and 90 days are shown in Table 3. In relation to the primary outcome of the study (Figure 2), macular sensitivity increased from a mean of 25.9±2.4 dB at baseline to 27.3±2.3 dB at 90 days (P=0.030) only in the DHA supplementation group (between-group differences P<0.19). Also, in the DHA supplementation group, the macular integrity index decreased from 71.2±33.2 at baseline to 51.6±35.9 at 90 days (P=0.002); differences between values at 45 days (63.5±36.4) and 90 days (51.6±35.9) were also statistically significant (P=0.03) (between-group differences P<0.05). Statistically significant changes in BCVA (Figure 3A) and CSMT (Figure 3B) throughout the study period were not found in any of the comparisons and in none of the study groups. In relation to ω-3 DHA on the erythrocyte membrane, a significant increase at 90 days as compared with baseline (5.6%±0.8% vs 3.9%±0.6% total fatty acids, P<0.001) was only observed in the DHA supplementation group. In this case, between-group differences were also statistically significant (P<0.05). Plasma TAC values increased significantly from baseline as compared with 90 days only in the DHA supplementation group (between-group differences P<0.05) (Figure 4A), whereas serum levels of IL-6 decreased significantly from baseline as compared to 90 days only in the DHA supplementation group (between-group differences P<0.015) (Figure 4B). Vision-related quality of life showed a trend toward improvement in the DHA supplementation group at 45 and 90 days as compared with baseline, and a trend toward worsening in the control group (between-group differences P=0.037) (Figure 5). The nutraceutical formulation was well tolerated and no adverse events were registered. In relation to compliance with the nutraceutical supplement, all patients in the DHA supplementation group reported having taking the three capsules each day of the study. BODY.DISCUSSION: Results of the present prospective controlled study performed in routine daily practice shows that oral supplementation based on a high-dose DHA formulation had a beneficial effect on macular function in asymptomatic patients with NPDR. After 3 months of oral supplementation with DHA, significant differences in macular sensitivity and macular integrity index as compared with baseline were observed only in the supplementation group. This finding is clinically relevant and may indicate that antioxidant and anti-inflammatory properties of DHA could play a contributing role on maintenance of macular function at early stages of DR. Also, maintenance and improvement of the quality of vision rather than simply visual acuity should be important in the prevention of visual loss in patients with diabetes. The favorable results of macular function obtained in the DHA supplementation group are further enhanced by biochemical findings, including a significant decrease of plasma IL-6 levels and significant increases of plasma antioxidant protection and level of DHA in the erythrocyte membrane. In all cases, significant between-group differences were found. Although evidence of dietary supplementation with PUFAs to reduce the risk of age-related macular degeneration and the progression of the disease is based on numerous studies published in the literature,44,45 data on the use of DHA supplementation in DR is scarce,46 particularly in asymptomatic patients with incipient retinopathy. In a cross-sectional study of 51 patients with type 2 diabetes and NPDR, oxidative deregulation as compared with healthy volunteers was found, with increased levels of lipid peroxidation products, nitrites and nitrates, erythrocyte catalase activity, and glutathione peroxidase activity and decreased levels of TAC.47 Other studies have shown that lipid peroxidation increases with the increase in severity and duration of diabetes.48 In studies of experimental DR in rats, DHA and lutein were capable of normalizing all the diabetes-induced biochemical, histological, and functional modifications,49,50 which allowed their proposal as potential adjuvant therapies to help prevent vision loss in diabetic patients.51 Newer technologies, such as microperimetry, are aimed at earlier detection of subtle deficits and enhancing diagnostic accuracy. In the last 15 years, microperimetry has been successfully used in the diagnosis and follow-up of different macular disorders, including age-related macular degeneration, myopic maculopathy, macular dystrophies, and DME. Different studies have shown a correlation between macular sensitivity determined by microperimetry and visual acuity in patients with clinically significant macular edema, as well as reduced macular sensitivity in relation to increasing macular thickness.52 Microperimetry has also been useful for detailed study of the macular region for the assessment of morphological and functional outcome after intravitreal ranibizumab in patients with clinically significant DME.53 However, as far as we are aware, studies assessing macular function by microperimetry in early stages of DR have not been previously published. In this respect, our study demonstrates the usefulness of microperimetry in this disease setting. The present results should be interpreted taking into account some limitations of the study, especially the few patients included in the study groups and the fact that duration of the study was limited to 90 days only. However, different studies have shown that 90 days is a sufficient period of time for incorporation of ω-3 PUFAs in the erythrocyte membrane.54,55 Also, in previous studies using the same nutraceutical formulation for 90 days in ocular surface disorders, reduced expression of inflammatory biomarkers (IL-6, IL-10, IL-1β, and TNFα) in tear samples as well as improvements of dry eye symptoms as compared with the nonsupplemented groups were found.56–58 The study was not designed as a masked study because patients in the control group were not treated with placebo. Therefore, this situation could have influenced the results of variables that are more dependent on the subject’s subjectivity, such as the results of the NEI-VFQ-25 questionnaire. Also, the small sample size of only 12 patients in each study group may account for baseline differences in the baseline scores of the NEI-VFQ-25 questionnaire. Compliance with the nutraceutical formulation was checked at the study visits by asking the patient to bring the empty box, but control over dietary intake of participants was lacking. No measure of dietary DHA was contemplated in the study protocol as it would be very unlikely that the usual Mediterranean diet consumed in our country would have a high DHA content of 1,050 mg/d similar to that administered with the nutraceutical supplementation. However, the significant increase in DHA content of the erythrocyte membrane is a reliable measure of good compliance. It may be argued that the fixed DHA daily dose of 1,050 mg/d does not fill all depending of body weight. In a study of 64 youth (7–14 years) with a diagnosis of mood disorder, a linear relationships between body weight and body mass index percentile with ω-3 PUFA accumulation has been reported.59 However, the daily dose of DHA of 200 mg was markedly lower to that used in our patients, and DHA levels were determined in blood samples rather than in the erythrocyte membrane. On the other hand, the rationale for use of DHA triglyceride form is based on a higher bioavailability of the compound.60,61 In addition, IL-6 was chosen because this cytokine has been found elevated in the vitreous fluid of diabetic patients with DR and has been shown to be a main contributor to the pathogenesis of DR.62,63 In a randomized, controlled clinical trial of patients with diabetes with no retinopathy or mild to moderate NPDR assigned to twice daily consumption of placebo (n=28) or a multi-component formula (n=39) containing vitamins, zinc oxide, eicosapentaenoic acid, DHA, α-lipoic acid, coenzyme Q10, mixed tocotrienols/tocopherols, zeaxanthin, lutein, benfotiamine, N-acetyl cysteine, and selected botanical extracts, visual function and macular pigment optical density improved significantly in the supplemented group after 6 months of treatment.64 The authors suggest that, theoretically, multiple and overlapping mechanisms implicated in DR may have been targeted by the various components of the test formula, although synergistic or inhibitory constituent effects were not analyzed. In our study, the synergistic influence of carotenoids on microperimetry was not evaluated, which is an interesting area of research for further studies. BODY.CONCLUSION: In asymptomatic patients with NPDR, dietary supplementation with a nutraceutical formulation based on high dose of DHA (1 g) plus antioxidant vitamins, minerals, and xanthophylls for 3 months was associated with a significant improvement of macular function as compared with controls. Changes of biochemical parameters were consistent with the antioxidant and anti-inflammatory effects of DHA and lutein/zeaxanthin. Further studies are needed to confirm these promising preliminary results. Replication of the present findings in a randomized trial coul

TITLE: Efficacy and Safety of Using Air Versus Alkalinized 2% Lignocaine for Inflating Endotracheal Tube Cuff and Its Pressure Effects on Incidence of Postoperative Coughing and Sore Throat ABSTRACT.BACKGROUND AND OBJECTIVE:: We wished to compare the endotracheal tube (ETT) cuff pressure inflated with air or alkalinized lignocaine during anesthesia and evaluate clinical symptoms such as coughing and sore throat (postoperative sore throat [POST]) following tracheal extubation. ABSTRACT.MATERIALS AND METHODS:: This was a prospective randomized controlled study conducted in a tertiary care set up over a period of 1 year. We included 100 patients in age group of 18–65 years posted for elective surgeries of duration more than 90 min under general anesthesia with N2O-O2 mixture. Patients were randomized using computer-generated randomization table into air and lignocaine group. The ETT cuff was inflated with air or alkalinized lignocaine (2% lignocaine with 7.5% sodium bicarbonate, in the proportions of 19.0:1.0 ml) to the volume that prevented air leak using cuff pressure manometer. After extubation, an independent observer blinded to study group recorded the presence or absence of coughing and POST at immediately, 1 h and 24 h postoperatively. ABSTRACT.RESULTS:: Demographic data, baseline characteristics (American Society of Anesthesiologists grade, intracuff volume/cuff pressure at start of surgery), and duration of anesthesia were comparable among study groups (P > 0.05). Cuff pressure and volume achieved in the end of surgery were much higher in air group as compared to lignocaine group (P < 0.05). Incidence of coughing and POST at immediately, 1 h and 24 h postoperatively was significantly higher in air group compared to lignocaine group. Impact of duration of anesthesia on rise in cuff pressure was significantly higher in air group and its effect on cuff-induced laryngotracheal morbidity was significant in both air and lignocaine group. ABSTRACT.CONCLUSION:: This study showed the significance of use of alkalinized 2% lignocaine in prevention of rise of cuff pressure and incidence of coughing and POST. Duration of anesthesia has also a significant effect on incidence of postoperative trachea-laryngeal morbidity. BODY.I:NTRODUCTION Coughing, sore throat, and hoarseness are most common postoperative complications after emergence from general anesthesia[1] which are very distressing and unpleasant and become more upsetting than surgery itself. Postoperative sore throat (POST) being the most undesirable symptom occurs in more than 50% of surgical patients.[2] Among the various methods of airway management such as the use of facial mask, laryngeal mask airways, and endotracheal tube (ETT), ETT is most frequently associated with such postoperative complications ranging from 30% to 70%.[34] The cause of these morbidities could be either patient's bucking or coughing or friction between the tracheal mucosa or increase in ETT cuff pressure during general anesthesia.[4] This has deleterious effects as it may increase intracranial, intrathoracic or intra-abdominal pressure, bronchospasm, wound dehiscence, bleeding, and laryngeal complication such as sore throat, hoarseness, or dysphonia.[2] The ETT cuff pressure is most important factor which when elevated compromises of the blood supply of tracheal mucosa followed by serious morbidities such as ciliary loss, inflammation, ulceration, hemorrhage, tracheal stenosis, and trachea-esophageal fistula.[15] Maintenance of ideal cuff pressure during whole surgery is also challenging as many factors govern it like anesthetic gases such as N2O, agent used to fill the cuff, and material and type of cuff[1678] Most commonly used anesthetic gas N2O in conjunction with other gases frequently causes rise in intracuff pressure with the progression of surgery. N2O and oxygen readily diffuse in air-filled cavities such as ETT cuff leading to gradual rise in volume and cuff pressure of ETT. Consequently, this pressure change results in more severe form of POST.[1910] Various literature in the past has proven the relation of rise in cuff pressure and volume with the usage of N2O. Intracuff use of saline and lignocaine is recently being reviewed for the effectiveness in preventing coughing, POST, and postoperative hoarseness (PH).[1811] Lignocaine being liquid in nature not only inhibits the entry of N2O in the cuff but also permeates through semipermeable cuff membrane to tracheal mucosa and provides direct anesthetic effect. Alkalinization of lignocaine with sodium bicarbonate (NaHCO3) increases the nonionized form of lignocaine which considerably increases the diffusion of lignocaine through polyvinyl cuff walls (63 folds).[1] Thus, lesser amount of lignocaine can provide rapid and prolong action over mucosa.[12] Hence, in our study, we have studied the effect and safety of use of intracuff alkalinized lignocaine over conventional air and evaluated incidence of coughing and POST after tracheal extubation. BODY.M:ATERIALS AND METHODS This was a prospective randomized control study conducted in tertiary care teaching public hospital after obtaining ethical permission and well informed written consent from the patients. The study was conducted over a period of 1 year from June 2014 to August 2015. We enrolled patients with age group 18–65 years with American Society of Anesthesiologists (ASA) Class I and II, Mallampatti classification 1 being posted for surgeries under general anesthesia with minimum surgical duration of 90 min. We excluded patients with laryngeal disease/surgery/tracheostomized, ASA Class III and IV, difficult intubation, or failed extubation. Standard routine balanced general anesthesia was given as per the attending senior anesthesiologist's protocol and dosage according to the body weight of the patient. ETTs with high residual volume, low-pressure cuff, with an inner diameter of 7.0 mm for female and 8.5 mm for male were used in all patients. Subjects were allocated in two groups as Air group (A) and Lignocaine group (L) according to computer-generated randomization sheet with fifty patients in each group. In A group, ETT cuff was filled with air to prevent air leak during positive pressure ventilation guided with cuff manometer. While in L group, ETT cuff was filled with 2% lignocaine with 7.5% NaHCO3, in the proportions of 19.0:1.0 ml. Care was taken to ensure that starting cuff pressure as approximately 20 cm H2O adequate enough to just prevent leak around cuff during positive pressure ventilation. Volume and cuff pressure of air and lignocaine injected in the cuff was noted at start and end of surgery. Total duration of anesthesia was also noted. Immediately after extubation, an independent observer blinded from the study group recorded the presence or absence of coughing. Similarly, in postoperative care unit, occurrence of coughing and POST at 1 h and 24 h were recorded. Similarly, in postoperative care unit, occurrence of coughing and POST at 1 h and 24 h was recorded. Coughing/POST was recorded as present or absent. Sample size is calculated with the help of the study of Navarro et al.[1] for a Type I error of 0.05 and a Type II error of 0.20 with a power equal to 80% and confidence interval of 95%. Thus, a total sample size was 100 patients with fifty patients in each group. Data analysis is done with the help of SPSS Software ver 15 (SPSS Inc. Released 2006. SPSS for Windows, Chicago, USA) and Sigma plot Ver. 11 (Systat Software, Inc., San Jose, California, USA). BODY.R:ESULTS AND ANALYSIS The anthropometric parameters in term of age, sex, weight, and ASA were comparable in both groups with P > 0.05 [Tables 1–3]. The volume and cuff pressure of A group and L group introduced into the cuffs at the start of surgery for adequate seal were recorded and on analysis both were comparable (P > 0.05), [Table 4]. The mean cuff pressures obtained at the end of surgery were 49.86 cm H2O and 19.66 cm H2O in A and L group, respectively, proving statistically significant difference between the two groups [Table 4]. The duration of anesthesia in both groups was comparable with average duration in A group was 2.67 ± 0.72 h and in L group was 2.66 ± 0.70 h [Table 4]. This was well above the mean duration of anesthesia as set in inclusion criteria required for the effect of lignocaine over tracheal mucosa. Analysis of change in volume and pressure of cuff during surgery revealed significant difference in A group than L [Table 5]. Table 1Association between the two study groups on the basis of age and weight Table 2Association between the study groups on the basis of sex Table 3Association between the study groups on the basis of American Society of Anesthesiologists classes Table 4Association between the two study groups on the basis of multiple variables Table 5Association between change in cuff volume and pressure between two groups The incidence of coughing immediately after extubation, postoperatively 1 h and 24 h after surgery is depicted in Figures 1 and 2. Significant difference determined by Pearson Chi-square test and Fisher's exact test showed P < 0.05 thereby substantiating the reduced incidence of coughing and POST in L group. Through the analysis, it was found that in A group, there was a high correlation between duration of anesthesia and cuff pressure achieved in the end of surgery with P < 0.001 [Figure 3] while no such correlation was obtained in lignocaine group [Figure 4]. Figure 1Incidence of coughing in air and lignocaine group Figure 2Incidence of postoperative sore throat among air and lignocaine group Figure 3Correlation between duration of anesthesia and cuff pressure at end in air group Figure 4Correlation between duration of anesthesia and cuff pressure at end in lignocaine group Further, association between duration of anesthesia with coughing and POST in A group revealed significant association at immediately after extubation, 1 h and 24 h postoperatively [Table 6]. In L group, such association was found only during coughing immediately after extubation and POST at 1 h and 24 h. This association was statistically significant with P < 0.05 (unpaired t-test) [Table 7]. Table 6Association between duration of anesthesia and incidence of coughing and postoperative sore throat in air group Table 7Association between duration of anesthesia and coughing and postoperative sore throat in lignocaine group BODY.D:ISCUSSION The most bothersome postoperative complaint in the setting of general anesthesia after extubation of ETT is coughing and sore throat affecting more than half of the patients.[124] Various factors contribute to the emergence of these symptoms that may adversely affect the outcome if not monitored adequately. ETT cuff pressure is the indirect measure of pressure exerted by cuff over tracheal mucosa that is not monitored routinely.[1] The critical function of ETT cuff is to provide adequate seal to airway during positive pressure ventilation to prevent aspiration due to under inflation. Long duration of cuff inflation can result in mucosal ischemia and further complications such as coughing, POST, PH, tracheal ulceration, stenosis, and tracheoesophageal fistula.[110] In humans, endotracheal (ET) cuff pressures at approximately 30 cm H2O can impair tracheal mucosal perfusion, and a critical perfusion pressure is reached at 50 cm H2O which has been demonstrated by endoscopic studies.[13] N2O anesthesia which is a common conjunct with other volatile anesthetics is the main factor, which increases the intracuff pressure by easily diffusing into the cuff with the advent of surgery.[18910] Various factors have been evaluated to reduce this rise in cuff pressure such as repeated inflation-deflation technique, use of O2-N2O mixture, polyurethane cuff, and liquid cuff media.[910] Lignocaine, an amide local anesthetic in its several preparations such as topical jelly, intracuff, aerosolized, or intravenous form has evolved as an effective measure in reducing POST. We compared the traditional practice of use of air as inflation media with lignocaine for determining the rise in cuff pressure and thereby incidence of signs of tracheal morbidity such as coughing and sore throat 24 h postoperatively. The basis of our study was that lignocaine inserted intracuff act as a reservoir of local anesthetic which being a liquid not only prevents the diffusion of N2O intracuff but also permeates through semipermeable membrane of polyvinyl chloride cuff to provide soothing effect on tracheal mucosa and helps in reducing pressure induced necrosis, cough reflex.[1414] The study of Estebe et al.[15161718] reported that alkalinization of intracuff lignocaine increases the diffusion of its nonionized neutral base across hydrophobic structure of cuff membrane from 1% to 65% within 6 h. They studied both in vitro and in vivo effect of alkalinized lignocaine across tracheal mucosa and showed significant decrease in dose requirement of lignocaine in alkalinized form to as low as 20–40 mg as compared to nonalkalinized lignocaine dose of 200–500 mg.[11418] This report encouraged us to design our study using alkalinized lignocaine for more safety and efficacy in the study subjects. No form of external lubrication in the form lignocaine jelly was used over the cuff during intubation for facilitation of cuff beyond the vocal cords. Multiple studies have shown that cuff lubrication in form of jelly or topical spray is associated with unfavorable phenomena at the time of emerging from anesthesia.[1419] The lignocaine spray contains additives such as l-methanol and ethanol which are more associated in causing POST and hoarseness.[19] The intravenous form of lignocaine is more associated with sedation or deepening plane of anesthesia which is not suitable at the time of extubation.[14] Demographic data (age, sex, weight) of study subjects, baseline characteristics (ASA class, intracuff volume/cuff pressure inflated at start of surgery), and duration of anesthesia were comparable among study groups (P > 0.05) [Tables 1–4]. Our study has demonstrated that using N2O anesthesia, filling of ET cuff with air despite initial cuff pressure set well below critical pressure of 30 cm H2O rises toward the end of surgery. Average volume of air injected to achieve adequate seal was 4.54 ± 0.63 ml with average cuff pressure at the start of surgery as 20.42 ± 0.73 cm H2O [Table 4]. Toward the end of surgery, mean volume of cuff reached to 6.00 ± 0.78 ml and mean cuff pressure rose to 49.86 ± 0.72 cm H2O [Table 4]. Our data confirmed the increased cuff pressure and cuff volume after air inflation with N2O-oxygen anesthesia.[1814202122] Significance of continuous cuff pressure monitoring to prevent POST and hoarseness was emphasized by Suzuki et al.,[23] Sengupta et al.,[24] Hoffman et al.,[13] Liu et al.,[6] and Manissery et al.[10] In comparative group, mean initial volume of alkalinized lignocaine required to inflate the ETT cuff was 4.40 ± 0.60 ml and initial cuff pressure as 20.60 ± 0.78 cm H2O [Table 4]. Both of the volume and cuff pressure at start of surgery in air and lignocaine group were similar and statistically not significant (Mann–Whitney test, P > 0.05), [Table 4]. Toward the end of surgery, mean volume obtained and average cuff pressure was 4.14 ml and 19.66 ± 0.74 cm H2O, respectively. This showed a highly significant difference in the two groups (Mann-Whitney test, P < 0.001), [Table 4]. In A group, the volume and cuff pressure during surgery increased by 1.46 ± 0.48 ml and 29.44 ± 7.16 cm H2O, respectively. On the other hand, in L group, volume and cuff pressure decreased by 0.26 ml and 0.94 cm H2O, respectively. This change in volume and pressure was statistically significant between both groups (Unpaired t-test, P < 0.05), [Table 5]. This clearly showed the cuff volume and pressure did not change with time in lignocaine group as compared with air group. Lignocaine, being liquid in media, prevents hyperinflation of cuff with N2O with the course of surgery.[1] Lignocaine diffuses through cuff membrane in time and concentration-dependent fashion and influence local tracheal receptors by inducing local anesthesia and increase ETT tolerance.[11] Moreover, incidence of coughing and POST at immediately, 1 h and 24 h postoperatively was significantly higher in air group in comparison to lignocaine group [Figures 1 and 2]. These results are coherent with the studies of Navarro et al. 2007,[1] Navarro et al. 2012,[21] Jaichandran et al. 2009,[25] Shroff and Patil, 2009.[26] Wetzel et al. could not find attenuation in coughing and POST when intracuff lignocaine group used where procedures lasted for <1.5 h in patients who smoke.[27] Thus, using these lignocaine instilled cuffs for longer duration, surgeries would result in better outcome as diffusion across the cuff membrane is a function of time.[20] Although lignocaine was instilled in the cuff, it does not cause any depression of swallowing reflex and other protective reflexes. This has been confirmed by other study done by Estebe et al. which stated that alkalinized intracuff lignocaine improves cuff tolerance; however, the local anesthetic effect does not depress the swallowing reflex so that the patient can protect the airway.[15] In an extensive review by Tanaka et al. (Cochrane 2009) concluded that topical and systemic lignocaine therapy reduces the prevalence and severity of sore throat after general anesthesia with ET intubation.[4] Recent meta-analysis by Lam et al. showed the beneficial effect of alkalinized and nonalkalinized lignocaine intracuff over conventional inflation medias. The incidence of early and late POST, agitation, coughing, hoarseness, dysphonia like post intubation emergence phenomena was significantly lower in lignocaine group.[2] It was clearly evident that with the prolongation of duration of anesthesia cuff pressure also increased significantly in air group in comparison to lignocaine group [Figures 3 and 4]. Similarly, the outcome of rise in cuff pressure in such patients whose duration of surgery was prolonged showed significant increase in coughing and POST in air group [Table 6]. Furthermore, the analysis showed the increase in duration of anesthesia influenced the incidence of coughing and POST in lignocaine group [Table 7]. Mitchell et al. demonstrated linear rise in cuff pressure with time in air group. They suggested that the rise in cuff pressure causes tracheal mucosa ischemia due to lateral contact pressure exerted by cuff.[28] In our study, all the cases were extubated without any complication. The present study was not without limitations as we did not included children and elderly in our study subjects. Severity of coughing and sore throat were not graded rather only incidence of its presence was evaluated. Measurement of concentration of plasma lignocaine was not done. BODY.C:ONCLUSION In the setting of general anesthesia with the use of N2O and O2 mixture, rise in cuff pressure with the progression of surgery is better overcome when ETT cuff is inflated with lignocaine as compared to air. Alkalinized 2% lignocaine provides an improved protective effect in preventing postoperative laryngotracheal morbidity in form of coughing and POST. Duration of anesthesia is another risk factor, which has significant impact on increase in cuff pressure and consequently increased the incidence of coughing and POST in ETT cuff filled with air. BODY.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: A phase I, randomized, controlled, dose-ranging study of investigational acellular pertussis (aP) and reduced tetanus-diphtheria-acellular pertussis (TdaP) booster vaccines in adultsG. LEROUX-ROELS ET AL.HUMAN VACCINES & IMMUNOTHERAPEUTICS ABSTRACT.ABSTRACT: Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012–June 2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913). 420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin [FHA] and pertactin [PRN] at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid [TT] and diphtheria toxoid [DT]), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination. Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group). This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated. ABSTRACT.INTRODUCTION: Pertussis, also known as whooping cough, is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis (B. pertussis). All age groups are susceptible to pertussis; however, the most severe symptoms occur in infants and young children, in whom potentially fatal complications such as convulsions, bronchopneumonia and encephalopathy may occur.1–3 Household exposure is considered to play an important role in the spread of the disease.4–6 A previous study demonstrated that 35% to 55% of infant cases could be prevented if immunity to pertussis in parents was maintained or boosted. Despite the widespread vaccine availability and high vaccination coverage of primary and booster tetanus, diphtheria and acellular pertussis (aP) vaccinations, the incidence of pertussis continues to rise in many countries, with the highest morbidity and mortality rates in infants too young to be vaccinated.7–10 It was estimated in 2008 that 16 million cases of pertussis occurred worldwide, and 195,000 children died from the disease, an incidence that owes to insufficient coverage or compliance in pediatric immunization,11–12 as well as to resurgence in countries with high vaccination coverage: a high incidence of pertussis was observed in some developed countries (Australia, Portugal, United Kingdom, United States) that switched vaccination programs from vaccines that contained whole cell pertussis (wP) to aP.13 Health agencies in the United States and European Union recommend booster vaccine administration to adults in close contact with infants to reduce the risk of the disease.14–15 As a result of resurgence and increased incidence of infant mortality in 2010–2012, several countries including the United States and United Kingdom recommend maternal vaccination during pregnancy to protect newborns against pertussis.14–16 Currently available aP vaccines with pertussis toxin (PT) and other B. pertussis antigens have proven their safety and efficacy in large-scale clinical trials.17–18 However, the increased incidence of pertussis despite high aP coverage suggests that current vaccines induce immunity that may not be long-lasting against circulating strains. The limited longevity of protection against pertussis is also observed following natural infection.19 Therefore, a new generation of vaccines (primary and booster combinations) capable of inducing enhanced and long-lasting immunity is warranted. Such vaccines would also be useful for those countries currently using wP combinations, as neither vaccination nor natural immunity are able to confer life-long protection. In order to improve disease control and induce enhanced immunity, an alternative method of detoxifying the PT was developed. The genetically detoxified PT (9K/129G) is an inactivated PT mutant that has been shown to be a superior immunogen as compared to the chemically detoxified PT that is currently used in licensed vaccines.20–21 The investigational aP booster vaccine comprising the genetically detoxified PT contains 2 other pertussis antigens (pertactin [PRN] and filamentous hemagglutinin [FHA]), and can be administered alone or in combination with tetanus toxoid (TT) and diphtheria toxoid (DT). The 3 pertussis components had already been included in a DTaP vaccine licensed for pediatric immunization in the 1990's (Triacelluvax, Chiron S.p.A.). Previous studies have shown that vaccine to be clinically efficacious in infants22 and to elicit long-lasting protection up through six years of life23–24 but it was withdrawn from the market in 2002 for commercial reasons.25 The phase I studies (main randomized clinical study and its extension study) presented here were conducted to assess the safety and antibody responses (with persistence up to 3 years) of different doses of investigational aP and tetanus-diphtheria-acellular pertussis (TdaP; adsorbed, reduced antigen content) vaccines, containing the genetically detoxified PT, as compared to a licensed TdaP vaccine (Boostrix, GSK) with chemically detoxified PT in healthy adults aged 18 through 40 years. Cell-mediated immunity (CMI) responses and PT neutralizing titers were evaluated in a subset of participants. ABSTRACT.RESULTS: ABSTRACT.ENROLMENT, STUDY FLOW AND DEMOGRAPHICS: A total of 420 participants (average age: 26.8 ± 5.5 years, 60% female) were enrolled and vaccinated in the main study. Of these participants, 407 (97%) completed the study protocol up to day 365 (Fig. 1). Reasons for premature study withdrawal were lost to follow-up (n = 7), withdrawal of consent (n = 5), and serious adverse event (SAE) (n = 1; participant from aP4 group withdrew herself after experiencing an SAE [premature labor] at day 364). Of the originally enrolled 420 participants in the main study, 315 participated in the extension study (range 27 to 37 of participants across groups). All participants completed the extension study (Fig. 1). Figure 1.Flowchart main study and extension study. Footnote: FU, follow-up; N, number of participants in each group; SAE, serious adverse event. Participants enrolled in the main study were randomized into 10 equally-sized study groups of 42 participants each as outlined in Table 1. The baseline demographic characteristics of the enrolled participants in the main study and extension study are presented in Table 2. Vaccine groups were similar with respect to age, weight and height, and almost all study participants were of white heritage. Overall, a higher percentage of female than male participants was enrolled. Table 1.Study groups and vaccine formulation.  Day 1Day 30 Antigen dose  PT (μg)FHA (μg)PRN (μg)DT (Lf)TT (Lf) aP Booster aP1 1 1 2 0 0 Td-pur aP2 2 2 4 0 0 Td-pur aP4 4 4 8 0 0 Td-pur TdaP Booster T5d2aP1* 1 1 2 2 5 Placebo T5d2aP2 2 2 4 2 5 Placebo T5d2aP4* 4 4 8 2 5 Placebo T5d4aP1 1 1 2 4 5 Placebo T5d4aP2 2 2 4 4 5 Placebo T5d4aP4 4 4 8 4 5 Placebo Comparator* 8 8 2.5 2.5 5 Placebo *Selected groups for CMI analyses. Licensed comparator is Boostrix (GSK). Td-pur: Licensed tetanus and diphtheria vaccine (Novartis Vaccines and Diagnostics) aP: acellular pertussis; CMI: cell-mediated immunity; DT: diphtheria toxoid; FHA: filamentous hemagglutinin; Lf: limit of flocculation; PRN: pertactin; PT: pertussis toxin; TdaP: tetanus, diphtheria, acellular pertussis booster vaccine; TT: tetanus toxoid. Table 2.Study population demographics. Main studyaP1N = 42aP2N = 42aP4N = 42T5d2aP1N = 42T5d2aP2N = 42T5d2aP4N = 42T5d4aP1N = 42T5d4aP2N = 42T5d4aP4N = 42ComparatorN = 42Age, years 26.6 ± 5.6 26.8 ± 5.1 27.4 ± 6.5 26.8 ± 5.7 27.7 ± 5.9 25.8 ± 5.2 25.1 ± 4.2 27.5 ± 5.1 27.2 ± 5.6 27.4 ± 6.4 Female, n (%) 24 (57) 28 (67) 25 (60) 23 (55) 27 (64) 24 (57) 29 (69) 19 (45) 27 (64) 24 (57) Male, n (%) 18 (43) 14 (33) 17 (40) 19 (45) 15 (36) 18 (43) 13 (31) 23 (55) 15 (36) 18 (43) Ethnicity, n (%)                      Asian 0 0 0 0 1 (2) 0 0 0 0 0  Black 0 0 0 0 1 (2) 1 (2) 0 0 0 0  White 42 (100) 42 (100) 42 (100) 42 (100) 40 (95) 41 (98) 41 (98) 42 (100) 42 (100) 42 (100)  Other 0 0 0 0 0 0 1 (2)       Weight, kg 68.4 ± 10.5 70.1 ± 9.9 69.0 ± 11.6 68.9 ± 12.8 65.4 ± 10.0 69.1 ± 11.4 67.7 ± 11.9 68.8 ± 13.5 69.7 ± 12.6 70.3 ± 13.5 Height, cm 173.9 ± 8.5 171.9 ± 8.3 172.3 ± 8.4 172.8 ± 7.8 172.3 ± 10.3 173.2 ± 8.2 169.9 ± 8.5 175.8 ± 8.6 171.8 ± 9.3 173.1 ± 7.9 Met entry criteria, n (%) 42 (100) 42 (100) 42 (100) 42 (100) 42 (100) 42 (100) 41 (98) 41 (98) 42 (100) 42 (100) Extension Study aP1N = 27 aP2N = 36 aP4N = 32 T5d2aP1N = 27 T5d2aP2N = 33 T5d2aP4N = 30 T5d4aP1N = 37 T5d4aP2N = 30 T5d4aP4N = 30 Comparator N = 33 Age, years 30.2 ± 5.7 29.5 ± 4.9 30.3 ± 6.4 31.3 ± 6.3 30.9 ± 5.7 30.0 ± 5.6 28.2 ± 4.4 30.1 ± 4.6 31.3 ± 5.9 31.5 ± 6.7 Female, n (%) 17 (63) 25 (69) 19 (59) 11 (41) 19 (58) 16 (53) 27 (73) 13 (43) 19 (63) 21 (64) Male, n (%) 10 (37) 11 (31) 13 (41) 16 (59) 14 (42) 14 (47) 10 (27) 17 (57) 11 (37) 12 (36) Ethnicity, n (%)                      Asian 0 0 0 0 1 (3) 0 0 0 0 0  Black 0 0 0 0 1 (3) 1 (3) 0 0 0 0  White 27 (100) 36 (100) 32 (100) 27 (100) 31 (94) 29 (97) 36 (97) 30 (100) 30 (100) 33 (100)  Other 0 0 0 0 0 0 1 (3)       Weight, kg 67.2 ± 8.4 71.1 ± 10.7 69.1 ± 11.5 72.4 ± 12.6 67.7 ± 10.7 69.9 ± 12.2 68.1 ± 12.2 70.6 ± 15.1 74.0 ± 11.7 72.5 ± 15.8 Height, cm 172.6 ± 9.0 171.1 ± 8.6 171.2 ± 8.5 174.2 ± 7.9 173.2 ± 10.7 172.9 ± 8.0 169.0 ± 8.0 175.8 ± 8.9 172.7 ± 9.6 172.1 ± 8.2 Met entry criteria, n (%) 27 (100) 36 (100) 32 (100) 27 (100) 33 (100) 30 (100) 37 (100) 30 (100) 30 (100) 33 (100) Age, weight and height are expressed as mean ± standard deviation. Licensed comparator is Boostrix (GSK). aP: acellular pertussis; N: number of participants in each group; n %: number (percentage). ABSTRACT.SAFETY AND REACTOGENICITY: All 420 enrolled participants were exposed to the study vaccines and 418 participants contributed to the safety analyses; 2 participants (1 in group aP4 and 1 in group T5d4aP1) were excluded for not providing any post-baseline safety data. Overall, the safety profile of the investigational aP and TdaP formulations was comparable to the licensed TdaP booster vaccine. At least one reactogenicity sign post-vaccination was reported by similar numbers of participants in the aP groups (81% to 88%) and in the licensed comparator group (86%); in the TdaP group, at least one reactogenicity sign post-vaccination was reported by 93% to 98% participants. Similarly, solicited local adverse events (AEs) were reported by 71% to 79% of participants in the aP groups, 83% to 98% in the TdaP groups and by 79% in the comparator group. Solicited systemic AEs were reported by similar numbers of participants in all groups, i.e., 48% to 64% of participants in the aP groups, 43% to 66% in the TdaP groups and 50% in the licensed comparator group. No relevant trends towards higher incidences of solicited AEs with increasing antigen doses were observed in the invest The most commonly reported local AE was injection site pain, experienced by 71% to 98% of participants in each group (Table 3). Severe injection site pain was reported by maximum 5% of participants across groups. Erythema was reported by 0% to 14% participants, induration by 2% to 22%; and pruritus by 0% to 14% across groups, with no reports of severe reactions. The most commonly reported systemic AEs occurring across all vaccine groups were fatigue (29% to 49%) and headache (19% to 40%) (Table 3). Severe fatigue was reported by maximum 7% of participants per group, severe headache by maximum 5%. Nausea was experienced by 2% to 17%, myalgia by 10% to 29%, and arthralgia by 0% to 17% of participants across groups, with only few severe reports (up to 5% across groups). Fever ≥38.0°C was rare, ranging from 0% to 7% across groups. There were no participants with fever ≥40C. Therapeutic use of analgesics and antipyretics was reported in 5% to 20% of participants across vaccine groups (Table 3). Table 3.Number (percentage) of participants experiencing solicited local* and systemic adverse events and other indicators of reactogenicity within 7 days of vaccination.  aP1N = 42aP2N = 42aP4N = 41T5d2aP1N = 42T5d2aP2N = 42T5d2aP4N = 42T5d4aP1N = 41T5d4aP2N = 42T5d4aP4N = 42ComparatorN = 42Erythema* 0 1 (2) 1 (2) 1 (2) 2 (5) 6 (14) 3 (7) 2 (5) 4 (10) 3 (7) Induration* 3 (7) 3 (7) 1 (2) 6 (14) 1 (2) 7 (17) 9 (22) 4 (10) 4 (10) 6 (14) Pain* 30 (71) 32 (76) 31 (76) 35 (83) 38 (90) 36 (86) 40 (98) 35 (83) 39 (93) 31 (74)  Severe 0 1 (2) 0 0 1 (2) 1 (2) 0 2 (5) 1 (2) 0 Pruritus* 0 4 (10) 1 (2) 3 (7) 2 (5) 5 (12) 3 (7) 3 (7) 6 (14) 5 (12) Nausea 3 (7) 6 (14) 2 (5) 1 (2) 2 (5) 5 (12) 4 (10) 6 (14) 7 (17) 1 (2)  Severe 1 (2) 0 1 (2) 0 0 0 1 (2) 1 (2) 0 0 Myalgia 10 (24) 8 (19) 4 (10) 8 (19) 10 (24) 11 (26) 12 (29) 8 (19) 9 (21) 11 (26)  Severe 1 (2) 1 (2) 0 1 (2) 0 0 0 0 2 (5) 0 Arthralgia 2 (5) 0 3 (7) 4 (10) 1 (2) 3 (7) 4 (10) 4 (10) 7 (17) 1 (2)  Severe 1 (2) 0 0 0 0 0 0 0 0 0 Headache 17 (40) 13 (31) 12 (29) 8 (19) 13 (31) 13 (31) 16 (39) 14 (33) 17 (40) 13 (31)  Severe 1 (2) 0 0 2 (5) 1 (2) 2 (5) 2 (5) 2 (5) 2 (5) 1 (2) Fatigue 19 (45) 14 (33) 13 (32) 13 (31) 15 (36) 18 (43) 20 (49) 15 (36) 16 (38) 12 (29)  Severe 3 (7) 2 (5) 0 2 (5) 0 1 (2) 2 (5) 0 2 (5) 0 Body temperature                      ≥ 38C 1 (2) 0 1 (2) 2 (5) 0 0 3 (7) 2 (5) 0 2 (5)  ≥ 40C 0 0 0 0 0 0 0 0 0 0 Use of analgesics and antipyretics                      Prophylactic 0 1 (2) 0 0 1 (2) 1 (2) 2 (5) 1 (2) 2 (5) 1 (2)  Therapeutic 6 (14) 7 (17) 2 (5) 6 (14) 8 (19) 3 (7) 8 (20) 7 (17) 7 (17) 8 (19) Erythema and induration with diameter > 100 mm were classified as severe. No cases of severe erythema, induration, or pruritus were reported. Values represent number of reported cases (percentage). Licensed comparator is Boostrix (GSK). aP: acellular pertussis; N: number of participants in each group. The overall mean numeric rating scale (NRS) scores26 in the groups were very low (range 0.68 to 1.51 at 60 minutes post-vaccination, 0.84 to 1.77 at 6 hours post-vaccination, on a 0 to 10 scale), with slightly higher mean values in the TdaP groups (0.87–1.51 at 1 hour and 1.10–1.77 at 6 hours post-vaccination in the TdaP groups, compared to 0.80–0.99 and 0.84–0.99 in the aP groups; Table S1) For the responses to the likelihood of vaccination, the majority of participants (67% to 90% at day 1, 55% to 88% at day 8) across vaccine groups ‘strongly agreed’ that they were likely to undergo repeated vaccination after being administered the study vaccination (Table S1). Unsolicited AEs occurred in 29% to 67% (17% to 31% considered at least possibly related) of participants in the aP groups, 31% to 50% (10% to 26% considered at least possibly related) in the TdaP groups and 43% (24% considered at least possibly related) in the licensed comparator group. The most frequently reported unsolicited AEs (by preferred term) were upper respiratory tract infection (up to 12%) and headache (up to 10%) for aP groups, upper respiratory tract infection, oropharyngeal pain, and headache (all up to 10%) for TdaP groups, and headache and injection site induration (both up to 5%) for the comparator group. Across groups, the most common at least possibly related AEs were injection site movement impairment, injection site pain, and fatigue (all up to 7%). A total of 15 SAEs were reported by 14 participants, none of which were considered related to the study vaccination (Table S2) No deaths occurred in this study. ABSTRACT.ANTIGEN-SPECIFIC ANTIBODY RESPONSES: Analyses of antibody responses were performed on the per-protocol (PP) data sets, which included 383 (91%) of the total of 420 participants at days 30, 372 (89%) at day 180 and 383 (91%) at day 365, and 300/315 (95%) of participants approximately 3 years after vaccine administration (on day 1 of the extension study) (Table S3). ABSTRACT.ANTIBODY RESPONSE AGAINST PERTUSSIS ANTIGENS (PT, PRN, FHA): Geometric mean concentrations (GMCs) against pertussis antigens PT, FHA and PRN from baseline up to 3 years post-vaccination are presented in Fig. 2. Baseline values were comparable across groups. Overall, antibody responses against pertussis antigens peaked at the day 30 timepoint after the booster dose and then waned over the following 3 years, but were still above baseline levels. Figure 2.Geometric mean concentrations and 95% confidence intervals against pertussis antigens PT, FHA and PRN in investigational aP and TdaP booster groups and licensed comparator group from day 1 through year 3 post-vaccination. Footnote: aP, acellular pertussis; CI, confidence interval; FHA, filamentous hemagglutinin; GMC, geometric mean concentration; IU/mL, International Units per milliliter; PRN, pertactin; PT, pertussis toxin; TdaP, tetanus diphtheria acellular pertussis. For PT, the highest antibody responses were observed in the investigational formulations containing 4 µg genetically detoxified PT (groups aP4, T5d2aP4, and T5d4aP4) at all timepoints, including day 365 and 3 years post-vaccination. Even formulations with 2 µg of genetically detoxified PT induced antibody responses similar to or higher than the licensed comparator containing 8 µg of chemically inactivated PT (Fig. 2). A statistically significant increase in antibody responses at day 30 post-vaccination (evidenced by the ratio of GMCs and their 95% confidence intervals [CIs]) against PT was observed in aP2, aP4, T5d2aP4 and T5d4aP4 groups, as compared to the licensed vaccine. Day 30 GMCs across groups ranged from 62 to 182, being lowest in the T5d2aP1 group and highest in the aP4 group. Percentages of participants with ≥2-fold increase in levels of antibodies against PT were 94% to 100% (aP formulations), 89% to 100% (TdaP formulations) and 100% (licensed comparator); ≥4-fold increase was observed in 92% to 100% (aP formulations), 76% to 100% (TdaP formulations) and 98% (licensed comparator) participants. For all vaccine groups, antibody levels against PT at 3 years post-vaccination were higher than at baseline (7.21 to 11-fold higher GMCs in the aP groups, 3.4 to 6.67-fold higher GMCs in the TdaP groups; 4.28-fold higher in the comparator group). Participants who received vaccine formulations containing 4 µg PT demonstrated statistically significant higher persistence of anti-PT antibody responses at 3 years post-vaccination than participants who received the licensed comparator containing the double dose of PT. For aP and TdaP formulations containing 1or 2 µg PT, persistence of antibody against PT was comparable or statistically higher (T5d4aP1 and T5d4aP2 groups, respectively) than for the licensed TdaP vaccine (Table 4). Table 4.Ratio of GMCs with 95% confidence intervals of aP and TdaP booster doses relative to licensed comparator against pertussis PT, FHA and PRN antigens approximately 3 years post-vaccination (day 1 extension study).  aP1:ComparatoraP2:ComparatoraP4:ComparatorT5d2aP1:ComparatorT5d2aP2:ComparatorT5d2aP4:ComparatorT5d4aP1:ComparatorT5d4aP2:ComparatorT5d4aP4:ComparatorPT 1.54(0.85-2.81) 1.70(0.97-2.98) 2.65(1.50-4.70) 0.92(0.56-1.52) 1.20(0.74-1.94) 1.86(1.13-3.05) 1.71(1.07-2.75) 1.69(1.03-2.77) 2.37(1.45-3.87) FHA 0.68(0.44-1.04) 0.86(0.57-1.28) 0.95(0.63-1.43) 0.64(0.42-0.99) 0.52(0.34-0.78) 0.81(0.53-1.23) 0.80(0.54-1.21) 0.76(0.50-1.17) 0.92(0.60-1.39) PRN 1.64(0.95-2.82) 2.19(1.31-3.65) 3.14(1.87-5.28) 0.74(0.40-1.36) 1.97(1.11-3.51) 2.96(1.63-5.36) 1.96(1.11-3.46) 2.11(1.17-3.82) 2.32(1.29-4.18) When the 95% confidence interval does not contain 1, there is an indication of a statistically significant difference. Licensed comparator is Boostrix (GSK). aP: acellular pertussis; GMC: geometric mean concentration; FHA: filamentous hemagglutinin; PRN: pertactin; PT: pertussis toxin; TdaP: tetanus diphtheria acellular pertussis. The investigational aP and TdaP groups demonstrated lower antibody responses against the FHA antigen at all timepoints than the licensed TdaP vaccine (Fig. 2). The aP and TdaP formulations contained 1/8 to 1/2 of the dose of FHA as compared to the licensed vaccine. In all aP and TdaP study groups, antibody responses against FHA at day 30 post-vaccination were statistically significant lower than those elicited by the licensed vaccine. GMCs at day 30 following booster vaccinations ranged from 83 (T5d2aP1) to 241 (licensed TdaP vaccine) international units (IU)/mL. Percentages of participants with ≥2-fold increase in the levels of antibodies against FHA were 85% to 94% for the aP groups, 76% to 95% for the TdaP groups and 95% for the licensed vaccine; ≥4-fold increase was observed in 59% to 83% (aP groups), 46% to 61% (TdaP groups) and 83% (licensed vaccine) participants. Antibody persistence 3 years post-vaccination against FHA was higher than at baseline for all vaccine groups (2.25 to 3.25-fold rise across the aP groups, 1.67 to 2.19-fold rise across the TdaP groups; 3.36-fold rise comparator). The level of persisting anti-FHA antibodies 3 years post-vaccination was lower in the aP and TdaP groups than in the licensed comparator group, with significant differences observed for T5d2aP1 and T5d2aP2 groups (Table 4). For PRN, antibody responses were 0.32–2.71-fold higher in aP4, 2.10–2.96-fold higher in T5d2aP4, and 1.98–2.60-fold higher in the T5d4aP4 study groups (PRN content 8 μg) at all timepoints, as compared with the licensed comparator (PRN content 2.5 μg) (Fig. 2). At 30 days after administration of the booster vaccine, significant increases in antibody responses against PRN were noted for aP2, aP4, T5d2aP2, T5d2aP4, T5d4aP1 and T5d4aP4 groups, as compared to the licensed vaccine. Post-vaccination day 30 GMCs ranged from 445 to 1384, with the lowest observed response in the T5d2aP1 group, and the highest observed response in the aP4 group. Percentages of participants with ≥2-fold increase in PRN antigen were 100% (all aP groups), 92% to 100% (TdaP groups) and 100% (licensed comparator); percentages of participants with ≥4-fold increase were 92% to 100% (aP groups), 70% to 98% (TdaP groups) and 90% (licensed comparator). At 3 years following vaccination, anti-PRN GMCs for all groups were higher than at baseline as indicated by an 11 to 20-fold (aP groups), 7.65 to 13-fold (TdaP group) and 6.61-fold (comparator) rise in year 3 GMCs relative to day 1. A higher persistence of antibody responses at 3 years post-vaccination was observed for aP and TdaP groups as compared to the licensed vaccine, with significant group differences for all investigational formulations except aP1 and T5d2aP1 (Table 4). ABSTRACT.ANTIBODY RESPONSE AGAINST TETANUS AND DIPHTHERIA (TT AND DT): On day 30 post-vaccination with TdaP or licensed comparator, participants had antibody concentrations above the seroprotection level (i.e., ≥0.1 IU/mL) against TT (97% to 100%) and DT antigens (95% to 100%). The 2 limit of flocculation (Lf) diphtheria antigen dose was sufficient to achieve seroprotection levels against diphtheria in 95% to 98% of participants. Percentages of participants with antibodies above cut-off values ≥1.0 IU/mL were 97% to 100% (investigational TdaP) and 98% (licensed comparator) against the TT antigen and 68% to 89% (investigational TdaP, highest in the groups with the 4Lf diphtheria antigen dose) and 78% (licensed comparator) against the DT antigen. GMCs against TT and DT from baseline up to 1 year post-vaccination are presented in Fig. 3. Baseline GMCs were similar in the TdaP and comparator groups. Values had increased on day 30 in all groups, with comparable GMCs for TdaP and comparator groups against the TT antigen. GMCs against the DT antigen were highest with the TdaP formulations with the 4Lf DT antigen dose. Antibody levels waned in all groups at day 180 and day 365, but remained above baseline levels across all groups. Figure 3.Geometric mean concentrations and 95% confidence intervals against tetanus and diphtheria antigens in investigational TdaP booster groups and licensed comparator group from day 1 up through day 365 post-vaccination. Footnote: CI, confidence interval; DT, diphtheria toxoid; GMC, geometric mean concentration; IU/mL, International Units per milliliter; n, maximum number of participants with available results, TT, tetanus toxoid; TdaP, tetanus diphtheria acellular pertussis. ABSTRACT.PT NEUTRALIZATION: PT neutralization analysis was performed post-hoc in the same subsets of participants from the same three groups selected for CMI analyses. PT neutralization analysis was performed to assess the functionality of anti-PT antibodies induced by the formulations with 1 and 4 μg PT in comparison to the licensed comparator containing 8 μg PT. Geometric mean titers at all timepoints are shown in Fig. 4. Analysis of neutralizing titers revealed that formulation with 4 μg resulted in a markedly higher anti-PT antibody functionality at all timepoints. High titers were observed already at day 8, with a following peak at day 30; titers then showed a slight decline at later timepoints high functional activity still persisted over 3 years. The 1 μg formulation and the licensed comparator lead to a similar path of neutralization titers: an increase of GMT was observed starting from day 8 with a peak at day 30; titers then waned over the following 3 years but still remained higher than the baseline values. Figure 4.Geometric mean anti-pertussis neutralizing titers and 95% confidence intervals from day 1 through year 3 post-vaccination. Footnote: PT, pertussis toxin. ABSTRACT.ANTIGEN-SPECIFIC T- AND B-CELL RESPONSES (CMI): Antigen-specific T- and B-cell responses were assessed in a subset of 20 participants from each of the T5d2aP1, T5d2aP4, and licensed comparator groups. Irrespective of timepoint and class of antibody (IgG or IgM), over 65% of participants across the 3 vaccine groups presented quantifiable frequencies of memory B cells (MBC) for each vaccine antigen. Higher frequencies of IgG-positive MBC were observed on day 30 relative to day 1, whereas no increased frequencies were observed for IgM-positive MBC. MBC frequencies decreased from day 30 to day 365, but remained generally still higher than those observed on day 1. A higher frequency of IgG-positive PT-specific MBC was observed in the high aP dose TdaP group (T5d2aP4) at all timepoints as compared with the lower dose groups and licensed comparator. Frequency of IgG-positive FHA-specific MBC at day 30 post-vaccination was higher in the licensed TdaP vaccine, with the lowest observed responses in the investigational TdaP formulation containing the lower aP dose. There was no significant difference in PRN responses between the different groups (Fig. 5). Figure 5.Median percentages of Memory B Cells (ELISpot) against pertussis antigens PT, FHA and PRN in high and low dose aP dose TdaP groups and licensed comparator from day 1 through day 365 post-vaccination. Footnote: aP, acellular pertussis; ELISpot, enzyme-linked immunospot assay; FHA, filamentous hemagglutinin; IgG, immunoglobulin G; IgM, immunoglobulin M; n, number of participants with available results; PRN, pertactin; PT, pertussis toxin; TdaP, tetanus diphtheria acellular pertussis. On day 8 post-vaccination, at least 85% of participants presented quantifiable frequencies (values greater than 0) of plasmablasts (PB) secreting IgG antibodies against each vaccine-related antigen. Although there were no major differences across the 3 vaccine groups in percentage of participants with measurable frequencies of IgG-secreting PB, there was a significantly higher frequency of PT-specific PB in the high aP dose Tdap group than in the low-dose group (p < 0.0015). Comparable frequencies of PB were observed in the high aP dose Tdap group and licensed comparator group against all vaccine antigens except FHA antigen; the IgG-secreting PB against FHA antigen were higher in the comparator group than in the T5d2aP1 group (p < 0.0004) and T5d2aP4 group (p < 0.0004). A low percentage (0% to 5%) of participants had IgM-secreting PB against DT and PT, while up to 35% of participants produced antibodies against FHA and PRN (data not shown). All participants (100%) had quantifiable frequencies of antigen-specific CD4+ T cells for all antigens at all analyzed timepoints (day 1, day 8 and day 30). The frequencies of CD4+ T cells against vaccine antigens were consistently higher at days 8 and 30 than on day 1. However, the T cell-specific responses against PT antigen were very weak (Fig. 6). Analyses of the functional profile of antigen-specific CD4+ T cells demonstrated that on day 1, the majority of the participants had detectable antigen-specific CD4+ T cells expressing at least one of the following cytokines: interferon gamma (IFN-γ), interleukin-2 (IL-2), IL-17, IL-21, and tumor necrosis factor alpha (TNF-α), excluding IL-13. On day 8 and day 30, the frequency of cytokine-positive CD4+ T cells increased in all vaccine groups and against all tested antigens except PT. In general, fewer or no IL-13-producing T cells were observed at all timepoints analyzed, demonstrating that the response induced by vaccination was principally Th-1-like (data not shown). Figure 6.Median percentages of antigen-specific CD4+ T cells in high and low aP dose groups and licensed comparator at baseline and days 8 and 30 post-vaccination. Footnote: CTK, cytokine; DT, diphtheria toxoid; FHA, filamentous hemagglutinin; n, maximum number of participants with available results; PRN, pertactin; PT, pertussis toxin; TdaP, tetanus, diphtheria, acellular pertussis; TT, tetanus toxoid. ABSTRACT.DISCUSSION: The present study evaluated the safety and antibody responses of different doses of investigational aP and TdaP vaccines, containing a genetically detoxified PT, as compared to a licensed TdaP vaccine containing a chemically detoxified PT, in a population of healthy adults. The results demonstrate that all investigational study formulations were well tolerated, with reactogenicity and safety profiles similar to those observed for the licensed comparator. Adverse reactions were transient, mainly mild to moderate in severity, and there was no evidence of increasing rates of events with increasing dose. The overall mean NRS scores were very low, and comparable across groups. On the NRS scale of 0–10 that was used, a value of 0 is no pain while 1–3 is mild pain.26 In chronic pain studies, a reduction of approximately two points or a reduction of approximately 30% in the pain intensity NRS has been reported to represent a clinically important difference.27 The most frequently reported local reaction to either vaccine formulation was pain at the injection site; the most commonly reported systemic reactions were fatigue and headache. Rates of fever ≥38°C were rare, with no cases of severe fever ≥40C. There was no vaccine-related SAE throughout the study period and none of the participants withdrew prematurely due to a non-serious AE; 1 participant from the aP4 group withdrew herself due to an SAE (premature labor on day 364). These findings are in line with the known safety profile of the licensed TdaP vaccines.28–29 All vaccine formulations induced an antibody response against the tested antigens. Antibody responses against B. pertussis peaked at day 30 after the booster vaccination and then waned in the following 3 years post-vaccination, but remained above baseline levels. The genetically detoxified PT dose of 4 μg/mL induced a statistically significantly higher antibody response and persistence of antibodies to the PT antigen as compared to the licensed vaccine containing the double PT dose; even 1/4th of the dose of genetically detoxified PT induced antibody responses similar to or higher than those observed for the licensed comparator. Of importance, the higher performance of genetically detoxified PT was markedly evident at functional level, giving a stronger neutralizing activity both at the level of early response and at antibody persistence level. While chemical detoxification is effective, it also greatly alters the immunological properties of the toxin through its impact on the structure of the toxin; in contrast, genetically detoxified PT maintains all functional and immunological properties, making it a superior antigen compared to chemically detoxified PT.20 The improved PT-specific antibody responses, despite lower antigen doses, are consistent with previous efficacy trials in infants showing that the genetically inactivated PT induced a stronger antibody response than the chemically detoxified toxin.22–24 These studies also demonstrated long-lasting protection through the first six years of life.23–24 Superiority of the genetically detoxified PT antigen was further supported by the significantly enhanced numbers of PT-specific IgG MBC cells, when compared to the licensed comparator. Antibody responses to FHA and PRN antigens reflected the quantity of vaccine per dose. For PRN, antibody responses and persistence of antibodies were especially higher with the investigational formulations containing 8 μg PRN dose (aP4, T5d2aP4, and T5d4aP4 groups) as compared with the licensed comparator containing 2.5 μg PRN dose. FHA doses in all investigational formulations were lower than in the licensed vaccine and were not sufficient to induce an immune response higher than or similar to that obtained with the licensed comparator, indicating the need to increase the FHA content. No established serological correlates of protection for pertussis have been identified, hampering estimation of the protective potential after pertussis booster vaccination.30 However, for the aP part of the vaccine, results can be compared with the efficacy trial of the licensed TdaP comparator in adults and adolescents, demonstrating that on the basis of primary pertussis case definition, vaccine protection was 92%.31 Also, previous studies reported persistence of antibodies above pre-booster values against all vaccine antigens up to 3 years after vaccination with the licensed TdaP comparator in adults and 10 years after vaccination in young adults,32–33 and pertussis-specific antibody and CMI levels above the pre-booster levels measured 5 years earlier.34 Analyses of B cell responses were in line with the findings of antibody responses. Increased frequencies of antigen-specific CD4+ T cells against vaccine antigens were detected on days 8 and 30 relative to baseline. The fast increase of the frequency of these cells supports the presence of a sizeable pool of memory antigen-specific CD4+ T cells which could be quickly expanded following vaccination. The results reported in this study do not allow discriminating whether the genetically detoxified and the chemically detoxified PT induced qualitatively different CD4+ T cell populations. This may be due to the low number of participants tested in each group and to the intrinsic variability of the results. Another explanation, although not mutually exclusive with the first, could be the fact that all participants were primed during their pediatric age with a wP vaccine, which is known to drive the immune response towards a Th1-type functional phenotype, as compared to aP vaccines,35 and probably to provide an “imprinting” of the response that would have not been changed upon boosting with the vaccines used in the study. IgG-positive MBC were highest on day 30 after the booster vaccination and then waned up to day 365, but remained above pre-booster levels. Frequencies of PB, MBC and CD4+ T cells in the high aP dose group were similar to or higher than those observed for the licensed comparator, which strengthens the arguments that the most appropriate dosage for aP is 4 μg for PT, 4 μg for FHA, and 8 μg for PRN. The high frequency of MBC and PB committed to produce antigen-specific IgG more than IgM speaks in favor of a persisting immunological memory originally primed by vaccination at the pediatric age, despite the lack of detectable antibodies before vaccination. The strong response induced with the genetically detoxified PT also suggests that the genetically detoxified toxin was able to boost a response after priming with cellular wP vaccine. The low CD4+ T cell response observed after in vitro stimulation with the PT, despite the strong antibody and B-cell response, may be due to the treatment PT underwent in order to avoid its intrinsic ability to stimulate T cells non-specifically. Study limitations include the absence of predefined hypotheses to be tested formally, despite the relatively large number of participants included for a phase I study. Moreover, no adjustment for multiple testing was performed, so some spurious statistically significant differences between groups may have been observed by chance. For the analysis of antigen-specific cellular responses, the small number of participants analyzed (20 for each group) and the frequently observed non-detectable values for cytokine expression suggested performing just a descriptive analysis, and made it difficult to draw generalizable conclusions. Additionally, the vaccination history of the participants has not been registered, but DTwP vaccination was introduced in Belgium in 1961–1962, and considering the age of participants at study enrolment, most — if not all — participants should have received in infancy the recommended scheme of three doses of DTwP at the ages of 3, 4 and 5 months. Acellular pertussis vaccination was introduced in Belgium in 2001, and since participants in our study were 18–40 years old, we could infer that none of the participants have received acellular pertussis vaccine in infancy. Finally, no comparisons were performed between responses from a primary schedule with wP-containing vaccine versus aP-containing vaccine, which would have allowed for further conclusions in terms of global value. A lay language graphical summary contextualizing the results and potential clinical research relevance and impact is displayed in the Focus on Patient Section (Fig. 7). Figure 7.Focus on Patient Section. Altogether, this study demonstrates that all study vaccines were well tolerated and confirmed the potential benefit of the genetically detoxified PT antigen. Next steps in development may include further dose finding, assessment of vaccine safety and immunogenicity in vulnerable populations such as elderly and pregnant women, and evaluation of the potential for co-administrations with other vaccines. Also, potential inclusion of genetically detoxified PT in primary childhood vaccines — similar to Triacelluvax (DTaP) that demonstrated a long lasting clinical protection22–24 or in more complex combination vaccines — could be considered. Boostability of the genetically detoxified PT formulation itself also remains to be explored. Further studies on adult booster vaccination will be required to assess impact of this vaccine on adult disease, on the transmission dynamics of pertussis in the whole population, and consequently on the protection it may provide for infants who are too young to be immunized. ABSTRACT.PATIENTS AND METHODS: ABSTRACT.STUDY DESIGN AND OBJECTIVES: The current phase I randomized, controlled, observer-blind, dose-ranging study and extension study was conducted at 1 site in Belgium (Centre for Vaccinology, Ghent University Hospital) between March 2012 and June 2015 (Clinicaltrials.gov identifiers: main study: NCT01529645; extension study: NCT02382913). The study was undertaken according to Good Clinical Practice and the Declaration of Helsinki. The ethic review committee of the participating center approved the study protocol, and written informed consent was obtained from every participant prior to enrolment. The safety objectives included assessment of the safety profiles of 3 aP booster vaccines with different antigen doses and 6 different Tdap booster vaccines to the licensed TdaP vaccine in terms of solicited local and systemic AEs and patient reported outcomes for the period of 7 days after vaccination; and unsolicited AEs for the period of 30 days after vaccination. The immunogenicity objective was to select 1or 2 study vaccines from each of the aP and TdaP investigational formulations by comparison of antibody responses with the licensed TdaP vaccine and to each other at 30 days after vaccination, provided that safety profiles were comparable. The exploratory objectives included persistence of antibody responses against each antigenic component at days 180 and 365 post-vaccination and persistence of anti-PT, anti-FHA and anti-PRN antibody levels approximately 3 years after vaccine administration (day 1 of the extension study); immune persistence as measured by CMI responses in subsets of participants from selected groups (Table 1). at day 1, day 30, and day 365 after vaccination; comparison of early onset of immune response as measured by CMI and antibody response in subsets of participants from selected groups (Table 1) to the licensed TdaP vaccine and to each other at day 8; evaluation of the correlation between NRS and the solicited local reaction of injection site pain in participants receiving study vaccination. PT neutralization activity was evaluated by a retrospective laboratory research at days 1, 8, 30 and 365 and approximately 3 years after vaccine administration (day 1 of the extension study) in the same subsets of participants selected for antigen-specific T and B cell analysis. ABSTRACT.STUDY PARTICIPANTS: A total of 420 healthy adults were randomized to 1 of 10 study groups: 3 investigational aP (groups: aP1, aP2, and aP4), 6 investigational TdaP (groups: T5d2aP1, T5d2aP2, T5d2aP4, T5d4aP1, T5d4aP2, T5d4aP4) and 1 licensed TdaP comparator (Table 1). Randomization was performed according to a validated web-based system (Biostatistics and Clinical Data Management department, Novartis). An equal number of participants (42) were included in each group. Subsets of 20 participants in each of the pre-selected groups — T5d2aP1 (low aP dose TdaP), T5d2aP4 (high aP dose TdaP) and licensed TdaP comparator groups — were included in the CMI analyses. Eligible study participants for the main study were healthy adults of either sex between 18 and 40 years of age at the time of enrolment. Participants were excluded if they had received any vaccines against tetanus, diphtheria or pertussis (aP or wP), if they had been diagnosed with pertussis disease or if they had a household exposure with pertussis within the past 8 years. Other exclusion criteria were contraindications to the licensed TdaP comparator or Td-pur (Novartis Vaccines and Diagnostics) as specified within the summary of product characteristics; a significant infection or oral body temperature ≥38°C within 3 days of the intended date of vaccination; known reactions to vaccine components; any progressive or severe neurologic disease, seizure disorder or Guillain-Barré syndrome; behavioral or cognitive impairment that might interfere with the person's ability to participate in the study; any medical history or (serious) illness likely to interfere with the results; known or suspected immune disease or impairment including the administration of steroids; abnormalities of splenic or thymic function; known bleeding diathesis or any condition associated with a prolonged bleeding time; a body mass index greater than 35kg/m2; previous receipt of any other vaccine within 14 days (inactivated vaccines) or 28 days (live vaccines) or intent to receive any other vaccines within 28 days from the study vaccines; participation or intent to participate in any clinical trial 30 days prior to study start or during the time of enrolment; substance or alcohol abuse within the past 2 years; family members of study staff and pregnant or breast-feeding women. Women of childbearing potential had to be committed to using birth control measures for the duration of the study, and they had to have used birth control measures for at least 2 months before study participation. Eligible study participants for the extension study included adults who had been previously enrolled and had completed the main study and who had received the appropriate booster vaccine according to the randomization group. ABSTRACT.VACCINATION: Study vaccines were prepared as a 0.5mL white suspension in a prefilled, glass syringe for a single intramuscular (IM) administration. The investigational vaccine formulations as listed in Table 1 included the following combinations of pertussis antigens (1, 2 or 4 μg PT; 1, 2 or 4 μg FHA and 2, 4 or 8 μg PRN), DT (0, 2 or 4 Lf) and TT (0 or 5 Lf). The licensed comparator TdaP vaccine (Boostrix) included the following active ingredients per dose: pertussis antigens (8 μg PT; 8 μg FHA; 2.5 μg PRN), DT (2.5 Lf) and TT (5 Lf). Boostrix is indicated for active booster immunization against TdaP as a single dose in individuals 10 years and older. All study participants received a single 0.5mL IM vaccine dose on day 1 in the deltoid region of the upper non-dominant arm. Designated unblinded study personnel, who otherwise did not participate in the evaluation of the participants during the trial, administered the vaccine formulations. To ensure Td booster vaccination in all groups, participants who received aP alone on day 1 received the licensed vaccine Td-pur on day 30, all other participants in the remaining groups who received TdaP on day 1 received placebo (saline) on day 30. ABSTRACT.SAFETY ANALYSES: Study participants were provided with diary cards and the frequency and severity of a predefined set of solicited local and systemic AEs and other reactogenicity indicators were recorded on a daily basis from day 1 to day 7 following vaccination. After vaccination, all participants were observed for at least 30 minutes at the study site to monitor for immediate AEs. Solicited local AEs included erythema, induration, pain and pruritus. Solicited systemic AEs included nausea, myalgia, arthralgia, headache and fatigue. Other indicators of reactogenicity were oral body temperature (≥38C) and the use of analgesics/antipyretics. Additional outcomes included the NRS to measure the participant's perception of pain at the injection site ranging from 0 (no pain) to 10 (worst imaginable pain) and the Likert scale to evaluate the participant's perception on ‘the likelihood to undergo repeat vaccination after receipt of study vaccination’ (1 strongly disagree, 5 strongly agree). All AEs were collected from day 1 through day 30. SAEs and AEs leading to study withdrawal were recorded from day 1 through day 365. The severity of unsolicited AEs was categorized as mild, moderate, or severe, if they resulted in no limitation, some limitation, or inability to perform normal daily activities, respectively. Assessments of the causal relationship of unsolicited AEs to the vaccination were classified by the investigator as not related, possibly related, or probably related. ABSTRACT.IMMUNOGENICITY ANALYSES: Blood samples were obtained for immunogenicity analyses at baseline (day 1, pre-vaccination), on days 8, 30, 180 and 365 post-vaccination and approximately 3 years after vaccine administration (i.e., on day 1 of the extension study). Sera were tested using validated methods at PPD, Inc., Vaccines & Biologics (Wayne, PA United States). Peripheral blood mononuclear cells (PBMC) were analyzed at the Novartis Vaccine and Diagnostics, Translational Medicine Laboratory, Siena, Italy. Antibody responses to PT, FHA and PRN were assessed by standard enzyme-linked immunosorbent assay (ELISA) and expressed as GMC, and percentages of participants with at least 2-fold and 4-fold antibody concentration increase from pre- to post-vaccination. Anti-diphtheria and anti-tetanus antibodies were determined using ELISA with protective levels set at ≥0.1 IU/mL.36–37 Results were expressed as percentages of participants with antibody levels ≥0.1 IU/mL, 1.0 IU/mL and GMCs. For tetanus, seroprotection was defined as antibody levels >0.1 IU/mL, while antibody levels >1.0 IU/mL were considered indicative of long term protection.38 To evaluate the persistence of pertussis antibody levels, geometric mean ratios (GMRs) of the post-vaccination to pre-vaccination concentrations were calculated as PT, FHA and PRN concentrations on day 1 of the extension study approximately 3 years after vaccination, relative to days 1, 8, 30, 180 and 365. Frequency and functional profile of CD4+ T cells specific for vaccine antigens were assessed by polychromatic flow cytometry as described in the literature.39 Antigen-specific CD3+ CD4+ T lymphocytes were analyzed by measuring the frequency of T cells that produced the following cytokines in response to the in vitro stimulation: IL-2, IL-13, IL-17, IL-21, IFN-γ and TNF-α. A subset of participants of the groups T5d2aP1 (low AP dose TdaP), T5d2aP4 (high AP dose TdaP) and licensed comparator were analyzed (20 participants/group) at days 1, 8 and 30. In addition, the B lymphocyte response was evaluated by assessing the frequencies of antigen-specific MBC at days 1, 30 and 365 and PB at day 8 through enzyme-linked immunospot assay (ELISpot).40 PT neutralizing titers were measured as described in the literature.41 Two-fold serially diluted sera were pre-incubated with active PT and added to Chinese hamster ovary-K1 cells, followed by evaluation of morphological alterations (clustered phenotype) by light microscopy. Endpoint titers are the reciprocal of the highest dilution able to inhibit cell clustering. ABSTRACT.STATISTICAL ANALYSES: Statistical analyses were performed using Statistical Analyses System (SAS) software version 9.1 (SAS Institute, Cary, NC, United States). A minimal sample of 40 evaluable participants per vaccine group was estimated to provide sufficient power to examine the primary study objective. As this was an exploratory study, no formal statistical hypothesis was tested. The primary immunogenicity objective was to select 1 or 2 study vaccines (out of 3 aP booster vaccines and out of 6 TdaP booster vaccines) by comparison of antibody responses with the licensed TdaP vaccine as well as to each other; comparisons were based on antibody responses to each antigenic component at 30 days after vaccination. Two dose groups were considered statistically different if the 2-sided 95% CI around the difference of their means of the log10 transformed data did not contain the value 0 for at least 1 antigen and, similarly, if the 2-sided 95% CI around the difference of group proportions did not include the value 0 for at least 1 antigen. GMCs, GMRs, percentages of participants with 2- or 4- fold changes for the aP antigens (PT, FHA, PRN) and percentages of participants with anti-diphtheria and anti-tetanus antibodies above cut-off values ≥0.1 IU/mL and ≥1.0 IU/mL were calculated. The estimation of GMTs for the PT neutralization activity at each timepoint and for each vaccine group was done using ANOVA models with fixed factor for regimen group. CIs were calculated using the same models. Safety data was summarized for each vaccine group, providing the frequency and proportion of participants reporting an event. Immunogenicity analyses were run on the PP set, which consisted of participants who received the relevant dose of vaccine correctly, provided at least one evaluable serum sample at the relevant timepoints, and had no major protocol violations (Supplementary. Table 3) A major deviation was defined as a protocol deviation that was considered to have a significant impact on the immunogenicity result of the participant. Safety was analyzed for all participants who provided post-vaccination data. Boostrix is a trade mark of the GSK group of companies. Td-pur is a trade mark of Novartis Vaccines and Diagnostics. ABSTRACT.SUPPLEMENTARY MATERIAL: ABSTRACT.KHVI_A_1385686_SUPPLEMENTAL.DOCX:

TITLE: Effectiveness of an educational video as an instrument to refresh and reinforce the learning of a nursing technique: a randomized controlled trial The Undergraduate Nursing Course has been using videos for the past year or so. Videos are used for many different purposes such as during lessons, nurse refresher courses, reinforcement, and sharing and comparison of knowledge with the professional and scientific community. The purpose of this study was to estimate the efficacy of the video (moving an uncooperative patient from the supine to the lateral position) as an instrument to refresh and reinforce nursing techniques. A two-arm randomized controlled trial (RCT) design was chosen: both groups attended lessons in the classroom as well as in the laboratory; a month later while one group received written information as a refresher, the other group watched the video. Both groups were evaluated in a blinded fashion. A total of 223 students agreed to take part in the study. The difference observed between those who had seen the video and those who had read up on the technique turned out to be an average of 6.19 points in favour of the first (P < 0.05). The results of the RCT demonstrated that students who had seen the video were better able to apply the technique, resulting in a better performance. The video, therefore, represents an important tool to refresh and reinforce previous learning. BODY.INTRODUCTION: Videos are widely used for supporting and stimulating student comprehension in various contexts: in the classroom, in the laboratory and in distance education [1–3]. Educators understand the benefit of combining auditory and visual enhancement within the traditional lecture. The more technologically advanced European universities have already been making wide use of videos since the 1990s [4–6]. Bradley University, in the USA, has been experimenting extensively with video production. The educational model of this university already foresees a tutoring system with or without face-to-face lessons [7]. Easy access to the web allows the scientific community to acknowledge and promote the exchange of opinions [8]. Skiba describes the transformation of higher education and its impact on nursing education. Nursing education is considered by many to be a pioneer in the use of educational technologies. A challenge is the use of emerging technologies, such as Web 2.0 tools, which will help to bridge the gap between the next generation and faculty in nursing schools. Nurse educators need to understand and use the power of technologies to prepare the next generation of nurses. Some authors are concerned about the risk that the use of computers will belittle rather than to improve education; moreover, they argue whether the use of technology will ever be able to replace the fundamental role of the teacher in student learning. Despite this, many studies suggest that the use of technology and internet is a useful instrument which favours education [1, 8, 9]. In the literature, various observational studies have demonstrated that the use of video streaming contributes to learning as a powerful instrument for education and for the acquisition of clinical competencies, reducing the gap between theory and practice [10–12]. It has also been highlighted that knowledge is not only acquired through video observation, but as part of the students’ learning process. Moreover, some authors suggest how the use of the video can support students in learning different types of techniques [11, 12]. There are also some observational studies which demonstrate that the introduction of video streaming in schools has been very positive in terms of effectiveness and facilitation of learning. [9, 13–16]. Some authors describe the simulation as a characteristic feature which may render the video particularly effective in stimulating learning and emphasizing the concepts. Watching videos is not a passive but an experiential process [17, 18]. Video use should help students to learn new skills and enhance learning processes in much the same way as experience is acquired during their practical training. The benefits of using videos are well documented and videos are considered a valid tool for student education [19–22]. Particular reference is given to the pedagogical aspects and the impact of the image, the interaction and integration as key elements of video streaming [23]. Currently, there are few experimental studies which demonstrate video effectiveness applied to traditional nursing techniques [24–26]. Studies carried out in critical care have demonstrated how teaching video use in the emergency situation can enhance performance quality. Einspruch [27] adopted this method with laypersons by estimating the efficacy in a randomized controlled trial (RCT). In an RCT, conducted by medical students, it was demonstrated that a 3 min video play produced a comparable or better performance than that used in the American Heart Association (AHA) course [28]. Cofield [29] carried out an experimental study measuring qualitative variables, suggesting the effectiveness of video streaming to reinforce and enhance learning. Indications of better video use have been analyzed by various experts, in particular by Race and Shephard [1–3]. Two key elements should prevail: keeping to the content and guaranteeing maximum interactivity. Technological progress and the use of streaming videos has virtually erased problems. Interactivity is, therefore, guaranteed by enabling change in real time of any sequence of the video [27]. The advantage of using YouTube clearly emerges as an instrument for sharing videos and allowing students to participate actively in the discussion on films. YouTube has grown during the last few years and is visited by almost 70 million persons a day. With their free access, the main search engines worldwide (Google, Yahoo) allow comparisons among the entire professional and scientific community, thus offering the possibility to comment and make suggestions, and for free and constructive debate, therefore becoming a reliable and updated point of reference. It is a powerful instrument which makes sharing and exchanging ideas possible [30]. Videos are surely among the most interesting technologies, adding value to learning content. Not only should the image be detailed, but it should offer students the possibility to use more channels, thus enhancing and facilitating learning processes. However, there is little evidence on the efficacy of video streaming to support student learning. The aim of this study is to verify the effectiveness of video streaming as an instrument for refreshing and reinforcing nursing education. BODY.METHODS: BODY.DESIGN OF THE STUDY: A two-arm RCT design was chosen and performed in December 2009 at the Undergraduate Nursing Course at Turin University. This is a two-group design: subjects were randomly assigned to the experimental and control groups. Both groups attended lessons in the classroom as well as in the laboratory; a month later the control group received written information to refresh the technique, while the experimental group saw the video. Both groups were given ten minutes to allow them to freshen up on the technique. Students were assessed individually by a blinded evaluator using observational rating scales. BODY.PARTICIPANTS: The population consisted of 250 undergraduate students attending the nursing course at Turin University. Students who consented to participate in the study were fully informed about their right to withdraw at any time and the fact that data were to undergo aggregate processing and would be stored safely. BODY.RANDOMIZATION: Participants were randomized by a computer-generated randomization scheme and the identification number from the randomized allocation schedule. Students were not told whether they would be seeing the video or reading up on the technique. To guarantee the effective homogeneity between the two groups, data variables influencing student performance, such as age, sex, qualifications, laboratory participation, reading information concerning the technique and development of a working activity in the field, were collected. BODY.THE VIDEO: The video chosen involves the technique dealing with ‘moving an uncooperative patient from the supine to the lateral position’. The health care professional uses a sliding sheet to move a mobility-impaired person. The written information was compared with the video, which turned out to be identical regarding both the words and the sequence. The duration of the video was 7.32 min. BODY.INSTRUMENT OF APPRAISAL: The appraisal instrument was an observational grid developed to assess student performance. The instrument was a 33-item observational form with the possibility to mark whether a single action had been carried out or not. Each item expresses single actions which together make up the technique (e.g. inform the patient about what is going to happen/wrap the cloth around both shoulders/lift the edge of the bed…). Less important items were given 1 point whereas core items were given 2 points. The assessment forms were anonymous and were identified through a code assigned at the time of enrolment. Performance was evaluated based on the weighted sum of all the actions making up the procedure. BODY.PILOT STUDY AND EVALUATION OF THE SAMPLE SIZE: A pilot study was carried out in May 2009 on 21 students with the aim of identifying potential shortcomings, testing the appraisal instrument for the collection of information and assessing the sample size necessary to perform the RCT. Considering the measuring instrument could supply a range of values between 0 and 53 and taking 5 as the minimum significant value which we would observe, we needed to enrol 100 students for each arm of the study for an 80 % statistical power. BODY.STATISTICAL ANALYSIS: The items that made up the technique were treated as qualitative variables: actions not performed vs actions done correctly. In comparing the experimental and control groups for qualitative variables, the Pearson’s Chi-square or the Fisher’s exact test were used: in particular, the latter was used if at least one cell showed less than five expected values. The Shapiro–Wilk test was performed to assess the normal distribution of quantitative variables: the Student’s t test was used for variables normally distributed, while the Mann–Whitney test was used if the variable showed a non-normal distribution (Shapiro–Wilk test statistically significant). All tests were conducted at a significance level of 5 %. The data collected were placed on Excel spreadsheets and analyzed with the Stata 9 statistical programme. BODY.RESULTS: Altogether, 223 students joined the study: 74 males and 129 females. They were randomized into the experimental group (watching video, n = 112) and the control group (written information, n = 111). Table 1 illustrates the characteristics of the study sample (experimental group vs. control group) for some variables: gender, age, whether the participant already had a degree, or was already a certified nursing assistant (CNA). The two groups were homogenous for all these variables so that we can presume that possible differences in their performance are attributable to the use of the video.Table 1Characteristics of the study sample: experimental group vs. control group  Experimental group (n = 112)Control group (n = 111)PSex (M/F) 40/72 34/77 N.s.^ Age (mean ± SD) 22.25 ± 5.9 21.92 ± 4.55 N.s.§ Other degree (yes/no) 9/103 12/99 N.s.^ CNA (yes/no) 3/109 2/109 N.s.° N.s. (P > 0.05) CNA certified nursing assistant ^ Pearson’s Chi-square test ° Fisher’s exact test § Student’s t test The results of the study are shown in Table 2.Table 2Overall performance: experimental comparison group vs. control group  Experimental group (n = 112)Control group (n = 111)Mean difference ± SE (CI 95 %)P*All itemsa 42.95 ± 6.7 36.76 ± 9.2 6.19 ± 1.08 (4.06–8.32) 0.00001 Minimum score 22 9 Maximum score 53 53 CI confidence interval, SE standard error * Mann–Whitney test aData are presented as mean ± SD unless otherwise stated Table 2 illustrates, relatively to the two groups (experimental and control), the results of the performance. The mean difference observed between those participants who had seen the video and those who had read about the technique was 6.19 points (±1.08 standard error (SE). The difference between the two groups was statistically significant (P < 0.05), so viewing the video led to a better performance in comparison with simply reading up on the technique. BODY.DISCUSSION: What emerges according to the data we obtained is that videos are beginning to draw serious attention to their learning potential. The result of this study is in conformity with the other experimental studies present in the literature [24–26]. We also demonstrated that those who saw the video were better able to apply the technique. The video lasts 7.32 min. The technical aspect of video duration conforms with what emerged from the literature, which recommends that videos should last no longer than 15 min [22]. The possibility of interrupting the film and seeing some sections again offers the advantage of maintaining a high attention level. However, this means that the user should be able to easily access and view the contents. Therefore, it is necessary for users to simply surf the Net, review, observe and retrieve the material of their choice. As described in the literature, and in particular by Bennet [21], students reported that the use of the video seemed a more effective learning tool since they could easily remember what they saw on the film; moreover, they had the possibility to stop, start and rewind the video to address their specific needs. Various descriptive studies and opinions of experts assert that the video represents an important opportunity for the student to increase and support learning, thus contributing to a better performance of the technique. But the video cannot completely replace face-to-face lessons, although it widely contributes to and complements education [13]. The video library will be available in additional languages, to allow comparison among the scientific and professional community all over the world. Moreover, it would be of support to those students who are not Italian. The educational use of the video surely offers remarkable advantages for online learning even if it clashes with the limits imposed by the web (the possibility of quickly linking to the web). Furthermore, even if effective, the video has to be part of an educational plan correlated with precise objectives which are reflection, verification, understanding and clarification. BODY.CONCLUSIONS: During the last few years, university education has undergone profound changes in the organisation and methodologies used. Video use and video streaming represent one of these instruments. What emerges from the results of this study is that videos are useful tools for refreshing and reinforcing concepts learnt during nursing courses. The use of educational videos could become a routine instrument for student training. A limitation of the research is the possibility that respondents in the intervention group sought more information on their own. A second limitation is that the difference between the two groups could be a short-term effect, because the refresher training was immediately followed up by the assessment. Follow-up research could explore the permanence of the effect of using video as a tool for refresher training. BODY.ESSENTIALS: The aim of the study is to verify the effectiveness of the video as an instrument for refreshing and reinforcing nursing education. The University’s need to provide a useful tool for students. The use of the video contributes to refresher training as a powerful instrument for the education and the acquisition of clinical competencies, The use of educational videos could become a routine instrument for student training.

TITLE: Effect of Lactobacillus gasseri BNR17 on Overweight and Obese Adults: A Randomized, Double-Blind Clinical Trial ABSTRACT.BACKGROUND: Lactobacillus gasseri BNR17 is a type of probiotic strain isolated from human breast milk. A study was reported regarding the fact that BNR17 was an inhibitor of obesity and diabetic activities in the human body through previous animal experiments. This study was furthered to investigate the effect of BNR17, a probiotic strain isolated from human breast milk, on obese and overweight adults. ABSTRACT.METHODS: Sixty-two obese volunteers aged 19 to 60 with body mass index ≥ 23 kg/m2 and fasting blood sugar ≥ 100 mg/dL participated in a placebo controlled, randomized, and double-blind trial. For 12 weeks, 57 participants were given either placebo or BNR17 and were tested by measuring body fat, body weight, various biochemical parameters, vital signs, and computed tomography at the start of the study and at weeks 4, 8, and 12. The subjects assumed usual daily activities without having to make behavioral or dietary modifications during the course of the study. ABSTRACT.RESULTS: At the 12th week, a slight reduction in body weight was noted in the BNR17 group, but there were no significant weight changes between groups. Decrease of waist and hip circumferences in the BNR17 group was more pronounced than those in the placebo group. The two groups had no special or severe adverse reactions. ABSTRACT.CONCLUSION: Despite there being no change in behavior or diet, administration of only the supplement of BNR17 reduced weight and waist and hip circumference. However, there were no significant differences between the two groups. These findings warrant a subsequent longer-term prospective clinical investigation with a large population. BODY.INTRODUCTION: Obesity is a major contributor to the global epidemic of type-2 diabetes,1) fatty liver disease,2) and cardiovascular diseases.3) Worldwide, at least 300 million individuals are clinically obese,4) and in Finland, out of those aged 25 to 74 (years), 25% are obese and over half are overweight.5) Obesity, a chronic disease characterized by excessive body weight, results from a complex interplay of multiple factors, including genetic, metabolic, social, behavioral, and cultural factors. Obesity is caused mainly by high energy intake with low energy consumption. Behavioral and cultural influences, represented by the excessive intake of sweet and salty foods combined with insufficient exercise, best explain the rising occurrence of obesity in developed countries. In recent years, growing awareness that obesity is closely linked to various adult diseases has prompted the rapid development of obesity management programs involving such aspects as drug therapeutics, surgical intervention, and dietary treatment. However, such management strategies often are accompanied by serious side effects or are found ineffective; and even more critically, obese people depend on such programs without changing their eating and exercise habits. In societies where being "underweight" wrongfully equates to being "attractive," many young, socially inept teenagers engage in excessive dietary restrictions that often give rise to anorexia, bulimia, and malnutrition, at a time when balanced nutrition is paramount for healthy growth. Therefore, development of a safe, yet effective dietary supplement to properly manage body weight is a natural calling in this world. The gut's microbial community has been regarded as one of the critical factors related to obesity and metabolic disorders.6-8) Conventionalization of germ-free mice with a distal gut microbiota harvested from conventionally raised mice leads to an increase in body fat.7) Other reports demonstrated that obese mice have a higher proportion of intestinal Firmicutes communities and extracted calories more efficiently from their diet than lean mice, which have a higher percentage of Bacteroidetes communities.9) It has also been demonstrated that the gut microbiota is involved in high-fat, diet-induced metabolic endotoxemia, adipose tissue inflammation, and metabolic disorders in mice.10-12) Lactobacillus (Lb.) and Bifidobacteria are major constituents of gut microbiota and include such probiotics as lactic acid bacteria (LAB), which have been shown to benefit to human health, making them natural subjects of investigation in obesity research. Some researchers reported that probiotics caused weight gain of livestock and children with diarrhea;13) meanwhile some did not demonstrate any link.14,15) Despite the arguments for the association between probiotics and obesity, many papers demonstrating various mechanisms of anti-obesity of LAB have been reported, such as regulation of lipid and glucose metabolism,7,16) production of conjugated linoleic acid,17,18) reduction of adipocyte size and increase of numbers of small adipocytes in white adipose tissue,19,20) and regulation of leptin.21) The Lb. gasseri BNR17 used in this research was isolated from human breast milk. Our previous studies have showed that BNR17 inhibited increase in body weight and white adipose tissue weight in rats fed a high sucrose-diet, and effectively improved various diabetic symptoms, including polydipsia, hyperplasia, polyuria, fasting and postprandial 2-hour blood-glucose levels, and oral glucose tolerance in a type 2 diabetes animal model.22,23) These results suggest that BNR17 may be a good candidate for probiotic bacteria to prevent obesity and its related disorders. In the present clinical study, we investigated the effects of BNR17 intervention on overweight or obese adults including their primary end point such as a rate of weight reduction, and second end point such as change in body fat, abdominal fat and blood glucose. This is the first clinical study on the anti-obesity effects of the Lb. strain isolated from human breast milk. BODY.METHODS: BODY.1. STUDY DESIGN: This study was a single center, randomized, double-blinded, and placebo-controlled clinical trial to examine the efficacy and safety of BNR17 in obese or overweight adults. The trial was approved by the institutional review board in Yeungnam University Medical Center. Of 83 registered obese adults screened, 62 were enrolled. The subjects included men and non-pregnant women aged between 19 and 60 with body mass index (BMI) ≥ 23 kg/m2 and fasting blood sugar (FBS) ≥ 100 mg/dL, and were divided into two groups, each with 31 subjects. Subjects were excluded if they were taking drugs that could affect weight change, including anti-diabetic drugs, lipid-lowering drugs, orantiphycotic drugs, or if they had endocrine, cardiovascular, thyroid, or chronic liver disease. Also subjects were excluded if they had received surgery to reduce weight, were taking probiotics or antibiotics within one month, or if their weight had changed over five percent within three months. During the 2nd week of the screening period, each subject was analyzed for the eligibility criteria after consenting to test, including demographic data, past medical history, medication use, vital signs, blood sugar rate, and body weight. On the last day of the 2nd week of the screening period, eligibility of the subjects was evaluated based on the collected information. Subject recruitment was stopped when all 62 subjects were recruited. No wash-out period was required. Sixty-two eligible subjects were randomly assigned to the BNR17 or the placebo group and prescribed supplements according to a random code (Figure 1). BODY.2. PREPARATION OF SAMPLES AND TREATMENT: BNR17 capsules were composed of 1010 cfu of Lb. gasseri BNR17 and filler powder (50% trehalose, 25% skim milk, and 25% fructooligosaccharide) and placebo capsules were packaged only with the filler produced by the pharmaceutical factory. BNR17 or placebo capsules were dispensed by the investigational pharmacist. Subjects were instructed to take 6 capsules per day (2 capsules 30 minutes before breakfast, lunch, and dinner) for 12 weeks. If the subject withdrew from the study, measurements were taken at the time of the last administration, and follow-up measurements were made within a month. We investigated subjects' diary of administration and pill count at every visit to improve compliance. BODY.3. MEASUREMENTS: Subjects returned to clinic at 0, 4, 8, and 12 weeks post randomization, and at each visit, vital signs such as blood pressure and pulse rate were measured after sitting comfortably for 5 minutes. We collected health-related behaviors (smoking, alcohol intake, and dietary patterns such as calory intake and regular exercise) at every visit by the dietitian. Subjects were recommended to engage in the same health-related behaviors during the study. Bowel habits were examined on the second and fifth visit. For common blood and urinary tests, subjects were asked to fast for at least 8 hours prior to the clinic visit. Electrocardiogram was obtained at screening and on the last visit. X-ray was taken only during screening. Abdominal obesity was determined by measuring waist (navel line) and hip circumferences using a measuring tape. Also body composition including body fat and muscle amount was determined by a bioelectrical impedance test (In-Body 3.0, Biospace, Seoul, Korea). On the second and fifth visit, abdominal obesity was directly measured using a computed tomography scan. Body weight was measured using the same scale throughout the study period. BMI was calculated by dividing body weight (kg) by height (m2, barefoot). Oxygen consumption and basal metabolic rate (BMR) were measured by indirect calorimetry (Sensorimedics Co., Yorba Linda, CA, USA) using a ventilated hood system while the subject was resting comfortably lying down on a bed. Insulin, hemoglobin A1c and FBS were measured at each visit. Two hour postprandial blood sugar (2PPBS) was examined only on the second and fifth visit. A pregnancy test was conducted on women of reproductive age on every visit. After measurements, subjects were given a supply of the test foods to permit dosing until the time of the next scheduled visit. Adverse reactions were observed for by monitoring anthropometric parameters and biochemical indices before the intake of test food and after completion of the 12 week trial. BODY.4. SAMPLE SIZE ESTIMATION: The intended sample of 62 recruited subjects provided a power of approximately 80%, assuming a significance level < 0.05, to detect a 2.5 kg placebo-adjusted treatment effect in body weight (change from baseline to week 12th) with a standard deviation of 3.5 kg. The formula for the sample size was as follows: n = 2(za/2 + zβ)2/{(µ0 - µ1)/σ}2 where two population means (µ0 and µ1), σ2 = common variance. BODY.5. STATISTICAL ANALYSIS: The data were analyzed using intention-to-treat (ITT) and per-protocol (PP) analysis. For ITT analysis, all data from the subjects who received the test food at least once were included. For missing or incomplete data, the most recently available data were used (last observation carried forward analysis). For the PP analysis, only the data obtained from the subjects who completed the study on schedule were included, and the analysis was performed only when the data were available at weeks 0 and 12. Of the 62 subjects enrolled in the study, 8 withdrew (2 from the placebo group and 6 from the BNR17 group), and none were omitted due to noncompliance. Efficacy-related data received ITT analysis while safety and side effect-related data underwent ITT & PP analysis. All side effects, regardless of their relevance to ingestion of the test food, were reported and noted in detail. Differences in baseline characteristics between groups were analyzed using a chi-square test or Fisher's exact test. For efficacy analysis, a paired t-test and independent t-test were used to detect the differences within and between groups in the ITT dataset, and a repeated measures analysis of variance was used in the PP dataset. The same statistical methods used in efficacy analysis for the ITT dataset were performed in safety analysis. BODY.RESULTS: BODY.1. BASELINE CHARACTERISTICS OF THE SUBJECTS: The subjects' baseline characteristics are summarized in Table 1. Although the overall number of males was less than that of females, there were no significant differences in the sex ratio between the two groups. Seventy-eight point nine percent of the subjects were nonsmokers, and 38.6% were nondrinkers. Smoking, drinking, dietary, and exercise patterns were similar in both groups. Past medical history and drug use history revealed no significant differences between the two groups. There were no non-compliance subjects except 2 persons in the placebo group and 6 persons in the anti obesity microorganism group who refused study procedures during the study. BODY.2. EFFICACY ANALYSES: BODY.1) REDUCTION OF BODY WEIGHT: In the placebo group, body weight showed little change after the 12th week (Table 2). In contrast, within-group comparisons showed a slight decrease in body weight (-1.1 ± 2.2 kg) in the BNR17 group. In the between group, however, there were no significant changes in body weight by ITT analysis. BODY.2) CHANGES OF CLINICAL PARAMETERS: There were no significant changes in fat (%) or muscle amount (kg) (Table 2). BMI in the BNR17 group decreased after the 12th week compared with week 0 (-0.5 ± 0.9 kg; P < 0.05). A within-group comparison showed a significant reduction in waist circumference in the BNR17 group (-2 ± 4.4 cm; P < 0.05). But there were no significant changes based on the between-group differences. In both groups, there were no significant differences in visceral adipose tissue and deep adipose tissue based on between- and within-group comparisons. The placebo groups showed an increase (0.05 ± 0.011 L/min; P < 0.01) in oxygen consumption and a decrease in BMR of borderline significance (-33 ± 22; P > 0.05), respectively; however, they were not of clinical significance. BODY.3) CHANGES IN METABOLIC RELATED PARAMETERS: The placebo group exhibited an increased level of 2PPBS of borderline significance (16.3 ± 34.1 mg/dL; P = 0.05) at week 12 (Table 3) compared with week 0; however the increase was not clinically significant. All serum lipids including total cholesterol, high density lipoprotein, low density lipoprotein (LDL), and triglyceride showed no significant clinical differences between the two groups. BODY.3. SAFETY ANALYSES: BODY.1) CHANGES IN BLOOD PRESSURE AND PULSE RATE: There were no significant changes in systolic or diastolic blood pressure or pulse rate between groups during the study period (Table 4). BODY.2) CHANGES IN HEMATOLOGY AND BLOOD CHEMISTRY: There were no significant changes in hematological or blood chemistry parameters except hematocrit changes in the BNR17 group (Table 5). Other adverse reactions were monitored during the course of the study. Of the 57 subjects, none reported adverse reactions directly related to the intervention. One subject in the BNR17 group reported diarrhea, and one in the placebo group had gastrointestinal symptoms (nausea) that were unrelated to the test food. No serious adverse reactions were reported. BODY.DISCUSSION: In this study, 1010 cfu of Lb. gasseri BNR17 were given daily before meals for 12 weeks and effects on obesity-related factors were investigated. There were no significant differences in ratio of gender, age, and history of personal habits including smoking, drinking, medical treatment, or medication use between the placebo and BNR17 group. Lb. gasseri SBT2055 also showed lowering effects on abdominal adiposity, body weight, and waist and hip circumferences in a randomized controlled human trial,19) which was similar with our results. The reduction in waist circumference is meaningful because it is a useful indicator of visceral and subcutaneous fat distribution, and is strongly correlated with atherogenic lipid profiles.24,25) However, there were no changes in visceral or superficial fat in this study. There were no changes in diabetic indices such as FBS or insulin. In animal experiments using db/db mouse,23) however, we observed more remarkable results than in the present study; administration of BNR17 not only reduced FBS and 2PPBS but also improved tolerance to oral glucose. It has been reported that obesity is accompanied by increased concentrations of serum total cholesterol.26,27) In many studies, reduction of serum total cholesterol or LDL-cholesterol by consumption of fermented milk containing probiotic strains was described, suggesting a hypocholesterolaemic effect.13,28) However, no reduction of cholesterol level was observed in the BNR17 group in this study, thus, it appears unlikely that BNR17 exerts a hypocholesterolaemic effect. Administration of BNR17 reduced coliform bacterial counts in the feces of subjects. This result indicates that BNR17 suppresses the proliferation of harmful bacteria in the gut, confirming its probiotic activity. It was reported that Lb. plantarum No. 14 may exert a beneficial effect on the onset of diet-induced obesity by reducing the cell size of white adipose tissues in mice.20) They considered that the inhibition of lipid absorption is a possible mechanism for those effects. We also observed the reduction of white adipose tissue weight by the administration of BNR17 in high-fat diet-fed rats.22) Adipose tissue serves as a metabolic buffer, sequestering fatty acids in the postprandial state, and releasing them under fasting conditions.28) It has been reported that gut microbiota have an essential role in obesity development. The mechanisms that gut microbiota may influence which affect weight gain include regulation of energy harvest from the gut,9) digestion of indigestible polysaccharides in diet,10) production or activation of signaling molecules involved in host metabolism,18) modification of gut permeability,10) and release of hormones and inflammation.21,29,30) However, whether typical probiotics such as Lb. and Bifidobacteria, which are members of gut microbiota, exert a similar effect in the human intestine is largely unknown. In a study that determined the influence of an obesity treatment program on gut microbiota and body weight of overweight adolescents,31) an increase in Lb. group counts in feces was correlated with weight loss and BMI z-score reductions in the high weight loss group, suggesting the role of this bacterial group in body weight management. Another researcher reported that high numbers of Bifidobacteria and low numbers of Staphylococcus aureus in infancy may provide protection against obesity development.9) The feeding of mice with a high-fat diet led to increased serum lipopolysaccharides (LPS) levels and metabolic endo toxemia, resulting in an increase in proinflammatory cytokine concentration in various tissues.4) Supplementation of the high-fat diet with oligofructose resulted in increased concentration of Bifidobacteria and normalized concentration of LPS and consequently improved glucose tolerance and insulin sensitivity.10) Supplementation with probiotic strains has the same effect as although endogenous bacteria is not completely clear,8) however it seems that BNR17 may have a similar effect on energy metabolism and inflammation, because Lb. gasseri is one of the major constituents of gut microbiota. This will be the first clinical study on the slight weight-reducing effects of Lb. gasseri BNR17, a probiotic strain originating from human breast milk, and could provide a new and safe means to manage body weight. Even though this study had limitations such as a short trial period and unevaluated indices, it still suggests that BNR17 is effective in reducing body weight in obese or overweight patients.

TITLE: Water Drinking Test: Intraocular Pressure Changes after Tube Surgery and Trabeculectomy ABSTRACT.PURPOSE:: To study the effects of filtration surgeries (tube and trabeculectomy) on changes in intraocular pressure after a water-drinking test. ABSTRACT.METHODS:: In this prospective, non-randomized, comparative clinical study, 30 patients who had tube surgery and 30 age- and sex-matched trabeculectomy patients underwent a water-drinking test. Only one eye of each patient was included. The baseline intraocular pressure was ≤21 mmHg in all enrolled eyes with or without adjunctive topical medications. After the water-drinking test, the intraocular pressure was measured and recorded at 15, 30, 45, and 60 minutes and the results were compared between the two groups. ABSTRACT.RESULTS:: In both groups, intraocular pressure significantly increased from baseline at all measured time-points (P < 0.001). In the trabeculectomy group, the average intraocular pressure increased from 14.8 ± 2.9 to 18.8 ± 4.7 mmHg at 30 minutes, but decreased at 60 min (18.0 ± 5.2 mmHg). In the Tube group, intraocular pressure increased incrementally until the last measurement (14.2 ± 3.9, 18.8 ± 5.6, and 19.7 ± 6.0 mmHg at baseline, 30, and 60 minutes, respectively). The end-pressure difference (intraocular pressure at 60 minutes vs. baseline) was significantly greater in the tube group (5.6 ± 3.6 mmHg; 41% change) than in the trabeculectomy group (3.2 ± 4.7; 23% change; P = 0.03). ABSTRACT.CONCLUSION:: Intraocular pressure significantly increased after the water-drinking test in both the groups. Intraocular pressure started to decline 30 minutes after the water-drinking test in the trabeculectomy group, while it continued to increase up to 60 minutes in the Tube group. This finding may have implications regarding the efficacy or safety of the procedures in advanced glaucoma patients. BODY.INTRODUCTION: The major goal in glaucoma management is lowering intraocular pressure (IOP) to a safe level (target IOP) in order to prevent further optic nerve damage. A lower target IOP is needed (due to fragility of the already damaged optic nerve) when a greater degree of damage is present.[1] In addition to IOP and IOP-independent factors that are important in glaucoma progression, the fluctuation of IOP has also been suggested to play a major role.[23] It was demonstrated that about one-third of patients with normal single IOP measurements at office hours had pressure peaks detected only during a 24-hour diurnal curve assessment.[4] Studies have shown that the IOP peak of most untreated and treated glaucoma patients occurs during the nocturnal/sleep period.[56] Relying on office IOP measurements will not be accurate in patients with highly variable IOP, particularly those with nocturnal peaks. Checking a 24-hour diurnal curve, modified diurnal tension curve (4 to 5 IOP measurements during office hours from 8 AM to 6 PM), home tonometry, contact lens sensor, and the water-drinking test (WDT) have been suggested as tools for determining the diurnal curve.[789] Most of the aforementioned tools are not always feasible, except the WDT. Five decades ago, the WDT was a popular diagnostic test for glaucoma, but later discontinued because of low sensitivity and specificity.[10] The emphasis on the value of this test has changed now. The IOP peaks detected during the WDT correlate well with the peaks detected during diurnal tension curves.[1112] It is believed that the WDT is an index of outflow facility representing the conductivity out of the anterior chamber.[13] A treatment that improves the outflow facility can be expected to result in smaller diurnal IOP fluctuation.[14] WDT has been suggested as a tool for assessing the efficacy of clinical or surgical therapy for the prevention of intraocular pressure spikes.[15] The “Tube Versus Trabeculectomy (TVT)” study demonstrated a higher success rate with the tube surgery when compared to that of the trabeculectomy with mitomycin C after five years.[16] Since the publication of the TVT study, a trend toward tube surgery with a proportional decrease in trabeculectomy surgery has been observed.[17] Currently, tube surgery is advocated as the primary surgery in fit candidate.[18] Recent Medicare data and surveys of glaucoma specialists demonstrates a trend toward tube surgery with a concurrent decrease in the frequency of trabeculectomy surgery since the publication of the TVT.[17] A review of the Medicare Current Procedural Terminology codes showed that between 1995 and 2004, the volume of trabeculectomy surgery decreased by 43%, whereas that for tube surgeries increased by 184%.[19] With respect to the rising popularity of tube surgery, it would be interesting to find out how the WDT response (as an index for outflow facility[2021]) for this procedure differs from that of the trabeculectomy. BODY.METHODS: BODY.STUDY POPULATION: This prospective, non-randomized, comparative clinical study was conducted in a tertiary eye care hospital on patients that were treated with either trabeculectomy or tube surgery and had an IOP equal to or below the established target pressure (21 mmHg) with or without glaucoma medications based on isolated office readings. All surgical procedures were performed by one surgeon (MRR). The tube group (Tube) had an Ahmed Glaucoma Valve (FP7, New World Medical, Rancho Cucamonga, LA, USA) and the trabeculectomy group (Trab) had a procedure with adjunctive mitomycin C (0.2 mg/ml for 2 minutes). Both groups had at least 6 months of follow-up after surgery. All patients underwent a complete ophthalmological examination including a Snellen visual acuity (VA) test, IOP measurement, and a dilated stereoscopic fundus examination to assess the amount of damage to the optic nerve head using Disc Damage Likelihood Scale.[22] The average thickness of the retinal nerve fiber layer by optical coherence tomography; mean deviation and pattern standard deviation of Humphrey visual field were recorded. The exclusion criteria were a previous refractive surgery or laser trabeculoplasty, presence of ocular infection at the time of study, any corneal abnormalities preventing a reliable IOP measurement, more than one trabeculectomy or tube surgery, follow-up less than 6 months after trabeculectomy or tube surgery, pregnancy, cardiac or renal diseases, and history of urinary retention. BODY.WATER-DRINKING TEST: The patients refrained from fluid or food ingestion 3 hours before the WDT. After checking the IOP (IOP baseline), patients ingested 1 liter of bottled water (15 mL/kg for patients <16 years, because of lower weight and body mass index compared to adults[2324]) in 5 minutes. Then, IOP was measured every 15 minutes for one hour. Overall, five IOP measurements were performed (baseline, 15, 30, 45, and 60 minutes). One examiner measured the IOPs with a non-contact tonometer (CT80; Topcon Co., Tokyo, Japan). The average of 3 measurements was recorded; the measurement was repeated if the difference between 3 measurements was greater than 3 mmHg. In a recent meta-analysis on a comparison of all available tonometers with a Goldmann Applanation Tonometer, the least amount of variability in IOP measurement (mean difference of 0.2 mmHg) was seen with non-contact tonometers.[25] The following parameters were obtained from both groups: IOP peak (highest IOP after drinking water), IOP trough (the lowest IOP after drinking water), IOP mean (the mean of the 4 IOPs after drinking water), IOP fluctuation (difference between IOP peak and baseline), IOP range (difference between IOP peak and IOP trough after drinking water), end-pressure difference (IOP at 60 minutes versus baseline). BODY.STATISTICAL ANALYSIS: IOP was measured in both eyes; if both eyes met the eligibility criteria, one eye was randomly selected for inclusion in the study. All data were recorded and analyzed using IBM SPSS statistics software version 21 (SPSS Inc., Chicago, IL). Descriptive results were presented as the mean ± standard deviation (SD). IOP fluctuation after WDT at different time-points in each group was analyzed using repeated-measures ANOVA. An independent T-test was used to compare the IOP changes between the two groups. AP value of <0.05 was considered statistically significant. BODY.RESULTS: Overall, data from 30 patients who underwent trabeculectomy and 30 patients who underwent tube surgery were compared. Baseline characteristics of the patients in each group are shown in Table 1. The two groups were not statistically different for most baseline parameters including age, sex, weight, height, body mass index, refractive error, severity of glaucoma, baseline IOP, and number of topical medications. However, compared to the Trab group, the Tube group had a higher proportion of cases with primary closed angle glaucoma (PCAG), pseuduoexfoliative glaucoma, and congenital glaucoma. In the Trab group, 66.7% were phakic, and in the Tube group, 56.7% were pseudophakic [Table 1]. However, in linear regression analysis, no statistically significant association between the primary diagnosis or lens status and any of the WDT-IOP parameters was observed. Table 1Baseline characteristics of patients in the trabeculectomy and tube groups IOP significantly increased from baseline at all measurement time points in both groups [Table 2]. In the Trab group, IOP increased up to 30 minutes, but decreased afterwards. In the Tube group, IOP increased in increments until the last measurement at 60 minutes [Figure 1]. The inter-group gaps in the IOP changes from baseline widened through time; the highest difference between the two groups was observed at 60 minutes after WDT (40.9% in Trab vs. 23% in Tube, P = 0.04; Figure 2 and Table 3). Table 2Comparison of intraocular pressure measurements at baseline and after the water drinking test between the trabeculectomy and tube groups Figure 1Comparison of the mean of the intraocular pressure at baseline and at each time point between the trabeculectomy and tube groups. Figure 2Comparison of the intraocular pressure change from baseline (in percent) at each time point between the trabeculectomy and tube groups. Table 3Comparison of changes in intraocular pressure after the water drinking test between the trabeculectomy and tube groups Table 4 presents the comparative results of different WDT-IOP parameters in the two groups. There was no significant difference for all studied parameters between both groups except for the end-pressure difference, which was significantly greater in the Tube group. There was a trend towards a greater frequency of IOP peaks >25 mmHg (23.3% vs. 13.3%) and >30 mmHg (6.7% vs. 3.3%) in the Tube group, when compared to that of the Trab group; however, the difference was not statistically significant [Figure 3]. Table 4Comparison of different IOP parameters between the trabeculectomy and tube groups Figure 3Comparison of the categorized IOP peak between the trabeculectomy and tube groups. BODY.DISCUSSION: The purpose of this study was to compare the response to a WDT in eyes that had undergone trabeculectomy or tube surgery. Previous reports have evaluated the WDT response in medically treated glaucoma eyes and after trabeculectomy.[26272829] In a recent study by Martinez et al,[30] 40 eyes of 34 open-angle glaucoma subjects who had undergone trabeculectomy (n = 20) or tube surgery (n = 20) underwent WDT. The IOP response to the WDT was similar in eyes previously treated with trabeculectomy or tube surgery. They included both eyes of some patient, but in the current study, only one eye of each patient is included. There was an upward trend in IOP up to 30 minutes after WDT for both groups; then, the IOP decreased in the Trab group, while the upward trend continued in the Tube group. The final IOP of the Tube group at 60 minutes was significantly higher than that of the Trab group. A similar trend was observed in the study by Martinez et al[30] in the first 30 minutes after the WDT; after 30 minutes, the IOP decreased in both groups. This difference and lack of increase in IOP in the Tube group after the first 30 minutes could be due to the inclusion of both eyes (having a similar IOP profile) of some patients. Additionally, the patient drank the water over 15 minutes instead of the usual five minutes reported in almost all previous WDT studies. The fluctuation of IOP during the test was also evaluated with no statistically significant differences observed between the Trab (mean of 6 mmHg) and Tube (mean of 6.8 mmHg) groups. This value in the Trab group was higher than previously reported. The IOP fluctuation in the Trab and Tube groups in the study by Martinez et al[30] were 3.95 and 3.6 mmHg, respectively. Medeiros et al[26] reported an IOP change of 1.4 ± 0.4 mmHg in 30 patients with one or two trabeculectomy, which was significantly lower than that of a group of patients with medically-controlled glaucoma (3.7 mmHg, P < 0.0001). Mansouri et al[29] reported the results of 20 subjects treated with latanoprost and compared 20 patients who underwent trabeculectomy and 20 patients who underwent non-penetrating glaucoma surgery. The patients in these surgical groups were not on any glaucoma medications. The WDT-IOP fluctuation was 5.2 mmHg in the latanoprost group, 2.4 mmHg in the trabeculectomy group, and 3.8 mmHg in the non-penetrating glaucoma surgery group (P < 0.05). The latanoprost group had the highest baseline IOP baseline (15.5 mmHg) followed by the non-penetrating glaucoma surgery (13.9 mmHg) and trabeculectomy (10.1 mmHg) groups. More IOP fluctuation was observed in the following groups with a higher baseline IOP: latanoprost (5.2 mmHg), non-penetrating glaucoma surgery (3.8 mmHg), and trabeculectomy (2.4 mmHg). Danesh-Meyer et al[31] compared 30 medically treated glaucoma patients (on different number of different class of anti-glaucoma medications) with 30 trabeculectomy patients who had a controlled IOP without glaucoma medications. The baseline IOP was 10.4 mmHg in the trabeculectomy group and 11.1 mmHg in the group that was treated with medications. WDT-IOP fluctuation was 6.2 mmHg in the medication group and 1.3 mmHg in the trabeculectomy group (P < 0.0001). In all the three aforementioned studies, the trabeculectomy groups had a lower IOP baseline compared to our Trab group and were not on glaucoma medication. A greater degree of IOP fluctuation in our Trab group, compared to the results observed in the aforementioned studies, may be due to a higher baseline IOP and their glaucoma medication. In another study by our group on 203 glaucoma patients, the IOP baseline had an independent association with the peak IOP (r = 0.51, P < 0.001) (unpublished data). The use of glaucoma medication indicates insufficient pressure control following the filtration procedure, suggesting a more limited outflow. In other words, a greater number of medications required to maintain the target IOP may be an indirect measure of an increased resistance of the outflow pathway. The IOP peak, IOP mean, IOP fluctuation, IOP range, end-pressure difference, and percentage of IOP increase at all measurement time points were greater in the Tube group; however, only the end-pressure difference showed statistical significance. The highest IOP in the Trab was detected at 30 minutes (average of a 28.8% increment in the IOP compared to the IOP baseline) with a subsequent decrease. In the Tube group, the percentage of IOP increase at 30, 45, and 60 minutes were 34.5%, 36.3%, and 40.9%, respectively. The exact mechanism of IOP increase after WDT has not been elucidated. A decrease in outflow secondary to the sympathetic activation after water intake due to the rise of vascular resistance, an increase in choroidal thickness, and blood osmotic changes are the proposed mechanisms.[273233] Irrespective of the mechanism, the ability of the eye to recover from the transient IOP rise after WDT depends on the outflow facility.[20] Brubake[1420] suggested using the WDT as an indirect measurement test of the outflow facility in order to compare the IOP responses of glaucomatous eyes to different drugs. The different nature of the bleb in the Tube group may explain the higher IOP change during the WDT when compared to that of Trab group. In a trabeculectomy, the diverted aqueous humor to the subconjunctival and sub-Tenon's spaces is absorbed primarily through vascular channels and secondarily through lymphatics and transconjunctival egress (oozing or bleb sweating).[34] The subepithelial connective tissue seems to be less dense and totally avascular in a trabeculectomy augmented with mitomycin C (MMC) compared to those without antimetabolites.[35] All our patients had a MMC trabeculectomy, which results in attenuated scar formation that facilitates aqueous egress. However, the blebs of the tube seemed to be thicker, which may affect the aqueous egress. The bleb surrounding the implant was as thick as the sclera in a case that underwent enucleation 11 months after tube surgery; histopathology showed a thick layer of collagenous tissue.[36] Anterior segment optical coherence tomography of the tube blebs revealed a relatively regular surface, homogenous and hyperreflective bleb wall, and a signal-void, fluid-filled cavity. Minimum bleb thickness in successful tube surgeries is reported to be approximately 0.56 mm by Jung et al.[37] There were no microcysts and no collection of multiloculated fluid, which are common in a trabeculectomy.[38] IOP peaks have been associated with the progression of glaucoma in the visual fields. In a study, about one-third of patients with progressive visual field loss had IOP peaks during home tonometry compared to 5% of patients with stable visual fields.[39] Yoshikawa et al[40] evaluated several clinical tests for predicting the progression of visual field loss in patients with normal tension glaucoma and concluded that the WDT was the most useful clinical predictor for progression of visual field defects. The Advanced Glaucoma Intervention Study Group suggests maintaining IOP between 10-15 mmHg, with IOP peaks maintained at <18 mmHg to prevent visual field deterioration in patients with moderate or advanced stage glaucoma.[41] The Early Manifest Glaucoma Trial showed that even a 1-mmHg increase in IOP was associated with an 11% increase in the hazard ratio for the progression of glaucoma.[42] A smooth IOP profile in Trab after WDT as an indicator of outflow facility may have a protective effect on a damaged optic nerve compared to Tube. One limitation of the present study was that we did not monitor the diurnal curve IOP in addition to the WDT. Earlier reports concluded that the WDT is a reliable tool to detect IOP peaks of 24-hour IOP profile.[264344454647] Additionally, healthy subjects without glaucoma and treated POAG patients do not manifest a repeatable diurnal IOP pattern from day to day.[4849] This observation limits the clinical value of 1-day diurnal IOP testing in clinical practice. Another limitation was receiving glaucoma medications in our Trab group. Including trabeculectomy patients with controlled IOP without using glaucoma medication may result in a lower WDT-IOP profile, because of lower outflow resistance. Generally, almost all tube patients need medication to maintain their target IOP[16] and recruiting patients with their IOP at target without medication into the tube group may not be practical. The type of glaucoma was not similar in both groups, and may be regarded as a limitation. Although WDT was initially used as a provocative test for detecting glaucoma, it has now been utilized as a fluid stress test that challenges the eyes with a damaged outflow system in any form of glaucoma. Therefore, it does not seem that different glaucoma types have different IOP profiles in the WDT. As mentioned in the Methods section, we used the average of 3 IOP measurements from an airpuff tonometer, which when compared to all available tonometers had only a 0.2-mmHg difference with the Goldmann Applanation Tonometer.[25] However, the ideal study would be one that uses the Goldmann tonometer. The WDT is not without its limitations; it should be avoided in patients with systemic conditions, such as cardiac, renal, prostatic, or respiratory illnesses. However, it can be used for a specific subgroup of glaucoma patients and is cheaper and more feasible than many other methods for evaluating the diurnal IOP profile. In conclusion, the present study suggests that both the Trab and Tube groups had IOP increases, despite showing seemingly stable IOPs in a standard clinical setting. The WDT-IOP profile was lower in the Trab group than in the Tube group; this difference became significant 60 minutes after drinking water. Further investigation on the 24-hour diurnal curve and WDT after trabeculectomy and tube surgeries, including those who have controlled IOPs without glaucoma medications, may provide more insights into the IOP profile after the filtration surgeries. BODY.FINANCIAL SUPPORT AND SPONSORSHIP: Nil. BODY.CONFLICTS OF INTEREST: There are no conflicts of interest.